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1.
Methods Mol Biol ; 2206: 47-56, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754810

RESUMO

The development and maintenance of a functioning vascular system is a critical function for many aspects of tissue growth and regeneration. Vascular endothelial cell in vitro co-culture spheroids are self-organized cell composites that have the capacity to recapitulate the three-dimensional tissue microenvironment. These spheroid testing platforms aim to better understand the mechanisms of functional tissue and how new therapeutic agents can drive these 3D co-culture processes. Here we describe direct cell-cell 3D endothelial co-culture spheroid methods, to examine the physiological spatial growth and cell-cell interaction of vascular cells and surrounding native tissue cells in the formation of vascular networks within spheroids and the potential to regenerate tissue.

2.
Int J Mol Sci ; 21(15)2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752092

RESUMO

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

3.
J Control Release ; 325: 335-346, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32629135

RESUMO

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like' printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.

4.
Biofabrication ; 12(3): 035010, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32259804

RESUMO

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cell-laden constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclay-based bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 d in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 d of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p < 0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p < 0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.

5.
Sci Rep ; 9(1): 17745, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31780671

RESUMO

Deficient bone vasculature is a key component in pathological conditions ranging from developmental skeletal abnormalities to impaired bone repair. Vascularisation is dependent upon vascular endothelial growth factor (VEGF), which drives both angiogenesis and osteogenesis. The aim of this study was to examine the efficacy of blood vessel and bone formation following transfection with VEGF RNA or delivery of recombinant human VEGF165 protein (rhVEGF165) across in vitro and in vivo model systems. To quantify blood vessels within bone, an innovative approach was developed using high-resolution X-ray computed tomography (XCT) to generate quantifiable three-dimensional reconstructions. Application of rhVEGF165 enhanced osteogenesis, as evidenced by increased human osteoblast-like MG-63 cell proliferation in vitro and calvarial bone thickness following in vivo administration. In contrast, transfection with VEGF RNA triggered angiogenic effects by promoting VEGF protein secretion from MG-63VEGF165 cells in vitro, which resulted in significantly increased angiogenesis in the chorioallantoic (CAM) assay in ovo. Furthermore, direct transfection of bone with VEGF RNA in vivo increased intraosseous vascular branching. This study demonstrates the importance of continuous supply as opposed to a single high dose of VEGF on angiogenesis and osteogenesis and, illustrates the potential of XCT in delineating in 3D, blood vessel connectivity in bone.

6.
J Bone Miner Res ; 34(11): 2117-2132, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31269275

RESUMO

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

7.
Adv Healthc Mater ; 8(9): e1800088, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-29756272

RESUMO

Decellularized matrices offer a beneficial substitute for biomimetic scaffolds in tissue engineering. The current study examines the potential of decellularized placental vessel sleeves (PVS) as a periosteal protective sleeve to enhance bone regeneration in embryonic day 18 chick femurs contained within the PVS and cultured organotypically over a 10 day period. The femurs are inserted into decellularized biocompatibility-tested PVS and maintained in an organotypic culture for a period of 10 days. In femurs containing decellularized PVS, a significant increase in bone volume (p < 0.001) is evident, demonstrated by microcomputed tomography (µCT) compared to femurs without PVS. Histological and immunohistological analyses reveal extensive integration of decellularized PVS with the bone periosteum, and enhanced conservation of bone architecture within the PVS. In addition, the expressions of hypoxia inducible factor-1 alpha (HIF-1α), type II collagen (COL-II), and proteoglycans are observed, indicating a possible repair mechanism via a cartilaginous stage of the bone tissue within the sleeve. The use of decellularized matrices like PVS offers a promising therapeutic strategy in surgical tissue replacement, promoting biocompatibility and architecture of the tissue as well as a factor-rich niche environment with negligible immunogenicity.

8.
FASEB J ; 33(3): 3279-3290, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30403537

RESUMO

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal-endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal-endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly ( P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies.-Inglis, S., Kanczler, J. M., Oreffo, R. O. C. 3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche.


Assuntos
Regeneração Óssea/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células-Tronco Mesenquimais/fisiologia , Animais , Embrião de Galinha , Técnicas de Cocultura , Fêmur/embriologia , Fêmur/lesões , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Imageamento Tridimensional , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Esferoides Celulares/citologia , Esferoides Celulares/fisiologia , Nicho de Células-Tronco/fisiologia , Microtomografia por Raio-X
9.
J Tissue Eng Regen Med ; 12(8): 1877-1890, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29893478

RESUMO

Traditionally used as an angiogenic assay, the chorioallantoic membrane (CAM) assay of the chick embryo offers significant potential as an in vivo model for xenograft organ culture. Viable human bone can be cultivated on the CAM and increases in bone volume are evident; however, it remains unclear by what mechanism this change occurs and whether this reflects the physiological process of bone remodelling. In this study we tested the hypothesis that CAM-induced bone remodelling is a consequence of host and graft mediated processes. Bone cylinders harvested from femoral heads post surgery were placed on the CAM of green fluorescent protein (GFP)-chick embryos for 9 days, followed by micro computed tomography (µCT) and histological analysis. Three-dimensional registration of consecutive µCT-scans showed newly mineralised tissue in CAM-implanted bone cylinders, as well as new osteoid deposition histologically. Immunohistochemistry demonstrated the presence of bone resorption and formation markers (Cathepsin K, SOX9 and RUNX2) co-localising with GFP staining, expressed by avian cells only. To investigate the role of the human cells in the process of bone formation, decellularised bone cylinders were implanted on the CAM and comparable increases in bone volume were observed, indicating that avian cells were responsible for the bone mineralisation process. Finally, CAM-implantation of acellular collagen sponges, containing bone morphogenetic protein 2, resulted in the deposition of extracellular matrix and tissue mineralisation. These studies indicate that the CAM can respond to osteogenic stimuli and support formation or resorption of implanted human bone, providing a humanised CAM model for regenerative medicine research and a novel short-term in vivo model for tissue engineering and biomaterial testing.


Assuntos
Reabsorção Óssea , Membrana Corioalantoide/metabolismo , Osteogênese , Idoso , Idoso de 80 Anos ou mais , Animais , Embrião de Galinha , Feminino , Xenoenxertos , Humanos , Masculino , Pessoa de Meia-Idade
10.
Tissue Eng Part C Methods ; 23(12): 938-952, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28877648

RESUMO

The fields of regenerative medicine and tissue engineering offer significant promise to address the urgent unmet need for therapeutic strategies in a number of debilitating conditions, diseases, and tissue needs of an aging population. Critically, the safety and efficacy of these pioneering strategies need to be assessed before clinical application, often necessitating animal research as a prerequisite. The growing number of newly developed potential treatments, together with the ethical concerns involved in the application of in vivo studies, requires the implementation of alternative models to facilitate such screening of new treatments. The present review examines the current in vitro and in vivo models of preclinical research with particular emphasis on the chorioallantoic membrane (CAM) assay as a minimally invasive, short-term in vivo alternative. Traditionally used as an angiogenic assay, the CAM of the developing chick embryo provides a noninnervated rapidly growing vascular bed, which can serve as a surrogate blood supply for organ culture, and hence a platform for biomaterial testing. This review offers an overview of the CAM assay and its applications in biomedicine as an in vivo model for organ culture and angiogenesis. Moreover, the application of imaging techniques (magnetic resonance imaging, microcomputed tomography, fluorescence labeling for tracking) will be discussed for the evaluation of biomaterials cultured on the CAM. Finally, an overview of the CAM assay methodology will be provided to facilitate the adoption of this technique across laboratories and the regenerative medicine community, and thus aid the reduction, replacement, and refinement of animal experiments in research.


Assuntos
Materiais Biocompatíveis/farmacologia , Bioensaio , Membrana Corioalantoide/metabolismo , Teste de Materiais , Engenharia Tecidual/métodos , Animais , Modelos Animais
11.
Sci Rep ; 6: 32168, 2016 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-27577960

RESUMO

Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (µCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by µCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.


Assuntos
Bioensaio , Regeneração Óssea , Membrana Corioalantoide/metabolismo , Fêmur/metabolismo , Modelos Biológicos , Engenharia Tecidual/métodos , Animais , Embrião de Galinha , Fêmur/transplante , Xenoenxertos , Humanos
12.
Stem Cell Res Ther ; 7: 13, 2016 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-26781715

RESUMO

BACKGROUND: A dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation. METHODS: Early osteogenic activities of the co-cultures (± VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (µCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31. RESULTS: ALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs. CONCLUSION: These studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF supplementation on markers of angiogenesis and osteogenesis in co-cultures and organ cultures, demonstrate the importance of the intricate temporal coordination of osteogenic and angiogenic processes during bone formation and implications therein for effective approaches to bone regenerative therapies.


Assuntos
Células Progenitoras Endoteliais/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Neovascularização Fisiológica , Osteogênese , Fosfatase Alcalina/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Feminino , Fêmur/citologia , Expressão Gênica , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/metabolismo
13.
PLoS One ; 10(12): e0145080, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26675008

RESUMO

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-ß3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.


Assuntos
Regeneração Óssea , Matriz Extracelular , Hidrogéis/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Osteoblastos/citologia , Alginatos/efeitos adversos , Alginatos/química , Animais , Condrogênese , Ácido Glucurônico/efeitos adversos , Ácido Glucurônico/química , Ácidos Hexurônicos/efeitos adversos , Ácidos Hexurônicos/química , Humanos , Hidrogéis/efeitos adversos , Ácido Láctico/efeitos adversos , Ácido Láctico/química , Camundongos , Pessoa de Meia-Idade , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/transplante , Osteogênese , Ácido Poliglicólico/efeitos adversos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Tecidos Suporte/efeitos adversos , Tecidos Suporte/química
14.
Stem Cell Res Ther ; 6: 251, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26684339

RESUMO

BACKGROUND: Adult skeletal stem cells (SSCs) often exhibit limited in vitro expansion with undesirable phenotypic changes and loss of differentiation capacity. Foetal tissues offer an alternative cell source, providing SSCs which exhibit desirable differentiation capacity over prolonged periods, ideal for extensive in vitro and ex vivo investigation of fundamental bone biology and skeletal development. METHODS: We have examined the derivation of distinct cell populations from human foetal femora. Regionally isolated populations including epiphyseal and diaphyseal cells were carefully dissected. Expression of the SSC marker Stro-1 was also found in human foetal femora over a range of developmental stages and subsequently utilised for immuno-selection. RESULTS: Regional populations exhibited chondrogenic (epiphyseal) and osteogenic (diaphyseal) phenotypes following in vitro and ex vivo characterisation and molecular analysis, indicative of native SSC maturation during skeletal development. However, each population exhibited potential for induced multi-lineage differentiation towards bone (bone nodule formation), cartilage (proteoglycan and mucopolysaccharide deposition) and fat (lipid deposition), suggesting the presence of a shared stem cell sub-population. This shared sub-population may be comprised of Stro-1+ cells, which were later identified and immuno-selected from whole foetal femora exhibiting multi-lineage differentiation capacity in vitro and ex vivo. CONCLUSIONS: Distinct populations were isolated from human foetal femora expressing osteochondral differentiation capacity. Stro-1 immuno-selected SSCs were isolated from whole femora expressing desirable multi-lineage differentiation capacity over prolonged in vitro expansion, superior to their adult-derived counterparts, providing a valuable cell source with which to study bone biology and skeletal development.


Assuntos
Células-Tronco Fetais/citologia , Mioblastos Esqueléticos/citologia , Adipogenia , Animais , Antígenos de Superfície/metabolismo , Regeneração Óssea , Diferenciação Celular , Separação Celular , Embrião de Galinha , Condrogênese , Diáfises/citologia , Epífises/citologia , Fêmur/citologia , Fêmur/embriologia , Células-Tronco Fetais/fisiologia , Feto/citologia , Humanos , Técnicas In Vitro , Mioblastos Esqueléticos/fisiologia , Osteogênese
15.
PLoS One ; 10(4): e0121653, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25835745

RESUMO

Transforming growth factor-beta3 (TGF-ß3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-ß3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-ß3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (µCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-ß3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-ß3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-ß3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.


Assuntos
Condrogênese/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta3/farmacologia , Vitamina D/análogos & derivados , Animais , Biomarcadores/metabolismo , Embrião de Galinha , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fêmur/citologia , Fêmur/crescimento & desenvolvimento , Fêmur/metabolismo , Expressão Gênica , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Técnicas de Cultura de Tecidos , Vitamina D/farmacologia
16.
Eur Cell Mater ; 26: 91-106; discussion 106, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24027022

RESUMO

Scientific research and progress, particularly in the drug discovery and regenerative medicine fields, is typically dependent on suitable animal models to develop new and improved clinical therapies for injuries and diseases. In vivo model systems are frequently utilised, but these models are expensive, highly complex and pose a number of ethical considerations leading to the development and use of a number of alternative ex vivo model systems. The ex vivo embryonic chick long bone and limb bud models have been utilised in the scientific research field as a model to understand skeletal development for over eighty years. The rapid development of avian skeletal tissues, coupled with the ease of experimental manipulation, availability of genome sequence and the presence of multiple cell and tissue types has seen such model systems gain significant research interest in the last few years in the tissue engineering field. The models have been explored both as systems for understanding the developmental bone niche and as potential testing tools for tissue engineering strategies for bone repair and regeneration. This review details the evolution of the chick limb organ culture system and presents recent innovative developments and emerging techniques and technologies applied to these models that are aiding our understanding of skeletal developmental and regenerative medicine research and application.


Assuntos
Regeneração Óssea , Fêmur/fisiologia , Técnicas de Cultura de Órgãos , Osteogênese , Medicina Regenerativa/métodos , Animais , Galinhas , Condrogênese , Engenharia Tecidual/métodos
17.
Arthritis Rheum ; 65(9): 2290-300, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23740547

RESUMO

OBJECTIVE: To generate doxycycline-inducible human tumor necrosis factor α (TNFα)-transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice. METHODS: A transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases. RESULTS: Doxycycline-inducible human TNFα-transgenic mice developed an inflammatory arthritis- and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of "sausage digits" with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3-6 weeks later, only minimal signs of disease were visible. CONCLUSION: Upon doxycycline administration, the doxycycline-inducible human TNFα-transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα-transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα.


Assuntos
Artrite Experimental/genética , Artrite Psoriásica/genética , Fator de Necrose Tumoral alfa/genética , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Psoriásica/metabolismo , Artrite Psoriásica/patologia , Cartilagem/metabolismo , Cartilagem/patologia , Inflamação/patologia , Articulações/metabolismo , Articulações/patologia , Camundongos , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/metabolismo
18.
Tissue Eng Part C Methods ; 18(12): 984-94, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22690868

RESUMO

Enhancement and application of our understanding of skeletal developmental biology is critical to developing tissue engineering approaches to bone repair. We propose that use of the developing embryonic femur as a model to further understand skeletogenesis, and the effects of key differentiation agents, will aid our understanding of the developing bone niche and inform bone reparation. We have used a three-dimensional organotypic culture system of embryonic chick femora to investigate the effects of two key skeletal differentiation agents, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), on bone and cartilage development, using a combination of microcomputed tomography and histological analysis to assess tissue formation and structure, and cellular behavior. Stimulation of embryonic day 11 (E11) organotypic femur cultures with PTH and PTHrP initiated osteogenesis. Bone formation was enhanced, with increased collagen I and STRO-1 expression, and cartilage was reduced, with decreased chondrocyte proliferation, collagen II expression, and glycosaminoglycan levels. This study demonstrates the successful use of organotypic chick femur cultures as a model for bone development, evidenced by the ability of exogenous bioactive molecules to differentially modulate bone and cartilage formation. The organotypic model outlined provides a tool for analyzing key temporal stages of bone and cartilage development, providing a paradigm for translation of bone development to improve scaffolds and skeletal stem cell treatments for skeletal regenerative medicine.


Assuntos
Desenvolvimento Ósseo , Fêmur/embriologia , Proteína Relacionada ao Hormônio Paratireóideo/fisiologia , Hormônio Paratireóideo/fisiologia , Animais , Embrião de Galinha
19.
Tissue Eng Part C Methods ; 18(10): 747-60, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22472170

RESUMO

Understanding the structural development of embryonic bone in a three dimensional framework is fundamental to developing new strategies for the recapitulation of bone tissue in latter life. We present an innovative combined approach of an organotypic embryonic femur culture model, microcomputed tomography (µCT) and immunohistochemistry to examine the development and modulation of the three dimensional structures of the developing embryonic femur. Isolated embryonic chick femurs were organotypic (air/liquid interface) cultured for 10 days in either basal, chondrogenic, or osteogenic supplemented culture conditions. The growth development and modulating effects of basal, chondrogenic, or osteogenic culture media of the embryonic chick femurs was investigated using µCT, immunohistochemistry, and histology. The growth and development of noncultured embryonic chick femur stages E10, E11, E12, E13, E15, and E17 were very closely correlated with increased morphometric indices of bone formation as determined by µCT. After 10 days in the organotpyic culture set up, the early aged femurs (E10 and E11) demonstrated a dramatic response to the chondrogenic or osteogenic culture conditions compared to the basal cultured femurs as determined by a change in µCT morphometric indices and modified expression of chondrogenic and osteogenic markers. Although the later aged femurs (E12 and E13) increased in size and structure after 10 days organotpypic culture, the effects of the osteogenic and chondrogenic organotypic cultures on these femurs were not significantly altered compared to basal conditions. We have demonstrated that the embryonic chick femur organotpyic culture model combined with the µCT and immunohistochemical analysis can provide an integral methodology for investigating the modulation of bone development in an ex vivo culture setting. Hence, these interdisciplinary techniques of µCT and whole organ bone cultures will enable us to delineate some of the temporal, structural developmental paradigms and modulation of bone tissue formation to underpin innovative skeletal regenerative technology for clinical therapeutic strategies in musculoskeletal trauma and diseases.


Assuntos
Desenvolvimento Ósseo , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/embriologia , Desenvolvimento Embrionário , Técnicas de Cultura de Órgãos/métodos , Microtomografia por Raio-X/métodos , Animais , Cartilagem/citologia , Cartilagem/diagnóstico por imagem , Cartilagem/embriologia , Embrião de Galinha , Fêmur/citologia , Fêmur/diagnóstico por imagem , Fêmur/embriologia , Coloração e Rotulagem , Fatores de Tempo
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