Adipose tissue-derived stem cells and their application in bone and cartilage tissue engineering

The adipose tissue was considered a reserve of energy until the ’80s, when it was found that this tissue was involved in the metabolism of sex steroids such as estrogens. From then on, the importance attributed to this tissue radically changed as it was then considered an active organ, involved in important functions of the human body. In 2001, for the first time, the existence of stem cells within this tissue was reported, and since then, this tissue has been gaining an increased importance as a stem cell source for a wide range of potential applications in cell therapies and=or tissue engineering and regenerative medicine strategies, mainly due to its wide availability and easy access. This manuscript provides an overview on adipose stem cells (i.e., adipose tissue–derived stem cells, ASCs) considering the tissue of origin, the niche of the ASCs, and their phenotype in all aspects. In this paper it is also discussed the markers that have been used for the characterization of these cells, their differentiation properties, and their immunological reactivity, reporting studies from 2001 until this date. The ASCs are also compared with bone marrow stem cells (BMSCs), until now considered as the gold standard source of stem cells, underlining the common characteristics and the differences between the stem cells obtained from these two sources, as well as the advantages and disadvantages of their potential use in different applications. Finally, this review will also focus on the potential application of ASCs in tissue engineering applications, particularly in the regeneration of bone and cartilage, commenting on the progress of this approach and future trends of the field.T. Rada thanks the European Marie Curie EST Project (Alea Jacta Est) for the Ph. D. fellowship. The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the partial financial support through funds from POCTI and/or FEDER programs and to the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and the European NoE EXPER-TISSUES (NMP3-CT-2004-500283)

Isolation of adipose stem cells (ASCs) subpopulations with distinct differentiation potential

[Excerpt] ASCs are becoming the elected cells for TE applications because ASCs have been easily isolated and have shown good differentiation potential. The aim of this work was to isolate the ASCs using immunomagnetic beads coated with different antibodies (Ab) markers and to test the differentiation potential of the different subpopulations isolated. [...]Marie Curie Actions Alea Jacta Est, Project HIPPOCRATES, NoE EXPERTISSUESinfo:eu-repo/semantics/publishedVersio

Expression, purification and bioactivity of recombinant human bone morphogenetic protein-4,-9,-10,-11 and-14 produced in Escherichia coli for tissue engineering applications

[Excerpt] Bone morphogenetic proteins (BMPs) are cytokines from the TGFb superfamily, with important roles during embryonic development and in inducing bone and cartilage in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b expression system. The BMPs were purified by affinity chromatography and its bioactivity accessed in C2C12 cell line, by screening the expression of osteogenic markers with RT-PCR. [...]Fundação para a Ciência e Tecnologia for PhD grant SFRH/BD/17049/2004 and project ElastM POCI/CTM/ 57177/2004 supported by FEDER and the Fundação para a Ciência e Tecnologia; European STREP Project HIPPOCRATES (NMP3-CT-2003-505758). The work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283).info:eu-repo/semantics/publishedVersio

Human dermal fibroblasts, keratinocytes and adipose stem cells behaviour on patterned electrospun nanofiber meshes

Natural extracellular Matrix (ECM) creates a unique cellular microenvironment. It acts as a support to organize cells in tissues, maintains their structure and works also as a reservoir for cytokines, thus controlling cell growth and differentiation. A well-defined biomaterial surface topography is believed to be adequate to mimic native ECM for guiding cell growth or tissue regeneration. This structure can be achieved by using an electrospinning technique, which allows producing a non-woven nanofibrous structure with topographic features mimicking the natural ECM. [...]info:eu-repo/semantics/publishedVersio

Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-b superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor- 11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.The author wishes to acknowledge the Sanger Institute for kindly offering the bacterial clones for cloning of human BMP-9 to -14. This work was supported by Fundacao para a Ciencia e Tecnologia (PhD grant SFRH/BD/17049/2004, project ElastM POCI/CTM/57177/2004 funded by FEDER and the Fundacao para a Ciencia e Tecnologia; and European STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Silk nanoparticles for delivery of human BMP-2 in bone regenerative medicine applications

[Excerpt] A tissue engineering approach combines the use of scaffold biomaterials, stem cells and growth factors. Bone morphogenetic proteins (BMPs) are growth factors that have sparked a great interest in tissue engineering due to their strong ability to promote new bone formation. Herein, we report the use of silk derived nanoparticles as carriers for delivery of human BMP-2. Silks are attractive biomaterials for tissue engineering due to its biocompatibility, slow biodegradability and excellent mechanical properties. Recombinant human BMP-2 was expressed in Escherichia coli and purified by affinity chromatography, showing bioactivity in human adipose stem cells. BMP2-containing silk particles were then prepared by a water-in-oil emulsion method. [...]info:eu-repo/semantics/publishedVersio

Dynamic culture of osteogenic cells in biomimetically coated poly(caprolactone) nanofibre mesh constructs

In our previous work, biomimetic calcium phosphate-coated poly(caprolactone) nanofibre meshes (BCP-NMs) were demonstrated to be more effective for supporting cell attachment and proliferation under static conditions, when compared with poly(caprolactone) nanofibre meshes (PCL-NMs). In many applications, in vitro cultivation of constructs using bioreactors that support efficient nutrition of cells has appeared as an important step toward the development of functional grafts. This work aimed at studying the effects of dynamic culture conditions and biomimetic coating on bone cells grown on the nanofibre meshes. BCP-NM and PCL-NM were seeded with osteoblast-like cells (MG63--human osteosarcoma-derived cell line). The cell-seeded constructs were cultured within a rotating bioreactor that simulated microgravity, at a fixed rotating speed, for different time periods, and then characterized. Cell morphology, viability, and phenotype were assessed. PCL-NM constructs presented a higher number of dead cells than BCP-NM constructs. Under dynamic conditions, the production of proteins associated with the extracellular matrix of bone was higher on BCP-NM constructs than in the PCL-NM ones, which indicates that coated samples may provide cells with a better environment for tissue growth. It is suggested that improved mass transfer in the bioreactor in combination with the appropriate substrate were decisive factors for this highly positive outcome for generating bone.This work was developed under the scope of the EU Project Network of Excellence "Expertissues'' (NMP3-CT-2004-500283) and supported by Alea jacta est Marie Curie Actions (MEST-CT-2004-008104). M. Alves da Silva would like to acknowledge the Portuguese Foundation for Science and Technology for her grant (SFRH-BD-28708-2006). Jose V. Araujo is grateful to S. Rathbone, H. Sura, I. Wimpenny, I. Dublon, G. Jones, and E. D. Pinho for useful technical discussions

Distinct stem cells subpopulations isolated from human adipose tissue exhibit different chondrogenic and osteogenic differentiation potential

Recently adipose tissue has become a research topic also for the searching for an alternative stem cells source to use in cell based therapies such as tissue engineer. In fact Adipose Stem Cells (ASCs) exhibit an important differentiation potential for several cell lineages such as chondrogenic, osteogenic, myogenic, adipogenic and endothelial cells. ASCs populations isolated using standard methodologies (i.e., based on their adherence ability) are very heterogeneous but very few studies have analysed this aspect. Consequently, several questions are still pending, as for example, on what regard the existence/ or not of distinct ASCs subpopulations. The present study is originally aimed at isolating selected ASCs subpopulations, and to analyse their behaviour towards the heterogeneous population regarding the expression of stem cell markers and also regarding their osteogenic and chondrogenic differentiation potential. Human Adipose derived Stem Cells (hASCs) subpopulations were isolated using immunomagnetic beads coated with several different antibodies (CD29, CD44, CD49d, CD73, CD90, CD 105, Stro-1 and p75) and were characterized by Real Time RT-PCR in order to assess the expression of mesenchymal stem cells markers (CD44, CD73, Stro-1, CD105 and CD90) as well as known markers of the chondrogenic (Sox 9, Collagen II) and osteogenic lineage (Osteopontin, Osteocalcin). The obtained results underline the complexity of the ASCs population demonstrating that it is composed of several subpopulations, which express different levels of ASCs markers and exhibit distinctive differentiation potentials. Furthermore, the results obtained clearly evidence of the advantages of using selected populations in cell-based therapies, such as bone and cartilage regenerative medicine approaches.EU funded Marie Curie Actions Alea Jacta Est for a PhD fellowship. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Prostaglandin F2-alpha receptor (FPr) expression on porcine corpus luteum microvascular endothelial cells (pCL-MVECs)

<p>Abstract</p> <p>Background</p> <p>The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.</p> <p>Methods</p> <p>Porcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.</p> <p>PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha.</p> <p>Results</p> <p>We obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures.</p> <p>Conclusion</p> <p>Our data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.</p

Adipose derived stem cells for bone and cartilage tissue engineering : novel isolation procedure in vitro characterization and in vivo functionality assessment

Tese de doutoramento em Ciência e Tecnologia de Materiais (área de conhecimento em Engenharia de Tecidos/Materiais Híbridos)Adult Stem cells are undifferentiated cells that can be found in various tissues or organs and that can renew itself and differentiate to yield some or all of the major specialized cell types and therefore have been widely studied for a variety of cellbased therapies, including tissue engineering approaches. Stem cells from the bone marrow, usually designated as Mesenchymal Stem Cells (MSCs), have been the elected cellular component, particularly in bone and cartilage tissue engineering strategies. This is mainly because they can be used in autologous approaches and because they are relatively easy to grow and to expand in vitro, presenting a great differentiation potential. Nevertheless, bone marrow harvesting is a painful procedure and the number of stem cells in the bone marrow is rather low. In recent years, many researchers have focused on alternative sources of stem cells that could overcome these limitations, such as the use of adipose tissue. In fact, the harvesting procedure to obtain adipose tissue is not painful, it is quite safe and the human body possesses several reserves of this tissue. As a consequence, since the Adipose-derived Stem Cells (ASCs) were isolated for the first time, a number of studies have been showing their high potential for application in tissue engineering, mainly because of the high number of stem cells per unit of tissue that can be obtained, the high replication rate and the great differentiation potential. Nevertheless, the knowledge on adipose stem cells is still very limited as compared with the bone marrow mesenchymal stem cells population. This thesis focused on the study of adipose derived stem cells as a potential cell source for bone and cartilage tissue engineering approaches. It was centered on the development and optimization of efficient isolation procedures and on the characterization of the in vitro and in vivo functionality of the different ASCs subpopulations meanwhile identified. The main objectives of this thesis were the following: - Development and optimization of an isolation procedure aiming at decreasing the presence of other cells types that might compromise the expansion and differentiation potential of the adipose derived stem cells. - Identify the presence of distinct subpopulations of ASCs (similarly to the findings that have been reported for the bone marrow mesenchymal stem cells) and characterize them, specifically regarding their osteogenic and chondrogenic differentiation potential. - Study the behavior of the adipose stem cells subpopulations in vitro and in vivo, in order to select those that, always combined with appropriate support materials, could be more efficient for bone and for cartilage tissue regeneration strategies. The first objective was accomplished through the development and optimization of a novel selective technique for the isolation of adipose stem cells that is based on the use of immunomagnetic beads coated with specific antibodies. Using this procedure, it was possible to isolate several subpopulations of rat and human ASCs, which were characterized towards the expression of the typical markers of stem cells and also regarding the osteogenic and chondrogenic potential. The results obtained in these studies provided evidence of the existence of a number of distinct ASCs subpopulations. Additionally, it was clearly shown that there are important differences in the behavior of the rat ASCs, as compared to human ASCs, which led to the exclusion of the rat model for subsequent studies. Further in vitro studies, focused on human ASCs, allowed selecting the subpopulations with the highest chondrogenic and osteogenic differentiation potential. In vivo studies, using a nude mice model, were designed to study the in vivo osteogenic and chondrogenic potential of these selected subpopulations, which were implanted after in vitro culture using appropriate templates for bone and cartilage TE applications, respectively. The data collected from these in vivo experiments highlighted the distinct behavior of the cells in vitro with respect to their behavior in vivo, and enabled for the further selection of the sub-populations with the highest chondrogenic or osteogenic differentiation potential. In summary, this work presents an alternative method to isolate adipose stem cells, that enables to obtain selected subpopulations with distinct characteristics, namely different osteogenic and chondrogenic differentiation potentials. The isolation method and consequently, the selected subpopulations for a given application, might allow for a more efficient use of ASCs, enhancing the potential of this cell source to become the “gold standard” in tissue engineering applications.As células estaminais adultas são células indiferenciadas presentes em vários tecidos ou órgãos com capacidade de se auto-regenerar e diferenciar em quase todos os principais tipos de células especializadas. Por esta razão, têm sido vastamente estudadas para um conjunto de terapias celulares, incluindo a engenharia de tecidos humanos. As células estaminais da medula óssea, geralmente designadas células estaminais do mesênquima (MSCs), têm sido um dos componentes celulares de eleição, particularmente em estratégias de engenharia de tecidos do osso e cartilagem. Isto deve-se sobretudo ao facto de poderem ser usadas de uma forma autóloga, mas também devido à sua fácil cultura e expansão in vitro e ao seu excelente potencial de diferenciação. No entanto, a extracção de medula óssea é um procedimento tipicamente doloroso, que muitas vezes origina um número reduzido de células estaminais. Nos últimos anos, muitos investigadores concentraram-se em encontrar fontes alternativas de células estaminais adultas que permitem ultrapassar estas limitações. O tecido adiposo é um dos exemplos de uma fonte alternativa de células com muito potencial. De facto, o processo de extracção de tecido adiposo não é doloroso, é seguro e o corpo humano possui várias reservas deste tecido. Por conseguinte, desde que as células estaminais do tecido adiposo (Adipose Stem Cells – ASCs) foram isoladas pela primeira vez, têm sido efectuados numerosos estudos que evidenciam o seu elevado potencial para aplicações em engenharia de tecidos, dada a elevada concentração de células estaminais e a sua alta taxa de replicação e elevado potencial de diferenciação. Contudo, o conhecimento sobre a população de células estaminais do tecido adiposo é ainda muito incipiente, particularmente quando comparado com o da população de células estaminais da medula óssea. Esta tese centrou-se no estudo de células estaminais do tecido adiposo como potencial fonte de células alternativa para aplicações em engenharia de tecidos de osso e cartilagem, nomedamente no desenvolvimento e optimização de métodos de isolamento eficientes e na caracterização da funcionalidade, in vitro e in vivo, das diferentes subpopulações identificadas entretanto. Dentro deste enquadramento, os principais objectivos propostos foram os seguintes: - Optimização de técnicas de isolamento visando diminuir a presença de outros tipos de células, susceptivies de comprometer o potencial de proliferação e diferenciação das células estaminais do tecido adiposo; - Identificar a presença de subpopulações em amostras de células estaminais do tecido adiposo e determinar o seu potencial osteogénico e condrogénico, à semelhança do que tem sido feito para as células estaminais da medula óssea; - Estudar diferenças no comportamento destas subpopulações de células in vitro e in vivo, de forma a selecionar as subpopulações que, combinadas com materiais de suporte adequados, possam ser mais apropriadas para aplicações especificas em engenharia de tecidos do osso ou cartilagem. O primeiro objectivo foi concretizado através do desenvolvimento e optimização de uma técnica inovadora para o isolamento selectivo de células estaminais do tecido adiposo, assim como de subpopulações destas. Esta técnica é baseado no uso de partículas imuno-magnéticas revestidas por anticorpos específicos. Usando este procedimento, foi possível isolar várias subpopulações de ASCs humanas e de rato, posteriormente caracterizadas quanto à expressão de marcadores típicos das células estaminais, bem como do seu potencial osteogénico e condrogénico. Estes estudos evidenciaram a existência de diferenças significativas entre os modelos humano e de rato. Os resultados obtidos in vitro permitiram seleccionar as subpopulações com maior potencial de diferenciação osteogénico e condrogénico. Os estudos in vivo foram baseados num modelo de rato atímico para avaliar o potencial osteogénico e condrogénico das subpopulações seleccionadas, as quais foram implantadas após um período de cultura in vitro em suportes apropriados para engenharia de tecidos de osso ou de cartilagem, respectivamente. Os resultados obtidos nas experiências in vivo permitiram evidenciar diferenças significativas entre o comportamento in vitro e in vivo e também permitiram realizar uma selecção adicional das subpopulações com o melhor potencial de diferenciação osteogenico e condrogenico. Resumindo, este trabalho descreve um método de isolamento alternativo de células estaminais do tecido adiposo, que permite obter várias subpopulações com características distintas, nomeadamente com potenciais de diferenciação. Este método de isolamento e consequentemente a possibilidade de selecionar subpopulações especificas para uma dada aplicação, pode permitir uma utilização mais eficiente das células estaminais do tecido adiposo, contribuindo para o seu uso mais alargado em Engenharia de Tecidos