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1.
Bioact Mater ; 38: 499-511, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38798890

RESUMO

The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regeneration, remaining a central challenge in tissue engineering. In this study, we present a novel (micro)vascularization strategy that explores the use of specialized "vascular units" (VUs) as building blocks to initiate blood vessel formation and create perfusable, stroma-embedded 3D microvascular networks from the bottom-up. We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels. This leads to the formation of VUs-derived capillaries, which fuse with adjacent capillaries to form stable microvascular beds within a supportive, extracellular matrix-rich fibroblastic microenvironment. Using a custom-designed biomimetic fibrin-based vessel-on-chip (VoC), we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused. Moreover, VUs can establish capillary bridges between channels, extending the microvascular network throughout the entire device. When VUs and intestinal organoids (IOs) are combined within the VoC, the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs. This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device. These findings underscore the remarkable potential of VUs as building blocks for engineering microvascular networks, with versatile applications spanning from regenerative medicine to advanced in vitro models.

2.
Carbohydr Polym ; 337: 122144, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38710569

RESUMO

In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.


Assuntos
Alginatos , Química Click , Matriz Extracelular , Hidrogéis , Maleimidas , Compostos de Sulfidrila , Humanos , Alginatos/química , Adesão Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Hidrogéis/química , Hidrogéis/síntese química , Maleimidas/química , Compostos de Sulfidrila/química
3.
Biomaterials ; 228: 119554, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31677395

RESUMO

Successful strategies to promote neovascularization of ischemic tissues are still scarce, being a central priority in regenerative medicine. Microparticles harboring primitive vascular beds are appealing cell delivery candidates for minimally-invasive therapeutic vascularization. However, dynamic cellular alterations associated with in vitro vascular morphogenesis are still elusive. Here, bioengineered microgels guided the assembly of entrapped outgrowth endothelial cells (OEC) and mesenchymal stem cells (MSC) into cohesive vascularized microtissues. During in vitro maturation, OEC formed capillary-like networks enveloped in newly-formed extracellular matrix. Gene expression profiling showed that OEC acquired a mesenchymal-like phenotype, suggesting the occurrence of partial endothelial-to-mesenchymal transition (EndMT), while MSC remained transcriptionally stable. The secretome of entrapped cells became more pro-angiogenic, with no significant alterations of the inflammatory profile. Importantly, matured microgels showed improved cell survival/retention after transplantation in mice, with preservation of capillary-like networks and de novo formation of human vascular structures. These findings support that in vitro priming and morphogenesis of vessel-forming cells improves their vasculogenic/angiogenic potential, which is of therapeutic relevance, shedding some light on the associated mechanisms.


Assuntos
Células-Tronco Mesenquimais , Microgéis , Animais , Células Endoteliais , Camundongos , Morfogênese , Neovascularização Fisiológica , Engenharia Tecidual
4.
J Tissue Eng Regen Med ; 12(7): 1567-1578, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29701896

RESUMO

Two decades after the first report on endothelial progenitor cells (EPC), their key role in postnatal vasculogenesis and vascular repair is well established. The therapeutic potential of EPC and their growing use in clinical trials calls for the development of more robust, reproducible, and safer methods for the in vitro expansion and maintenance of these cells. Despite many limitations associated with its usage, fetal bovine serum (FBS) is still widely applied as a cell culture supplement. Although different approaches aiming at establishing FBS-free culture have been developed for many cell types, adequate solutions for endothelial cells, and for EPC in particular, are still scarce, possibly due to the multiple challenges that have to be faced when culturing these cells. In this review, we provide a brief overview on the therapeutic relevance of EPC and critically analyse the available literature on FBS-free endothelial cell culture methods, including xeno-free, serum-free, and chemically defined systems.


Assuntos
Técnicas de Cultura de Células/métodos , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Neovascularização Fisiológica , Animais , Bovinos , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Humanos
5.
Sci Rep ; 8(1): 230, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29321569

RESUMO

Spheroid culture has gained increasing popularity, arising as a promising tool for regenerative medicine applications. Importantly, spheroids may present advantages over single-cell suspensions in cell-based therapies (CT). Unfortunately, most growth media used for spheroid culture contain animal origin-components, such as fetal bovine serum (FBS). The presence of FBS compromises the safety of CT and presents economic and ethical constraints. SCC (supplement for cell culture) is a novel xeno-free (XF) industrial cell culture supplement, derived from well-controlled pooled human plasma and processed under good manufacturing practice rules. Here, we developed a XF SCC-based formulation for 2D-culture of outgrowth endothelial cells (OEC), and then used it for generating co-culture spheroids of OEC and mesenchymal stem cells (MSC). XF MSC-OEC spheroids were characterized in detail and compared to spheroids cultured in FBS-supplemented medium. XF spheroids presented comparable integrity, size and morphology as the reference culture. The use of both media resulted in spheroids with similar structure, abundant extracellular matrix deposition and specific patterns of OEC distribution and organization. Notably, XF spheroids presented significantly enhanced angiogenic potential, both in vitro (fibrin sprouting assay) and in vivo (CAM assay). These findings are particularly promising in the context of potential therapeutic applications.


Assuntos
Técnicas de Cultura de Células , Terapia Baseada em Transplante de Células e Tecidos , Esferoides Celulares , Terapia Baseada em Transplante de Células e Tecidos/métodos , Meios de Cultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Matriz Extracelular , Sangue Fetal/citologia , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Esferoides Celulares/citologia , Esferoides Celulares/ultraestrutura
6.
Biomaterials ; 154: 34-47, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29120817

RESUMO

Efficient cell delivery strategies are urgently needed to improve the outcome of cell-based pro-angiogenic therapies. This study describes the design of an injectable cell delivery platform, based on biomaterial-guided morphogenesis principles. Soft high-mannuronic acid alginate microgels, oxidized and functionalized with integrin-binding peptides, provided adequate biochemical/biomechanical cues for the co-assembly of mesenchymal stem cells and outgrowth endothelial cells (OEC) into pre-vascularized microtissues. In vitro priming conditions regulated OEC tubulogenesis, which only occurred under normoxia (+O2) in the presence of angiogenic factors (+GF) and, importantly, did not revert in an ischemic-like environment. Primed (+O2+GF) microgel-entrapped cells secreted a large variety of angiogenesis-related proteins and produced endogenous extracellular-matrix, rich in fibronectin and collagen type I, fostering cell-cell/cell-matrix interactions and establishing a stable angiogenic niche. Extending the pre-culture time resulted in higher cell outward migration and in vivo angiogenic potential. Microgels partially disintegrated upon implantation in chick embryos, promoting interaction between pre-vascularized microtissues and the host. Preserved human vascular structures were still detected in vivo, and human cells showed the ability to migrate and integrate with the chick vasculature. Our results suggest that an integrated approach combining pro-angiogenic cells, cell-instructive microgels and adequate in vitro priming may provide the basis for successful therapeutic angiogenesis.


Assuntos
Géis/química , Morfogênese , Neovascularização Patológica/terapia , Alginatos/química , Animais , Movimento Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Embrião de Galinha , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/citologia , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Oxigênio/farmacologia
7.
J Tissue Eng Regen Med ; 12(3): e1785-e1795, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29024519

RESUMO

Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) are considered promising therapeutic agents in the field of cell therapy and regenerative medicine, mainly due to their relative facility to be isolated, multi-differentiation potential, and immunomodulatory role. However, their application in clinics requires a crucial step of in vitro expansion. Most of the protocols for hMSCs in vitro culture use foetal bovine serum as medium supplement that, being from animal origin, presents several safety concerns and may initiate xenogeneic immune responses after cells transplantation. This work reports the optimization of a pharmaceutical-grade xeno-free strategy for hMSCs in vitro expansion based on the supplementation of basal medium with a pharmaceutical-grade human plasma-derived supplement for cell culture (SCC) and 2 human growth factors (bFGF and TGFß1), plus a coating of human plasma fibronectin (Fn). After 4 weeks in culture, this strategy improves hMSCs expansion yield about 4.3-fold in comparison with foetal bovine serum supplementation and 4.5-fold compared with a commercially available xeno-free medium. hMSCs expanded in SCC-based formulation maintained their phenotype and differentiation capacity into osteogenic, adipogenic, and chondrogenic lineages, without alterations in cell karyotype. Overall, the SCC-based medium appears to be an excellent alternative for the xeno-free expansion of hMSCs as therapeutic agents for clinical applications.


Assuntos
Meios de Cultura/farmacologia , Células-Tronco Mesenquimais/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células CHO , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Fibronectinas/farmacologia , Humanos , Imunofenotipagem , Cariótipo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Adulto Jovem
8.
Stem Cells Int ; 2017: 6597815, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29158740

RESUMO

Human mesenchymal stem/stromal cells (hMSCs) have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiring in vitro expansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS) as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

9.
Biomaterials ; 124: 211-224, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28209528

RESUMO

Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-ß and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.


Assuntos
Quimiocinas CC/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Matriz Extracelular/química , Matriz Extracelular/imunologia , Macrófagos/imunologia , Microambiente Tumoral/imunologia , Polaridade Celular , Sistema Livre de Células , Neoplasias Colorretais/química , Humanos , Invasividade Neoplásica , Alicerces Teciduais , Células Tumorais Cultivadas
10.
Sci Rep ; 6: 27072, 2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27255191

RESUMO

Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Intermediate states, arising from inter-conversion between epithelial (E) and mesenchymal (M) states, are characterized by phenotypic heterogeneity combining E and M features and increased plasticity. Hybrid EMT states are highly relevant in metastatic contexts, but have been largely neglected, partially due to the lack of physiologically-relevant 3D platforms to study them. Here we propose a new in vitro model, combining mammary E cells with a bioengineered 3D matrix, to explore phenotypic and functional properties of cells in transition between E and M states. Optimized alginate-based 3D matrices provided adequate 3D microenvironments, where normal epithelial morphogenesis was recapitulated, with formation of acini-like structures, similar to those found in native mammary tissue. TGFß1-driven EMT in 3D could be successfully promoted, generating M-like cells. TGFß1 removal resulted in phenotypic switching to an intermediate state (RE cells), a hybrid cell population expressing both E and M markers at gene/protein levels. RE cells exhibited increased proliferative/clonogenic activity, as compared to M cells, being able to form large colonies containing cells with front-back polarity, suggesting a more aggressive phenotype. Our 3D model provides a powerful tool to investigate the role of the microenvironment on metastable EMT stages.


Assuntos
Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Esferoides Celulares/citologia , Fator de Crescimento Transformador beta1/farmacologia , Actinas/genética , Actinas/metabolismo , Alginatos/química , Animais , Biomarcadores/metabolismo , Caderinas/genética , Caderinas/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Expressão Gênica , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Camundongos , Ocludina/genética , Ocludina/metabolismo , Fenótipo , Esferoides Celulares/metabolismo
11.
J Mater Sci Mater Med ; 17(5): 455-63, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16688586

RESUMO

In the present work, a novel route for the preparation of porous ceramic microspheres is described. Two ceramic powders, calcium-titanium-phosphate (CTP) and hydroxyapatite (HAp), were mixed with a sodium alginate solution that enabled the preparation of spherical particles, using the droplet extrusion method combined with ionotropic gelation in the presence of Ca(2+). The spherical particles were subsequently sintered, to burn-off the polymer and obtain calcium-phosphate microspheres with a uniform size and an interconnected porous network. CTP microspheres with diameters ranging from 513 +/- 24 microm to 792 +/- 35 microm and with pores of approximately 40 microm were obtained. HAp microspheres presented diameters of 429 +/- 46 microm and 632 +/- 40 microm and pores of ca. 2 microm. Depending on the formulations tested, the structure of both calcium phosphates may become altered during the sintering process, suggesting that the ratio between the ceramic phase and the polymer solution is a critical parameter. Porous microspheres prepared using the described methodology are promising candidates as bone defect fillers and scaffolds for bone tissue regeneration.


Assuntos
Materiais Biocompatíveis/química , Fosfatos de Cálcio/química , Microesferas , Alginatos/química , Substitutos Ósseos/química , Cálcio/química , Cálcio/metabolismo , Cerâmica/química , Durapatita/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Teste de Materiais , Microscopia Eletrônica de Varredura , Polímeros/química , Pós , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Titânio/química , Difração de Raios X
12.
Blood Cells Mol Dis ; 35(3): 348-54, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16125984

RESUMO

Bone lesions are a major cause of morbidity in Gaucher disease (GD) type I. Enzyme replacement therapy (ERT) has been successful in treating many symptoms of type I GD but skeletal response lags behind. Local exogenous glucocerebrosidase supplementation in bone lesions via a drug delivery system may overcome this limitation. Although local enzyme supplementation aims to target lipid-engorged macrophages (Gaucher Cells) in bone compartment, enzyme uptake by osteoblasts is not excluded. To investigate the ability of human osteoblasts to internalize recombinant glucocerebrosidase (rGCR), we have used an artificial GD human osteoblasts cell culture system. MG63 human osteoblasts were treated with conduritol B epoxide (CBE) to induce complete and prolonged inhibition of endogenous glucocerebrosidase activity of cells. rGCR uptake by glucocerebrosidase-inactivated osteoblasts was examined using (125)I-radiolabelling, Western blot analysis and measurement of glucocerebrosidase activity. Analysis of radiolabeled enzyme uptake by CBE treated osteoblasts showed 67.9% of internalized protein in cell extract. Enzyme internalization was also observed by Western blot analysis where the amount of mature form of glucocerebrosidase protein recognized by the glucocerebrosidase antibody was increased following the administrations of rGCR. Moreover, enzymatic activity measurement showed 23.9% of glucocerebrosidase activity of control cells. The rGCR internalization by MG63 osteoblast seems to be partially mediated by mannose receptors. These data provide evidence that MG63 human osteoblasts are able to internalize rGCR.


Assuntos
Doença de Gaucher/enzimologia , Glucosilceramidase/farmacocinética , Osteoblastos/enzimologia , Proteínas Recombinantes/farmacocinética , Células Cultivadas , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/metabolismo , Humanos , Inositol/análogos & derivados , Inositol/farmacologia , Osteoblastos/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo
13.
J Biomed Mater Res A ; 74(4): 545-52, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16028235

RESUMO

Gaucher disease (GD) is caused by the decreased activity and/or stability of the lysosomal enzyme glucocerebrosidase (GCR). The available treatment consists in the intravenous administration of exogenous GCR, and is effective in reverting most of the symptoms. However, in terms of bone pathology, which is among the most disabling manifestations, a slow and incomplete response is observed, indicating that adjuvant therapies are necessary to consistently restore GCR activity in bone and accelerate regeneration. In this study, calcium alginate microspheres were analyzed as a vehicle for localized GCR delivery to bone. Results demonstrated that the entrapped enzyme retained full activity and exhibited a broader pH-dependent activity profile, compared to that of free-GCR, with improved stability at physiological pH. GCR release profile was established, and it was demonstrated that GCR could be released in a sustained manner. The biological behavior of the system was evaluated by analyzing the uptake of released GCR by GCR-deficient cells from GD patients, using different techniques: GCR activity measurements, radiolabeling, and cellulose acetate electrophoresis. Results demonstrated that GCR was internalized by cells significantly enhancing the residual enzymatic activity. To achieve an activity reconstitution level comparable to that obtained using free-GCR, only half of the dose was required with entrapped-GCR.


Assuntos
Alginatos/farmacologia , Portadores de Fármacos/farmacologia , Fibroblastos , Glucosilceramidase/farmacologia , Regeneração Óssea/efeitos dos fármacos , Células Cultivadas , Enzimas Imobilizadas/farmacologia , Fibroblastos/enzimologia , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/enzimologia , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/farmacologia , Humanos , Microesferas
14.
Acta Biomater ; 1(4): 411-9, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16701822

RESUMO

Poly(alpha-hydroxy acids), and in particular polylactic acid (PLA), are nowadays amongst the most used bioabsorbable materials. However, this polymer may not meet some application requirements due to inadequate mechanical properties and or its degradation characteristics. A possible strategy to tackle this problem is the incorporation of an inorganic phase into the polymeric matrix. In this work a new fully biodegradable composite material made with PLA and calcium phosphate soluble glass particles has been developed. The behaviour of the PLA/glass composite has been analysed during its degradation in simulated physiological conditions by means of weight loss, molecular weight and thermal properties analysis and electron microscopy observation. The results showed that the incorporation of phosphate glass particles into the polymer significantly accelerated the degradation of the PLA and induced the formation of calcium phosphate precipitates at the composite surface.


Assuntos
Implantes Absorvíveis , Materiais Biomiméticos/química , Líquidos Corporais/química , Substitutos Ósseos/química , Fosfatos de Cálcio/química , Ácido Láctico/química , Polímeros/química , Absorção , Vidro/química , Teste de Materiais , Peso Molecular , Poliésteres , Solubilidade , Propriedades de Superfície
15.
Biomaterials ; 25(18): 4363-73, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15046927

RESUMO

The present study concerns the preparation and initial characterisation of novel calcium titanium phosphate-alginate (CTP-alginate) and hydroxyapatite-alginate (HAp-alginate) microspheres, which are intended to be used as enzyme delivery matrices and bone regeneration templates. Microspheres were prepared using different concentrations of polymer solution (1% and 3% w/v) and different ceramic-to-polymer solution ratios (0.1, 0.2 and 0.4 w/w). Ceramic powders were characterised using X-ray diffraction, laser granulometry, Brunauer, Emmel and Teller (BET) method for the determination of surface area, zeta potential and Fourier transform infrared spectroscopy (FT-IR). Alginate was characterised using high performance size exclusion chromatography. The methodology followed in this investigation enabled the preparation of homogeneous microspheres with a uniform size. Studies on the immobilisation and release of the therapeutic enzyme glucocerebrosidase, employed in the treatment of Gaucher disease, were also performed. The enzyme was incorporated into the ceramic-alginate matrix before gel formation in two different ways: pre-adsorbed onto the ceramic particles or dispersed in the polymeric matrix. The two strategies resulted in distinct release profiles. Slow release was obtained after adsorption of the enzyme to the ceramic powders, prior to preparation of the microspheres. An initial fast release was achieved when the enzyme and the ceramic particles were dispersed in the alginate solution before producing the microspheres. The latter profile is very similar to that of alginate microspheres. The different patterns of enzyme release increase the range of possible applications of the system investigated in this work.


Assuntos
Alginatos/química , Fosfatos de Cálcio/química , Sistemas de Liberação de Medicamentos/métodos , Glucosilceramidase/administração & dosagem , Glucosilceramidase/química , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Absorção , Adsorção , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/química , Microesferas , Conformação Molecular , Tamanho da Partícula , Pós
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