The use of mesenchymal stromal stem cells (MSCs) in the field of tissue engineering for cartilage repair is a very promising tool since these cells are readily expandable and capable of differentiating into chondrocytes. However, in vitro manipulation of these cells for the production of an implantable construct for cartilage defect healing presents still many challenges [1]. In this regard, several studies are aimed at developing alternative protocols to obtain chondrogenic differentiation from cells able to express those proteins required for optimal in vivo chondrogenesis and subsequent tissue repair. The use of MSCs traditionally requires the use of TGF since in vitro treatment with TGF is a well accepted system by which MSCs become chondrocyte-like cells. However, it is taken into account that this is a forced system and, according to recent evidences, it may give rise to unexpected side effects which are worth some considerations. On the other hand, clinical outcome of the use of mature chondrocytes in conventional autologous chondrocyte implantation (ACI) is not entirely satisfactory since stable hyaline-like cartilage in vivo is not convincing produced. Recent evidences suggest that the employment of heterogeneous populations of uncharacterized cells may also account for the disparate results in cell therapy studies. In search of new strategies for the assessment of chondrogenic differentiation we focused on the potential of human de-differentiated chondrocytes (p3Ch) obtained after subculturing chondrocytes (Ch) from human nasal septum. We demonstrated, by immunocytochemical analysis, that p3Ch lost cartilaginous phenotype and acquired an expression profile really close to that of hMSCs [2]. In addition, FACS analysis of specific markers showed that p3Ch population had a degree of homogeneity higher than Ch from which they derived. These evidences prompted us to use p3Ch cells in combination with alginate based scaffolds to generate a construct to be proposed for cartilage tissue engineering. We produced alginate microfibers (2% w/v) in combination or not with gelatin (2.25% w/v) or urinary bladder matrix (UBM) (0.5% w/v), in the purpose of taking advantage from association of different biomaterials and improving the differentiation ability of encapsulated cells without adding chondrogenic inducers. Microfibers were produced by an extrusion method [3] where biomaterial solution containing p3Ch has let been flowed by a syringe-pump in BaCl2 gelling solution. Interestingly, microfibrous shape makes our scaffold manageable and adaptable to the structure and size of damaged tissue site for a possible in vivo application. Cells were successfully embedded in all tested conditions, showing rounded morphology and mantaining specific alignment and high viability. Scaffolds containing cells were maintained in culture without adding chemical inducers and monitored for 14 days. After an adequate set up of cell recovery, we were able to analyse gene expression by immunocytochemistry, RT-PCR and FACS. An effective redifferentiation ability of encapsulated cells was obtained as demonstrated by increase of typical chondrogenic markers including collagen type II and aggrecan, AlcianBlue staining of cartilage specific proteoglycans, associated with a desirable low level of collagen type I and X (a typical marker of hypertrophic cartilage), since day 7 of culture. In addition, transmission electron microscope (TEM) analysis showed that encapsulated p3Ch during redifferentiation process presented an appreciable production of secretory vesicles, containing extracellular matrix (ECM) dense materials, and collagen fibers with their typical banding pattern, in the surrounding lacuna, remembering the natural microenvironment of chondrocytes. As a whole, our data indicate that the cell-microfiber combinations here produced may be efficient in the production of the ECM in its correct form in a short time to support cartilage repair and regeneration. 1. Johnstone B, Alini M, Cucchiarini M, Dodge GR, Eglin D, et al. (2013) Tissue engineering for articular cartilage repair - the state of the art. European Cells & Materials 25: 248-267. 2. Lolli A, Lambertini L, Penolazzi L, Angelozzi M, Morganti C, et al. Pro-chondrogenic effect of miR-221 and Slug depletion in human MSCs. Stem Cell Rev and Reports, in press. 3. Mazzitelli S, Capretto L, Quinci F, Piva R, Nastruzzi C. (2013) Preparation of cell-encapsulation devices in confined microenvironment. Adv Drug Deliv Rev 65:1533-1555.

Encapsulation of dedifferentiated nasal septal chondrocytes in alginate based scaffolds for cartilage tissue engineering

ANGELOZZI, MARCO;PENOLAZZI, Maria Letizia;LAMBERTINI, Elisabetta;GALLERANI, Eleonora;VEZZALI, Federica;NASTRUZZI, Claudio;PIVA, Maria Roberta
2014

Abstract

The use of mesenchymal stromal stem cells (MSCs) in the field of tissue engineering for cartilage repair is a very promising tool since these cells are readily expandable and capable of differentiating into chondrocytes. However, in vitro manipulation of these cells for the production of an implantable construct for cartilage defect healing presents still many challenges [1]. In this regard, several studies are aimed at developing alternative protocols to obtain chondrogenic differentiation from cells able to express those proteins required for optimal in vivo chondrogenesis and subsequent tissue repair. The use of MSCs traditionally requires the use of TGF since in vitro treatment with TGF is a well accepted system by which MSCs become chondrocyte-like cells. However, it is taken into account that this is a forced system and, according to recent evidences, it may give rise to unexpected side effects which are worth some considerations. On the other hand, clinical outcome of the use of mature chondrocytes in conventional autologous chondrocyte implantation (ACI) is not entirely satisfactory since stable hyaline-like cartilage in vivo is not convincing produced. Recent evidences suggest that the employment of heterogeneous populations of uncharacterized cells may also account for the disparate results in cell therapy studies. In search of new strategies for the assessment of chondrogenic differentiation we focused on the potential of human de-differentiated chondrocytes (p3Ch) obtained after subculturing chondrocytes (Ch) from human nasal septum. We demonstrated, by immunocytochemical analysis, that p3Ch lost cartilaginous phenotype and acquired an expression profile really close to that of hMSCs [2]. In addition, FACS analysis of specific markers showed that p3Ch population had a degree of homogeneity higher than Ch from which they derived. These evidences prompted us to use p3Ch cells in combination with alginate based scaffolds to generate a construct to be proposed for cartilage tissue engineering. We produced alginate microfibers (2% w/v) in combination or not with gelatin (2.25% w/v) or urinary bladder matrix (UBM) (0.5% w/v), in the purpose of taking advantage from association of different biomaterials and improving the differentiation ability of encapsulated cells without adding chondrogenic inducers. Microfibers were produced by an extrusion method [3] where biomaterial solution containing p3Ch has let been flowed by a syringe-pump in BaCl2 gelling solution. Interestingly, microfibrous shape makes our scaffold manageable and adaptable to the structure and size of damaged tissue site for a possible in vivo application. Cells were successfully embedded in all tested conditions, showing rounded morphology and mantaining specific alignment and high viability. Scaffolds containing cells were maintained in culture without adding chemical inducers and monitored for 14 days. After an adequate set up of cell recovery, we were able to analyse gene expression by immunocytochemistry, RT-PCR and FACS. An effective redifferentiation ability of encapsulated cells was obtained as demonstrated by increase of typical chondrogenic markers including collagen type II and aggrecan, AlcianBlue staining of cartilage specific proteoglycans, associated with a desirable low level of collagen type I and X (a typical marker of hypertrophic cartilage), since day 7 of culture. In addition, transmission electron microscope (TEM) analysis showed that encapsulated p3Ch during redifferentiation process presented an appreciable production of secretory vesicles, containing extracellular matrix (ECM) dense materials, and collagen fibers with their typical banding pattern, in the surrounding lacuna, remembering the natural microenvironment of chondrocytes. As a whole, our data indicate that the cell-microfiber combinations here produced may be efficient in the production of the ECM in its correct form in a short time to support cartilage repair and regeneration. 1. Johnstone B, Alini M, Cucchiarini M, Dodge GR, Eglin D, et al. (2013) Tissue engineering for articular cartilage repair - the state of the art. European Cells & Materials 25: 248-267. 2. Lolli A, Lambertini L, Penolazzi L, Angelozzi M, Morganti C, et al. Pro-chondrogenic effect of miR-221 and Slug depletion in human MSCs. Stem Cell Rev and Reports, in press. 3. Mazzitelli S, Capretto L, Quinci F, Piva R, Nastruzzi C. (2013) Preparation of cell-encapsulation devices in confined microenvironment. Adv Drug Deliv Rev 65:1533-1555.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2363970
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