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1.
Acta Biomater ; 32: 161-169, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26773464

ABSTRACT

The interactions between cells and an underlying biomaterial are important for the promotion of cell adhesion, proliferation, and function. Mesenchymal stem cells (MSCs) have great clinical potential as they are an adult stem cell population capable of multilineage differentiation. The relationship between MSC behavior and several material properties including substrate stiffness and pore size are well investigated, but there has been little research on the influence of porous architecture in a three-dimensional scaffold with a well-controlled architecture. Here, we investigate the impact of two different three-dimensionally printed, pore geometries on the enrichment and differentiation of MSCs. 3D printed scaffolds with ordered cubic pore geometry were supportive of MSC enrichment from unprocessed bone marrow, resulting in cell surface marker expression that was comparable to typical adhesion to tissue culture polystyrene, the gold standard for MSC culture. Results also show that scaffolds fabricated with ordered cubic pores significantly increase the gene expression of MSCs undergoing adipogenesis and chondrogenesis, when compared to scaffolds with ordered cylindrical pores. However, at the protein expression level, these differences were modest. For MSCs undergoing osteogenesis, gene expression results suggest that cylindrical pores may initially increase early osteogenic marker expression, while protein level expression at later timepoints is increased for scaffolds with ordered cubic pores. Taken together, these results suggest that 3D printed scaffolds with ordered cubic pores could be a suitable culture system for single-step MSC enrichment and differentiation. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) have great therapeutic potential, as they are capable of multilineage differentiation. MSC behavior, including lineage commitment, may be influenced by biomaterial properties including substrate stiffness and pore size. With three-dimensional (3D) printing, we can investigate these relationships in 3D culture systems. Here, we fabricated scaffolds with two different well-controlled pore geometries, and investigated the impact on MSC enrichment and differentiation. Results show that scaffolds with ordered cubic pore geometry were supportive of both MSC enrichment from unprocessed bone marrow as well as MSC differentiation, resulting in increased gene expression during adipogenesis and chondrogenesis. These results suggest that 3D printed scaffolds with ordered cubic pores could be a suitable culture system for single-step MSC enrichment and differentiation.


Subject(s)
Cell Differentiation , Mesenchymal Stem Cells/cytology , Printing, Three-Dimensional , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Proliferation , Cell Survival , Flow Cytometry , Gene Expression Regulation , Humans , Porosity , Tissue Scaffolds
2.
Tissue Eng Part C Methods ; 22(4): 348-59, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26797048

ABSTRACT

Despite great promise surrounding mesenchymal stem cells (MSCs), their implementation for tissue engineering strategies remains in the development phases. Many of the concerns regarding the clinical use of MSCs originate from population heterogeneity, during both isolation and differentiation. In this study, we utilize our previously developed centrifugation cell adhesion protocol for the separation of MSCs. Our findings reveal that MSCs can be isolated from whole bone marrow using a 200 g (700 pN) centrifugal force after 24 h of culture on polystyrene with cell surface marker expression equivalent to positive controls. During differentiation, a centrifugation protocol with identical force parameters could be applied 14 days into chondrogenic differentiation to isolate differentiated chondrocytes, which exhibited increased expression of chondrogenic markers compared to controls. In summary, the use of our developed centrifugation cell adhesion protocol has proven to be an effective means to separate MSC populations, decreasing the heterogeneity of subsequent cell therapy products.


Subject(s)
Antigens, Differentiation/biosynthesis , Bone Marrow Cells , Cell Differentiation , Cell Separation/methods , Chondrogenesis , Mesenchymal Stem Cells , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Centrifugation , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism
3.
Ann Biomed Eng ; 44(7): 2103-13, 2016 07.
Article in English | MEDLINE | ID: mdl-26577256

ABSTRACT

We have recently developed a bioreactor that can apply both shear and compressive forces to engineered tissues in dynamic culture. In our system, alginate hydrogel beads with encapsulated human mesenchymal stem cells (hMSCs) were cultured under different dynamic conditions while subjected to periodic, compressive force. A customized pressure sensor was developed to track the pressure fluctuations when shear forces and compressive forces were applied. Compared to static culture, dynamic culture can maintain a higher cell population throughout the study. With the application of only shear stress, qRT-PCR and immunohistochemistry revealed that hMSCs experienced less chondrogenic differentiation than the static group. The second study showed that chondrogenic differentiation was enhanced by additional mechanical compression. After 14 days, alcian blue staining showed more extracellular matrix formed in the compression group. The upregulation of the positive chondrogenic markers such as Sox 9, aggrecan, and type II collagen were demonstrated by qPCR. Our bioreactor provides a novel approach to apply mechanical forces to engineered cartilage. Results suggest that a combination of dynamic culture with proper mechanical stimulation may promote efficient progenitor cell expansion in vitro, thereby allowing the culture of clinically relevant articular chondrocytes for the treatment of articular cartilage defects.


Subject(s)
Antigens, Differentiation/biosynthesis , Cell Culture Techniques/methods , Cell Proliferation , Chondrogenesis , Compressive Strength , Gene Expression Regulation , Mesenchymal Stem Cells/metabolism , Humans , Mesenchymal Stem Cells/cytology
4.
J Biomed Mater Res A ; 103(5): 1864-74, 2015 May.
Article in English | MEDLINE | ID: mdl-25195680

ABSTRACT

Tissue engineering is an alternative method for articular cartilage repair. Mechanical stimulus has been found to be an important element to the healthy development of chondrocytes and maintenance of their native phenotype. To enhance nutrient transport and apply mechanical stress, we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture chondrocytes in three-dimensional scaffolds. In our design, chondrocytes are encapsulated in alginate scaffolds and placed into a tubular growth chamber, which is perfused with media to enhance nutrient transfer and expose cells to fluid flow. Results demonstrate that TPS culture promotes the proliferation of chondrocytes compared to static culture as shown by DNA content and histochemical staining. After 14 days of culture, low messenger RNA expression of proinflammatory and apoptotic markers in TPS bioreactor culture confirmed that a flow rate of 3 mL/min does not damage the chondrocytes embedded in alginate scaffolds. Additionally, cells cultured in the TPS bioreactor showed increased gene expression levels of aggrecan, type II collagen, and superficial zone protein compared to the static group, indicative of the emergence of the superficial zone specific phenotype. Therefore, the TPS bioreactor is an effective means to enhance the proliferation and phenotype maintenance of chondrocytes in vitro.


Subject(s)
Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Chondrocytes/cytology , Perfusion/instrumentation , Perfusion/methods , Proteins/metabolism , Alginates , Animals , Apoptosis , Cattle , Cell Survival , Cells, Cultured , Chondrocytes/metabolism , Cytokines/metabolism , DNA/metabolism , Glucuronic Acid , Hexuronic Acids , Immunohistochemistry , Inflammation Mediators/metabolism , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Staining and Labeling
5.
Mol Pharm ; 11(7): 2172-81, 2014 Jul 07.
Article in English | MEDLINE | ID: mdl-24620713

ABSTRACT

The importance of providing a physiologically relevant environment for cell culture is well recognized. The combination of proper environmental cues which are provided in vivo by the bloodstream and extracellular matrix must be reproduced to properly examine cell response in vitro, and cannot be recapitulated using traditional culture on polystyrene. Here, we have developed a device, the dynamic stem cell culture platform (DSCCP), consisting of a biomimetic scaffold cultured within the dynamic environment of a perfusion bioreactor. By varying scaffold parameters including stiffness and protein inclusion at the material surface, we found that human mesenchymal stem cells (hMSCs) were able to adhere to modified substrates, while still maintaining multipotency. Culture in a perfusion bioreactor showed cell survival and proliferation, particularly on modified substrates. The DSCCP represents a complete platform for cell adhesion and subsequent evaluation, including the response of a cell population to drug treatment.


Subject(s)
Cell Adhesion/physiology , Cell Culture Techniques/methods , Mesenchymal Stem Cells/physiology , Biomimetics/methods , Bioreactors , Cell Differentiation/physiology , Cell Proliferation/physiology , Cell Survival/physiology , Extracellular Matrix/physiology , Humans , Perfusion/methods , Tissue Engineering/methods , Tissue Scaffolds
6.
Biomaterials ; 35(16): 4636-45, 2014 May.
Article in English | MEDLINE | ID: mdl-24631250

ABSTRACT

Biomaterial-supported culture methods, allowing for directed three-dimensional differentiation of stem cells are an alternative to canonical two-dimensional cell cultures. In this paper, we evaluate the suitability of alginate for three-dimensional cultures to enhance differentiation of mouse embryonic stem cells (mESCs) towards neural lineages. We tested whether encapsulation of mESCs within alginate beads could support and/or enhance neural differentiation with respect to two-dimensional cultures. We encapsulated cells in beads of alginate with or without modification by fibronectin (Fn) or hyaluronic acid (HA). Gene expression analysis showed that cells grown in alginate and alginate-HA present increased differentiation toward neural lineages with respect to the two-dimensional control and to Fn group. Immunocytochemistry analyses confirmed these results, further showing terminal differentiation of neurons as seen by the expression of synaptic markers and markers of different neuronal subtypes. Our data show that alginate, alone or modified, is a suitable biomaterial to promote in vitro differentiation of pluripotent cells toward neural fates.


Subject(s)
Alginates/chemistry , Biocompatible Materials/chemistry , Embryonic Stem Cells/cytology , Tissue Scaffolds/chemistry , Animals , Cell Culture Techniques , Cell Differentiation , Cell Line , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Hyaluronic Acid/chemistry , Mice
7.
Tissue Eng Part C Methods ; 18(7): 537-44, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22235797

ABSTRACT

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.


Subject(s)
Cell Adhesion/physiology , Cell Culture Techniques , Centrifugation , Chondrocytes/cytology , Chondrocytes/metabolism , Polystyrenes/metabolism , Animals , Blotting, Western , Cattle , Cell Differentiation , Cell Proliferation , Cells, Cultured , Collagen Type II/genetics , Collagen Type II/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Immunoenzyme Techniques , Mice , Phenotype , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Serial Passage
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