Novel Highly Organized Three-Dimensional Scaffold for Cartilage Tissue Engineering Prepared by Microfluidic Technology
Date Issued
2011
Date
2011
Author(s)
Wang, Chen-Chie
Abstract
【Study Design】A novel highly organized alginate scaffold was developed by microfluidic technology. Porcine chondrocytes were cultured in this three-dimensional scaffold in vitro. The cell proliferation and extracellular matrix secretion were measured by cell viability test, 1,9-dimethylmethylene blue (DMMB) assay, and real-time reverse-transcriptase polymerase chain reaction (real-time PCR). The 2nd stage is transplanted porcine cells/scaffold constructs into the back of SCID mice. Mice were sacrificed later and analyzed the results of cartilage regeneration.
【Objective】 To evaluate the effectiveness and feasibility of the new highly organized alginate scaffold for cartilage tissue engineering.
【Background】Osteoarthritis is a degenerative disease and frequently involved knee and hip joint. The annual medical care expenditures in the U.S. are more than 150 billion. Current treatment includes multiple drilling and abrasion arthroplasty etc. for small cartilage defect. Mosaicplasty and autogenous chondrocyte transplantation are new options but gap existence and fibrocartilage formation are still problems. Combination of cells, scaffold and growth factor are considering a promising tissue engineering method for cartilage regeneration.
【Materials and Methods】Initially we developed a new method to produce a highly organized alginate scaffold by microfluidic device. The swelling ratio, porosity and mechanical strength of the new alginate scaffold were analyzed. The microstructure of the scaffold was examined by confocal laser scanning microscope. Porcine chondrocytes were seeded into the alginate scaffold and being cultured for 1, 2 and 3 weeks, respectively. The cells/scaffold constructs were analyzed with cell viability, cell toxicity, extracellular matrix, DNA quantification, gene expression (real- time PCR), SEM and confocal microscopy. In the in vivo study, cells/scaffold constructs were transplanted into the subcutaneous portion of the back of the SCID mice. The mice were sacrificed at 2, 4, and 6 weeks. The constructs were examined with histological studies, immunohistochemical staining and gene expression (real- time PCR).
【Results】The alginate scaffold revealed a highly organized porous structure with interconnection like a honeycomb. Comparing with the scaffold fabricated by freeze drying methods, the results revealed similar mechanical strength, higher porosity and swelling ratio. In vitro study, the chondrocytes can keep phenotype, highly expressed of aggrecan and collagen type II and secret much extracellular matrix (sGAG). SEM and confocal laser scanning microscope showed the chondrocytes were attached firmly on the scaffold. The results demonstrated the new scaffold is effective in chondrocyte culture. In vivo study, the morphology of harvested cells/scaffolds from the mice became harder and turned to white color like cartilage. Cells produced glycosaminoglycans can be proved by alcian blue stain. Immunohistochemical staining revealed cells secreted type II collagen and expressed S-100 protein. PCR showed that the mRNA expressions of aggrecan and type II collagen were up-regulated. The results demonstrated the regenerated cartilage tissue kept the phenotype of hyaline cartilage.
【Conclusions】The new highly organized alginate scaffold fabricated by microfluidic technology has positive effects on cell proliferation and extracellular matrix production. The in vitro and in vivo studies both revealed that the alginate scaffold can maintain normal phenotypes of chondrocytes. The pores mimic the shape and environment of the lacuna so that the chondrocytes will stay and proliferate. We believe the alginate scaffold may provide new possibilities for cartilage tissue engineering in the near future.
【Future works】In spite of the fact that this in vivo animal experiment, in combination with the previous in vitro study, revealed this alginate scaffold is a good candidate for cartilage tissue engineering, the chondrogenesis of this scaffold requires more large animal study to demonstrate for chondral defect in articular cartilage before application to human body. Currently, we also try other biomaterials to fabricate microbubble scaffolds and have the positive results. We suggest addressing mesenchymal stem cell or other source stem cell cultured in this scaffold to increase the chondrogenic properties and is a theoretically valuable research aim.
Subjects
alginate
chondrocyte
microfluidic
cartilage regeneration
tissue engineering
SDGs
Type
thesis
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