Effect of dual growth factor delivery on chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in injectable hydrogel composites.

Contains fulltext : 80847.pdf (publisher's version ) (Open Access)An injectable hydrogel composite consisting of oligo(poly(ethylene glycol)fumarate) (OPF) and gelatin microparticles has been developed as a novel carrier system for cells and growth factors. Rabbit marrow mesenchymal stem cells (MSCs) and gelatin microparticles (MPs) loaded with insulin-like growth factor-1 (IGF-1), transforming growth factor-beta1 (TGF-beta1), or a combination of both growth factors were mixed with OPF, a poly(ethylene glycol)-diacrylate crosslinker and the radical initiators ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, and then crosslinked at 37 degrees C for 8 min to form hydrogel composites. Hydrogel composites encapsulating rabbit marrow MSCs and blank MPs served as controls. At day 14, confocal fluorescent images of OPF hydrogels showed a strong aggregation of rabbit marrow MSCs when encapsulated with IGF-1-loaded MPs with or without TGF-beta1-loaded MPs. Quantitative RT-PCR results showed that rabbit marrow MSCs encapsulated with MPs loaded with TGF-beta1 or both TGF-beta1 and IGF-1 had a significant increase in the expression of chondrocyte-specific genes such as collagen type II and aggrecan at day 14 as compared with the control group. Specifically, samples with both TGF-beta1-loaded MPs and IGF-1-loaded MPs exhibited a 121 +/- 20-fold increase of type II collagen gene expression and a 71 +/- 24-fold increase of aggrecan gene expression after 14 days of in vitro culture as compared with controls at day 0. These results suggest that hydrogel composites based on OPF and gelatin microparticles have great potential as carriers for MSCs and multiple growth factors for cartilage tissue engineering applications

Development and characterization of enhanced green fluorescent protein and luciferase expressing cell line for non-destructive evaluation of tissue engineering constructs.

Item does not contain fulltextThis study investigates the utility of genetically modified cells developed for the qualitative and quantitative non-destructive evaluation of cells on biomaterials. The Fisher rat fibroblastic cell line has been genetically modified to stably express the reporter genes enhanced green fluorescence protein (EGFP) and luciferase. These reporter genes provide two unique opportunities to evaluate cell growth on materials without destruction of the sample. Utilizing the fluorescence of EGFP expressed in the cells, we were able to demonstrate distribution of cells in a oligo(poly(ethylene glycol) fumarate) hydrogel material and on a titanium fiber mesh scaffold using an inverted fluorescent light microscope. In addition, we were able to utilize a molecular light imaging system to macroscopically image the cells on these materials both with fluorescence and luminescence, as well as quantify the signal from the samples. Quantification of cell growth on the titanium mesh material for a period of 28 days was accomplished using the molecular light imaging system. Imaging was extended in vivo to cells on the titanium mesh scaffolds subcutaneously implanted in Fisher rats for a period of 28 days. This study outlines a non-destructive method to evaluate cells growing on biomaterials in vitro and in vivo

Supplementary Material for: Chondroitin Sulfate Microparticles Modulate Transforming Growth Factor-β₁-Induced Chondrogenesis of Human Mesenchymal Stem Cell Spheroids

Mesenchymal stem cells (MSCs) have been previously explored as a part of cell-based therapies for the repair of damaged cartilage. Current MSC chondrogenic differentiation strategies employ large pellets; however, we have developed a technique to form small MSC aggregates (500-1,000 cells) that can reduce transport barriers while maintaining a multicellular structure analogous to cartilaginous condensations. The objective of this study was to examine the effects of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within small MSC spheroids cultured in the presence of transforming growth factor (TGF)-β₁ on chondrogenesis. Spheroids with MPs induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids without MPs, although no large differences in immunostaining for these matrix molecules were observed by day 21 between these groups. Collagen I and X were also detected in the extracellular matrix (ECM) of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. This study demonstrates the use of CSMA materials to further examine the effects of matrix molecules on MSC phenotype as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific musculoskeletal tissues