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A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair.

Snyder TN, Madhavan K, Intrator M, Dregalla RC, Park D - J Biol Eng (2014)

Bottom Line: This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ.However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength.Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioengineering Department, University of Colorado, Anschutz Medical Campus, Mail Stop 8607, 12700 East 19th Avenue, Aurora, CO 80045, USA ; Regenerative Sciences, 403 Summit Blvd, Suite 201, Broomfield, CO 80021, USA.

ABSTRACT

Background: Osteoarthritis (OA) is a degenerative joint disease affecting approximately 27 million Americans, and even more worldwide. OA is characterized by degeneration of subchondral bone and articular cartilage. In this study, a chondrogenic fibrin/hyaluronic acid (HA)-based hydrogel seeded with bone marrow-derived mesenchymal stem cells (BMSCs) was investigated as a method of regenerating these tissues for OA therapy. This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ. However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength. This was addressed by modifying HA with methacrylic anhydride (MA) (HA-MA), which reinforces the fibrin gel, thereby improving mechanical properties. In this study, a range of fibrinogen (the fibrin precursor) and HA-MA concentrations were explored to determine optimal conditions for increased mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro.

Results: Increased mechanical strength was achieved by HA-MA reinforcement within fibrin hydrogels, and was directly correlated with increasing HA-MA concentration. Live/dead staining and metabolic assays confirmed that the crosslinked fibrin/HA-MA hydrogels provided a suitable 3D environment for BMSC proliferation. Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis.

Conclusion: Fibrin/HA-MA hydrogel may be a suitable delivery method for BMSCs, inducing BMSC differentiation into chondrocytes and potentially aiding in articular cartilage repair for OA therapy.

No MeSH data available.


Related in: MedlinePlus

Flow cytometry verifies BMSC phenotype via positive and negative surface markers. Unlabeled BMSCs: (A) FSC vs. SSC and (B-C) Absence of fluorescence detected through any channels. BMSCs treated with CD34-FITC indicate no CD34 expression: (D) FSC vs. SSC, (E) all cells were FITC-negative as detected through channels FL1 and FL2 (100.0% in Q1-LL) and (F) through channels FL3 and FL4 (100.0% in Q2-LL). BMSCs treated with CD105-PE, CD73-PerCP, CD90-APC, CD44-FITC indicate expression of all four surface proteins: (G) FSC vs. SSC, (H) 99.8% of cells (Q1-UR) were PE, PerCP, APC and FITC-positive as detected through FL1 and FL2 and (I) 99.6% of cells (Q2-UR) were detected positive through FL3 and FL4.
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Figure 1: Flow cytometry verifies BMSC phenotype via positive and negative surface markers. Unlabeled BMSCs: (A) FSC vs. SSC and (B-C) Absence of fluorescence detected through any channels. BMSCs treated with CD34-FITC indicate no CD34 expression: (D) FSC vs. SSC, (E) all cells were FITC-negative as detected through channels FL1 and FL2 (100.0% in Q1-LL) and (F) through channels FL3 and FL4 (100.0% in Q2-LL). BMSCs treated with CD105-PE, CD73-PerCP, CD90-APC, CD44-FITC indicate expression of all four surface proteins: (G) FSC vs. SSC, (H) 99.8% of cells (Q1-UR) were PE, PerCP, APC and FITC-positive as detected through FL1 and FL2 and (I) 99.6% of cells (Q2-UR) were detected positive through FL3 and FL4.

Mentions: Phenotypical surface protein expression of BMSCs was confirmed by flow cytometry (Figure 1) after incubating BMSCs with the antibodies unlabeled with fluorescent dyes (Figure 1A, B and C), anti-CD34 antibody (Figure 1D, E and F) and fluorescent-labeled antibodies, CD105-PE, CD73-PerCP, CD-90APC, CD44-FITC & CD34-FITC (Figure 1G, H and I). The first graph of each case (Figure 1A, D and G) is the side scatter channel (SSC) vs. forward scatter channel (FSC). SSC is a measure of cell granularity, while FSC represents overall cell size. The next two graphs of each case represent the fluorescent intensity through channels FL1 (fluorescein isothiocyanate, FITC) and FL2 (phycoerythrin, PE) (Figure 1B, E and H), and FL3 (peridinin chlorophyll protein complex, PerCP) and FL4 (allophycocyanin, APC) (Figure 1C, F and I). Based on the fluorescent tag of the antibodies, positive markers display a shift in their respective fluorescent channel. In the four-color graphs (Figure 1G, H and I), there is a shift to the upper right quadrant of both fluorescence graphs, which signifies a positive result for all four markers. All markers were run individually with positive results. The unlabeled cell population was used to determine the placement of the quadrants. Thus, the BMSCs were verified to possess the typical phenotype necessary for them to undergo chondrogenesis.


A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair.

Snyder TN, Madhavan K, Intrator M, Dregalla RC, Park D - J Biol Eng (2014)

Flow cytometry verifies BMSC phenotype via positive and negative surface markers. Unlabeled BMSCs: (A) FSC vs. SSC and (B-C) Absence of fluorescence detected through any channels. BMSCs treated with CD34-FITC indicate no CD34 expression: (D) FSC vs. SSC, (E) all cells were FITC-negative as detected through channels FL1 and FL2 (100.0% in Q1-LL) and (F) through channels FL3 and FL4 (100.0% in Q2-LL). BMSCs treated with CD105-PE, CD73-PerCP, CD90-APC, CD44-FITC indicate expression of all four surface proteins: (G) FSC vs. SSC, (H) 99.8% of cells (Q1-UR) were PE, PerCP, APC and FITC-positive as detected through FL1 and FL2 and (I) 99.6% of cells (Q2-UR) were detected positive through FL3 and FL4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Flow cytometry verifies BMSC phenotype via positive and negative surface markers. Unlabeled BMSCs: (A) FSC vs. SSC and (B-C) Absence of fluorescence detected through any channels. BMSCs treated with CD34-FITC indicate no CD34 expression: (D) FSC vs. SSC, (E) all cells were FITC-negative as detected through channels FL1 and FL2 (100.0% in Q1-LL) and (F) through channels FL3 and FL4 (100.0% in Q2-LL). BMSCs treated with CD105-PE, CD73-PerCP, CD90-APC, CD44-FITC indicate expression of all four surface proteins: (G) FSC vs. SSC, (H) 99.8% of cells (Q1-UR) were PE, PerCP, APC and FITC-positive as detected through FL1 and FL2 and (I) 99.6% of cells (Q2-UR) were detected positive through FL3 and FL4.
Mentions: Phenotypical surface protein expression of BMSCs was confirmed by flow cytometry (Figure 1) after incubating BMSCs with the antibodies unlabeled with fluorescent dyes (Figure 1A, B and C), anti-CD34 antibody (Figure 1D, E and F) and fluorescent-labeled antibodies, CD105-PE, CD73-PerCP, CD-90APC, CD44-FITC & CD34-FITC (Figure 1G, H and I). The first graph of each case (Figure 1A, D and G) is the side scatter channel (SSC) vs. forward scatter channel (FSC). SSC is a measure of cell granularity, while FSC represents overall cell size. The next two graphs of each case represent the fluorescent intensity through channels FL1 (fluorescein isothiocyanate, FITC) and FL2 (phycoerythrin, PE) (Figure 1B, E and H), and FL3 (peridinin chlorophyll protein complex, PerCP) and FL4 (allophycocyanin, APC) (Figure 1C, F and I). Based on the fluorescent tag of the antibodies, positive markers display a shift in their respective fluorescent channel. In the four-color graphs (Figure 1G, H and I), there is a shift to the upper right quadrant of both fluorescence graphs, which signifies a positive result for all four markers. All markers were run individually with positive results. The unlabeled cell population was used to determine the placement of the quadrants. Thus, the BMSCs were verified to possess the typical phenotype necessary for them to undergo chondrogenesis.

Bottom Line: This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ.However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength.Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioengineering Department, University of Colorado, Anschutz Medical Campus, Mail Stop 8607, 12700 East 19th Avenue, Aurora, CO 80045, USA ; Regenerative Sciences, 403 Summit Blvd, Suite 201, Broomfield, CO 80021, USA.

ABSTRACT

Background: Osteoarthritis (OA) is a degenerative joint disease affecting approximately 27 million Americans, and even more worldwide. OA is characterized by degeneration of subchondral bone and articular cartilage. In this study, a chondrogenic fibrin/hyaluronic acid (HA)-based hydrogel seeded with bone marrow-derived mesenchymal stem cells (BMSCs) was investigated as a method of regenerating these tissues for OA therapy. This chondrogenic hydrogel system can be delivered in a minimally invasive manner through a small gauge needle, forming a three-dimensional (3D) network structure in situ. However, an ongoing problem with fibrin/HA-based biomaterials is poor mechanical strength. This was addressed by modifying HA with methacrylic anhydride (MA) (HA-MA), which reinforces the fibrin gel, thereby improving mechanical properties. In this study, a range of fibrinogen (the fibrin precursor) and HA-MA concentrations were explored to determine optimal conditions for increased mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro.

Results: Increased mechanical strength was achieved by HA-MA reinforcement within fibrin hydrogels, and was directly correlated with increasing HA-MA concentration. Live/dead staining and metabolic assays confirmed that the crosslinked fibrin/HA-MA hydrogels provided a suitable 3D environment for BMSC proliferation. Quantitative polymerase chain reaction (qPCR) of BMSCs incubated in the fibrin/HA-MA hydrogel confirmed decreased expression of collagen type 1 alpha 1 mRNA with an increase in Sox9 mRNA expression especially in the presence of a platelet lysate, suggesting early chondrogenesis.

Conclusion: Fibrin/HA-MA hydrogel may be a suitable delivery method for BMSCs, inducing BMSC differentiation into chondrocytes and potentially aiding in articular cartilage repair for OA therapy.

No MeSH data available.


Related in: MedlinePlus