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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

mRNA expression for BMSCs. (A) collagen type 1 alpha 1 gene and (B) SOX9 gene. All data has been normalized to fibrin/HA-MA with 10% FBS condition. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is the housekeeping gene. * – Statistical significance from the fibrin/HA-MA with 10% FBS condition (p < 0.05) and † – Statistical significance from the Fibrin with PL condition (p < 0.05).
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Figure 7: mRNA expression for BMSCs. (A) collagen type 1 alpha 1 gene and (B) SOX9 gene. All data has been normalized to fibrin/HA-MA with 10% FBS condition. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is the housekeeping gene. * – Statistical significance from the fibrin/HA-MA with 10% FBS condition (p < 0.05) and † – Statistical significance from the Fibrin with PL condition (p < 0.05).

Mentions: To determine chondrogenic potential, the cells were incubated within as-selected hydrogel for 12 days, and, the gene expression of collagen type 1 alpha 1 (COL1A1), collagen type 2 alpha 1 (COL2A1), SOX9 and aggrecan (ACAN) was assayed using qPCR (Figure 7). A platelet lysate (PL) was used as a supplementation to mimic real-life applications using BMSCs, since it is known to contain the growth factors such as transforming growth factor (TGFβ) that promote chondrogenesis [25]. While latent TGFβ was not quantified, active TGFβ concentration was quantified at 8.97 ng/mL using the Quantikine ELISA kit and other researchers have reported supplementation in the range of 5-20 ng/mL [53,54]. In the compared data set, one-way ANOVA confirmed that the data was statistically different (p < 0.05). The fold increase of mRNA transcripts was normalized to cells within fibrin/HA-MA gels supplemented with 10% fetal bovine serum (FBS). There was a downregulation of COL1A1 in all conditions with a significant downregulation in BMSCs in the fibrin/HA-MA containing PL (Figure 7A). SOX9 upregulation was observed for all conditions with a statistically significant difference in the fibrin/HA-MA with PL (Figure 7B). COL2A1 and ACAN genes were also upregulated in certain cases yet these genes were not expressed as strongly as SOX9 or COL1A1 (Refer to Additional file 1: Figure S1). A decrease in COL1A1 expression and an increase in SOX9 are good indicators of chondrogenesis [55]. Previous research has also indicated that the expressions of SOX9 and COL1A1 are very strong in comparison to the expression of COL2A1 and ACAN when BMSCs are cultured in a fibrin-based or hyaluronic acid-based scaffold [54,56-58]. Furthermore, SOX9 is by itself a favorable marker that indicates the production of COL2A1 and ACAN since it promotes the production of these extracellular components during chondrogenesis in vivo[59].


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)

mRNA expression for BMSCs. (A) collagen type 1 alpha 1 gene and (B) SOX9 gene. All data has been normalized to fibrin/HA-MA with 10% FBS condition. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is the housekeeping gene. * – Statistical significance from the fibrin/HA-MA with 10% FBS condition (p < 0.05) and † – Statistical significance from the Fibrin with PL condition (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4109069&req=5

Figure 7: mRNA expression for BMSCs. (A) collagen type 1 alpha 1 gene and (B) SOX9 gene. All data has been normalized to fibrin/HA-MA with 10% FBS condition. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) is the housekeeping gene. * – Statistical significance from the fibrin/HA-MA with 10% FBS condition (p < 0.05) and † – Statistical significance from the Fibrin with PL condition (p < 0.05).
Mentions: To determine chondrogenic potential, the cells were incubated within as-selected hydrogel for 12 days, and, the gene expression of collagen type 1 alpha 1 (COL1A1), collagen type 2 alpha 1 (COL2A1), SOX9 and aggrecan (ACAN) was assayed using qPCR (Figure 7). A platelet lysate (PL) was used as a supplementation to mimic real-life applications using BMSCs, since it is known to contain the growth factors such as transforming growth factor (TGFβ) that promote chondrogenesis [25]. While latent TGFβ was not quantified, active TGFβ concentration was quantified at 8.97 ng/mL using the Quantikine ELISA kit and other researchers have reported supplementation in the range of 5-20 ng/mL [53,54]. In the compared data set, one-way ANOVA confirmed that the data was statistically different (p < 0.05). The fold increase of mRNA transcripts was normalized to cells within fibrin/HA-MA gels supplemented with 10% fetal bovine serum (FBS). There was a downregulation of COL1A1 in all conditions with a significant downregulation in BMSCs in the fibrin/HA-MA containing PL (Figure 7A). SOX9 upregulation was observed for all conditions with a statistically significant difference in the fibrin/HA-MA with PL (Figure 7B). COL2A1 and ACAN genes were also upregulated in certain cases yet these genes were not expressed as strongly as SOX9 or COL1A1 (Refer to Additional file 1: Figure S1). A decrease in COL1A1 expression and an increase in SOX9 are good indicators of chondrogenesis [55]. Previous research has also indicated that the expressions of SOX9 and COL1A1 are very strong in comparison to the expression of COL2A1 and ACAN when BMSCs are cultured in a fibrin-based or hyaluronic acid-based scaffold [54,56-58]. Furthermore, SOX9 is by itself a favorable marker that indicates the production of COL2A1 and ACAN since it promotes the production of these extracellular components during chondrogenesis in vivo[59].

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