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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

Representative images of live/dead staining of BMSCs in fibrin/HA-MA hydrogels. Live cells were stained with Calcein-AM (Red) and dead cells with EthD-1 (Green). Formulation with 4 mg/mL fibrinogen with 0 mg/mL HA-MA at (A) day 2, (B) day 4, and (C) day 6. Formulation with 4 mg/mL fibrinogen with 1 mg/mL HA-MA at (D) day 2, (E) day 4, and (F) day 6. Formulation with 6 mg/mL fibrinogen with 1 mg/mL HA-MA at (G) day 2, (H) day 4, and (I) day 6. Scale bar: 100 μm.
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Figure 4: Representative images of live/dead staining of BMSCs in fibrin/HA-MA hydrogels. Live cells were stained with Calcein-AM (Red) and dead cells with EthD-1 (Green). Formulation with 4 mg/mL fibrinogen with 0 mg/mL HA-MA at (A) day 2, (B) day 4, and (C) day 6. Formulation with 4 mg/mL fibrinogen with 1 mg/mL HA-MA at (D) day 2, (E) day 4, and (F) day 6. Formulation with 6 mg/mL fibrinogen with 1 mg/mL HA-MA at (G) day 2, (H) day 4, and (I) day 6. Scale bar: 100 μm.

Mentions: BMSC activity was examined using a metabolic assay after 2, 4, and 6-day culture in different fibrin/HA-MA hydrogel formulations (Figure 3). No significant statistical differences between 4 mg/mL (Figure 3A) and 6 mg/mL (Figure 3B) fibrinogen groups were observed. However, the HA-MA concentration in each group influenced cellular activity. Within the two groups, there was significant statistical difference (p < 0.05) as calculated by one-way ANOVA. We, further, calculated the p values between individual group-pairs by student’s t-Test. With the introduction of HA-MA into the hydrogels, both 4 mg/mL and 6 mg/mL fibrinogen concentrations supported substantial cell activity with HA-MA concentrations below 1 mg/mL. Gel compaction occurred due to the stresses imposed by the BMSC contraction in the gel matrix [48]. Gel compaction could have been increased due to the presence of TGFβ [49,50]. Based on preliminary observations (data not shown), fibrinogen concentrations less than 3 mg/mL resulted in gel compaction after only two days in culture, after which cells migrated from inside the gel to the bare plate. This suggests that the hydrogels with fibrinogen concentrations below 3 mg/mL may be mechanically unreliable. 6 mg/mL was selected as the upper limit due to the fact that higher concentrations became too viscous to easily mix with the HA-MA solution. The BMSC viability was also visualized by live/dead staining (Figure 4), which showed increasing numbers of viable cells and cell density at successive time points for both 4 mg/mL and 6 mg/mL fibrinogen concentrations. It was evident that there were a greater number of cells outlined in day 6 compared to day 2 in both the 4 mg/mL and 6 mg/mL conditions. The use of both the metabolic assay and live/dead staining is a strong indication of cellular proliferation. Metabolic activity alone is not reliable enough to ensure cell division; however, the quantitative measure of cellular activity with the increase in cell density as determined by live/dead staining strongly suggests cellular proliferation. A decrease in cellular proliferation as a function of mechanical strength has been recently shown in similar hydrogels. Fibrin constructs containing HA modified with tyramine capable of crosslinking in the presence of hydrogen peroxide revealed a decrease in proliferation of endothelial cells with increased mechanical strength [51].


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)

Representative images of live/dead staining of BMSCs in fibrin/HA-MA hydrogels. Live cells were stained with Calcein-AM (Red) and dead cells with EthD-1 (Green). Formulation with 4 mg/mL fibrinogen with 0 mg/mL HA-MA at (A) day 2, (B) day 4, and (C) day 6. Formulation with 4 mg/mL fibrinogen with 1 mg/mL HA-MA at (D) day 2, (E) day 4, and (F) day 6. Formulation with 6 mg/mL fibrinogen with 1 mg/mL HA-MA at (G) day 2, (H) day 4, and (I) day 6. Scale bar: 100 μm.
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Related In: Results  -  Collection

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Figure 4: Representative images of live/dead staining of BMSCs in fibrin/HA-MA hydrogels. Live cells were stained with Calcein-AM (Red) and dead cells with EthD-1 (Green). Formulation with 4 mg/mL fibrinogen with 0 mg/mL HA-MA at (A) day 2, (B) day 4, and (C) day 6. Formulation with 4 mg/mL fibrinogen with 1 mg/mL HA-MA at (D) day 2, (E) day 4, and (F) day 6. Formulation with 6 mg/mL fibrinogen with 1 mg/mL HA-MA at (G) day 2, (H) day 4, and (I) day 6. Scale bar: 100 μm.
Mentions: BMSC activity was examined using a metabolic assay after 2, 4, and 6-day culture in different fibrin/HA-MA hydrogel formulations (Figure 3). No significant statistical differences between 4 mg/mL (Figure 3A) and 6 mg/mL (Figure 3B) fibrinogen groups were observed. However, the HA-MA concentration in each group influenced cellular activity. Within the two groups, there was significant statistical difference (p < 0.05) as calculated by one-way ANOVA. We, further, calculated the p values between individual group-pairs by student’s t-Test. With the introduction of HA-MA into the hydrogels, both 4 mg/mL and 6 mg/mL fibrinogen concentrations supported substantial cell activity with HA-MA concentrations below 1 mg/mL. Gel compaction occurred due to the stresses imposed by the BMSC contraction in the gel matrix [48]. Gel compaction could have been increased due to the presence of TGFβ [49,50]. Based on preliminary observations (data not shown), fibrinogen concentrations less than 3 mg/mL resulted in gel compaction after only two days in culture, after which cells migrated from inside the gel to the bare plate. This suggests that the hydrogels with fibrinogen concentrations below 3 mg/mL may be mechanically unreliable. 6 mg/mL was selected as the upper limit due to the fact that higher concentrations became too viscous to easily mix with the HA-MA solution. The BMSC viability was also visualized by live/dead staining (Figure 4), which showed increasing numbers of viable cells and cell density at successive time points for both 4 mg/mL and 6 mg/mL fibrinogen concentrations. It was evident that there were a greater number of cells outlined in day 6 compared to day 2 in both the 4 mg/mL and 6 mg/mL conditions. The use of both the metabolic assay and live/dead staining is a strong indication of cellular proliferation. Metabolic activity alone is not reliable enough to ensure cell division; however, the quantitative measure of cellular activity with the increase in cell density as determined by live/dead staining strongly suggests cellular proliferation. A decrease in cellular proliferation as a function of mechanical strength has been recently shown in similar hydrogels. Fibrin constructs containing HA modified with tyramine capable of crosslinking in the presence of hydrogen peroxide revealed a decrease in proliferation of endothelial cells with increased mechanical strength [51].

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