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. 2024;1(2):58-83.
doi: 10.37349/ebmx.2024.00006. Epub 2024 Apr 23.

A biomimetic approach to modulating the sustained release of fibroblast growth factor 2 from fibrin microthread scaffolds

Meagan E Carnes  1 , Cailin R Gonyea  1 , Jeannine M Coburn  1 , George D Pins  1
Affiliations

A biomimetic approach to modulating the sustained release of fibroblast growth factor 2 from fibrin microthread scaffolds

Meagan E Carnes et al. Explor Biomat X. 2024.

Abstract

Aim: The pleiotropic effect of fibroblast growth factor 2 (FGF2) on promoting myogenesis, angiogenesis, and innervation makes it an ideal growth factor for treating volumetric muscle loss (VML) injuries. While an initial delivery of FGF2 has demonstrated enhanced regenerative potential, the sustained delivery of FGF2 from scaffolds with robust structural properties as well as biophysical and biochemical signaling cues has yet to be explored for treating VML. The goal of this study is to develop an instructive fibrin microthread scaffold with intrinsic topographic alignment cues as well as regenerative signaling cues and a physiologically relevant, sustained release of FGF2 to direct myogenesis and ultimately enhance functional muscle regeneration.

Methods: Heparin was passively adsorbed or carbodiimide-conjugated to microthreads, creating a biomimetic binding strategy, mimicking FGF2 sequestration in the extracellular matrix (ECM). It was also evaluated whether FGF2 incorporated into fibrin microthreads would yield sustained release. It was hypothesized that heparin-conjugated and co-incorporated (co-inc) fibrin microthreads would facilitate sustained release of FGF2 from the scaffold and enhance in vitro myoblast proliferation and outgrowth.

Results: Toluidine blue staining and Fourier transform infrared spectroscopy confirmed that carbodiimide-conjugated heparin bound to fibrin microthreads in a dose-dependent manner. Release kinetics revealed that heparin-conjugated fibrin microthreads exhibited sustained release of FGF2 over a period of one week. An in vitro assay demonstrated that FGF2 released from microthreads remained bioactive, stimulating myoblast proliferation over four days. Finally, a cellular outgrowth assay suggests that FGF2 promotes increased outgrowth onto microthreads.

Conclusions: It was anticipated that the combined effects of fibrin microthread structural properties, topographic alignment cues, and FGF2 release profiles will facilitate the fabrication of a biomimetic scaffold that enhances the regeneration of functional muscle tissue for the treatment of VML injuries.

Keywords: Fibroblast growth factor 2; fibrin; fibrin microthreads; myoblast; skeletal muscle; tissue engineering.

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Conflict of interest statement

Conflicts of interest The authors declare that they have no conflicts of interest.

Figures

Figure 1.
Figure 1.
Transwell®-based proliferation assay. Myoblasts were seeded in 6-wells 24 h prior to the addition of the Transwell® insert, which contained the microthread constructs. Medium was switched from CM to SFM 4 h prior to the addition of the Transwell®. After 24 h in a specific well, the Transwell® insert was moved to a new well, which was seeded 24 h prior. The well from which Transwell® was moved was immediately fixed with ice-cold methanol until assay completion when all wells were stained
Figure 2.
Figure 2.
3D myoblast outgrowth assay. Side and top view of myoblast outgrowth assay, where PDMS rings anchoring three fibrin microthreads are placed on top of an elevated Thermanox coverslip, onto which a myoblast-seeded fibrin hydrogel is cast. The distance of the leading cell from the gel-microthread interface is measured over the course of four days. The representative fluorescent micrograph shows cellular outgrowth analysis on a fibrin microthread. The scale bar is 100 mm
Figure 3.
Figure 3.
Toluidine blue analysis of heparin-conjugated fibrin films. (A) Pixel intensity analysis of UNX passively adsorbed (EDC−) and covalently conjugated (EDC+) fibrin films with varying heparin concentrations stained with toluidine blue; (B) stereo microscope images of toluidine blue dyed fibrin films, where the blue dye uptake increases with increasing heparin concentration. Statistical significance is indicated by * P < 0.05 and ** P < 0.01 between corresponding groups determined by one-way ANOVA with Tukey’s multiple comparisons post hoc analysis (N ≥ 3 experimental replicates)
Figure 4.
Figure 4.
FTIR analysis of heparin-conjugated fibrin films. (A) Representative FTIR spectra of fibrin films passively adsorbed or covalently conjugated with heparin; (B) spectral regions of amide I and amide II; (C) spectral regions of amide III and sulfonated groups (−SO3). Representative data from N ≥ 3 experimental replicates
Figure 5.
Figure 5.
FGF2 release kinetics from fibrin microthreads. (A) Cumulative sustained release of FGF2 over one week from heparin conjugated and co-inc fibrin microthreads; (B) linear regression of FGF2 release from co-inc microthreads through day five (dashed line) showed that co-inc scaffolds achieved zero-order release kinetics of FGF2 (R2 = 0.94); (C) total FGF2 released from fibrin microthreads after one week reveals that UNX-HEP 1000 microthreads had the highest total FGF2 release at seven days. Statistical significance is indicated by * (P < 0.05) between corresponding groups determined by one-way ANOVA with Tukey’s multiple comparisons post hoc analysis (N ≥ 3 experimental replicates). EDCn: microthreads EDC crosslinked in neutral buffer
Figure 6.
Figure 6.
Transwell®-based proliferation assay to determine FGF2 bioactivity and its effect on in vitro percent Ki67+ myoblasts. (A) Percentage of Ki67+ cells is presented as a function of time and demonstrates trends in increasing percent Ki67+ cells with fibrin microthreads passively adsorbed with FGF2; (B) the same data presented as a function of FGF2 incorporation strategy demonstrates that the percent Ki67+ myoblasts significantly increase with time for all conditions passively adsorbed with FGF2. Statistical significance is indicated by * (P < 0.05) between corresponding groups determined by two-way ANOVA with Tukey’s multiple comparisons post hoc analysis (N ≥ 3 experimental replicates)
Figure 7.
Figure 7.
Transwell®-based proliferation assay to determine FGF2 bioactivity and its effect on in vitro myoblast normalized cell number. (A) Fold change in cell number normalized to SFM cell number as a function of time; (B) the same data presented as a function of FGF2 incorporation strategy indicate increased cell numbers in all FGF2 loaded microthreads. Statistical significance is indicated by * (P < 0.05) between corresponding groups determined by two-way ANOVA with Tukey’s multiple comparisons post hoc analysis (N ≥ 3 experimental replicates)
Figure 8.
Figure 8.
Representative images of myoblast outgrowth on fibrin microthreads. Outgrowth on fibrin microthreads with no FGF2 (UNX no FGF2), co-inc with FGF2 (co-inc), or passively adsorbed with FGF2 (EDC, UNX-HEP 1000). Myoblast outgrowth (visualized with DiI staining) was observed as several “leading” myoblasts furthest out on the microthread, which was followed by a more confluent layer of cells. White dotted lines show the microthread-gel interface on day one. White arrows indicate the leading cell position. The scale bar is 100 μm
Figure 9.
Figure 9.
Myoblast outgrowth on FGF2-loaded fibrin microthreads. (A) C2C12 myoblast outgrowth distance on fibrin microthreads as a function of time. Linear regression analysis constrained through the origin revealed that myoblast outgrowth rate was linear on all microthread conditions (0.92 < R2 < 0.99); (B) myoblast outgrowth was calculated as the linear slope over a period of four days and indicates that myoblasts on co-inc and UNX-HEP 1000 microthreads had the highest rate of outgrowth; (C) similar trends were observed when evaluating total distance traveled of the leading cell at day four (N ≥ 3 experimental replicates)
Figure 10.
Figure 10.
Evaluating myoblast proliferation on fibrin microthreads from the 3D outgrowth assay. (A) At the terminal day four timepoint of the myoblast outgrowth assay, microthreads were fixed, removed from outgrowth assays, and stained with Hoechst and Ki67 to determine the extent of myoblast proliferation; (B) Ki67 stained microthreads representative image of UNX with no FGF2; (C) Ki67 stained microthreads representative image of microthreads co-inc with 1 μg/mL F1GF2; (D) Ki67 stained microthreads representative image of UNX microthreads co-inc with 0 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (E) Ki67 stained microthreads representative image of UNX microthreads co-inc with 10 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (F) Ki67 stained microthreads representative image of UNX microthreads co-inc with 100 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (G) Ki67 stained microthreads representative image of UNX microthreads co-inc with 1,000 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (H) Ki67 stained microthreads representative image of EDC crosslinked microthreads co-inc with 0 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (I) Ki67 stained microthreads representative image of EDC crosslinked microthreads co-inc with 10 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (J) Ki67 stained microthreads representative image of EDC crosslinked microthreads co-inc with 100 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2; (K) Ki67 stained microthreads representative image of EDC crosslinked microthreads co-inc with 1,000 μg/mL heparin and passively adsorbed with 1 μg/mL FGF2. The scale bar is 100 μm
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