Equine Tenocyte Seeding on Gelatin Hydrogels Improves Elongated Morphology

Abstract

(1) Background: Tendinopathy is a common injury in both human and equine athletes. Representative in vitro models are mandatory to facilitate translation of fundamental research into successful clinical treatments. Natural biomaterials like gelatin provide favorable cell binding characteristics and are easily modifiable. In this study, methacrylated gelatin (gel-MA) and norbornene-functionalized gelatin (gel-NB), crosslinked with 1,4-dithiotreitol (DTT) or thiolated gelatin (gel-SH) were compared. (2) Methods: The physicochemical properties (¹H-NMR spectroscopy, gel fraction, swelling ratio, and storage modulus) and equine tenocyte characteristics (proliferation, viability, and morphology) of four different hydrogels (gel-MA, gel-NB85/DTT, gel-NB55/DTT, and gel-NB85/SH75) were evaluated. Cellular functionality was analyzed using fluorescence microscopy (viability assay and focal adhesion staining). (3) Results: The thiol-ene based hydrogels showed a significantly lower gel fraction/storage modulus and a higher swelling ratio compared to gel-MA. Significantly less tenocytes were observed on gel-MA discs at 14 days compared to gel-NB85/DTT, gel-NB55/DTT and gel-NB85/SH75. At 7 and 14 days, the characteristic elongated morphology of tenocytes was significantly more pronounced on gel-NB85/DTT and gel-NB55/DTT in contrast to TCP and gel-MA. (4) Conclusions: Thiol-ene crosslinked gelatins exploiting DTT as a crosslinker are the preferred biomaterials to support the culture of tenocytes. Follow-up experiments will evaluate these biomaterials in more complex models.

Keywords: cell proliferation; gel fraction; gelatin; hydrogels; morphology; storage modulus; swelling ratio; tenocytes; viability.

Figure 1

Reaction scheme depicting the development of gel-MA, gel-NB and gel-SH precursors. The crosslinked hydrogel films are obtained through chain growth polymerization for gel-MA and via step growth polymerization when gel-NB is combined with either DTT or gel-SH. DTT: 1,4-dithiotreitol; EDC: N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; NHS: N-hydroxysuccinimide.

Figure 2

Visualization of nuclear shape on DAPI stained nuclei. If the ratio of the major nuclear axis to the minor nuclear axis, the aspect ratio (AR), is bigger than 1, a more elongated shape is seen. Circularity (Circ.) is measured between 0 and 1, the value being close to 1 represents a rounder cell shape.

Figure 3

To confirm the degree of substitution, ¹H-NMR spectra were assessed of (A) Gel-MA DS 97%, (B) Gel-NB DS 85%, and (C) Gel-NB DS 55%. Gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin.

Figure 4

Physicochemical properties of the obtained hydrogel films: (A) gel fraction, (B) mass swelling ratio, and (C) mechanical properties of 10 w/v% films containing 2 mol% LAP photoinitiator. Significant differences are shown with adjusted p-values and error bars representing standard deviation. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; LAP: lithium(2,4,6-trimethylbenzoyl) phenylphosphinate.

Figure 5

Tenocyte characteristics as determined on thiol-ene based hydrogels and compared to both TCP and gel-MA: (A) cell proliferation: cell numbers per image are shown, (B) viability assay: the percentage of the area occupied by living cells is shown in relation to the total cell area cultured per image. Significant differences are shown with adjusted p-values. After 14 days in culture the number of cells on all thiol-ene based hydrogels were comparable and significantly higher than on gel-MA. It can therefore be concluded that gel-MA is not optimal to support tenocyte cultures. Gel-NB55/DTT also significantly differed from TCP which indicates superior biocompatibility of gel-NB55/DTT. Viability on all hydrogels exceeded 95%, indicating excellent biocompatibility (>70%, ISO 10993-5 (2009)). The decreased viability on gel-NB55/DTT can be explained by contact inhibition. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; TCP: tissue culture plastic.

Figure 6

Representative images of the qualitative evaluation of tenocyte morphology and cell alignment based on fluorescent staining of the actin filaments (orange) with DAPI nuclear counterstaining (blue). Experiment was performed in triplicate. Tenocytes should be spindle-shaped, elongated and organized in a parallel fashion (as seen on gel-NB55/DTT days 7 and 14, and gel-NB85/DTT day 14). A rounder cell morphology on gel-MA discs is seen (both timepoints) and an intermediate morphology (between round and spindle-shaped) on TCP and gel-NB85/SH75 (both timepoints). DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; TCP: tissue culture plastic.

Figure 7

Tenocyte characteristics as determined on thiol-ene based hydrogels and compared to both TCP and gel-MA: (A) nuclear aspect ratio (AR): ratio of the major nuclear axis to the minor nuclear axis. An elongated cell shape is represented by a higher aspect ratio, and (B) circularity: a perfect circle has a value of 1. Significant differences are shown with adjusted p-values. Favorable tenocyte characteristics are high AR and low circularity, as expressed on all thiol-ene based hydrogels, most clearly pronounced on day 14. DTT: 1,4-dithiotreitol; gel-MA: methacrylated gelatin; gel-NB: norbornene-functionalized gelatin; gel-SH: thiolated gelatin; TCP: tissue culture plastic.