Enhancing volumetric muscle loss (VML) recovery in a rat model using super durable hydrogels derived from bacteria

Abstract

Bacteria can be programmed to deliver natural materials with defined biological and mechanical properties for controlling cell growth and differentiation. Here, we present an elastic, resilient and bioactive polysaccharide derived from the extracellular matrix of Pantoea sp. BCCS 001. Specifically, it was methacrylated to generate a new photo crosslinkable hydrogel that we coined Pantoan Methacrylate or put simply PAMA. We have used it for the first time as a tissue engineering hydrogel to treat VML injuries in rats. The crosslinked PAMA hydrogel was super elastic with a recovery nearing 100 %, while mimicking the mechanical stiffness of native muscle. After inclusion of thiolated gelatin via a Michaelis reaction with acrylate groups on PAMA we could also guide muscle progenitor cells into fused and aligned tubes - something reminiscent of mature muscle cells. These results were complemented by sarcomeric alpha-actinin immunostaining studies. Importantly, the implanted hydrogels exhibited almost 2-fold more muscle formation and 50 % less fibrous tissue formation compared to untreated rat groups. In vivo inflammation and toxicity assays likewise gave rise to positive results confirming the biocompatibility of this new biomaterial system. Overall, our results demonstrate that programmable polysaccharides derived from bacteria can be used to further advance the field of tissue engineering. In greater detail, they could in the foreseeable future be used in practical therapies against VML.

Graphical abstract

Fig. 1

Chemistry behind the developed hydrogels and the corresponding characterizations. (a) Chemical structure of pantoan before and after methacrylation. (b) Photographic images of PAMA before and after UV cross-linking. (c) Zeta potential measurements of pantoan and PAMA with two methacrylation degrees. (d) Nuclear magnetic resonance (NMR) spectroscopy of pantoan and PAMA with different methacrylation degrees. (e–g) The thermal stability and crystallization temperature of pantoan and PAMA₂₁ and PAMA₃₇ were examined with TGA and DSC.

Fig. 2

Morphological, stability assays and rheological properties. (a) Scanning electron microscopy (SEM) of the 2, 3 and 4% PAMA₂₁ hydrogel variants developed herein. (b) Pore sizes within the imaged hydrogels were analyzed with ImageJ and are displayed. (c) Weight loss and (d) swelling profile of PAMA₂₁ hydrogels with different concentration (2, 3 and 4%). (e) The rheological properties (storage modulus (G′), loss modulus (G″) and Viscosity) of PAMA₂₁ (2, 3, and 4% w/v). (f) Quantification of the printability of PAMA₂₁ hydrogels. (g) Snapshots of the printed PAMA scaffolds like the native shapes of various muscles.

Fig. 3

Mechanical analysis. (a) Brief explanation of the working principle behind the mechanical analysis of hydrogels. (b) Cyclic stress−strain curves (up to five cycles) corresponding to 2, 3 and 4 % PAMA₂₁ hydrogels cross-linked in the presence of 0.5% photoinitiator. (c) Mechanical recovery and total energy dissipated after 70% strain were retrieved from the stress−strain curves. (d) Stress at 70% strain for 2, 3 and 4 % PAMA₂₁ hydrogels. (e) Ultimate stress and compressive modulus of the respective hydrogels as shown here. (f) Strain at breaking point of the respective hydrogels.

Fig. 4

C2C12 viability evaluation after encapsulating in PEMA₂₁UV crosslinked hydrogels. (a) Schematic showing the steps behind the mold preparation, C2C12 encapsulation, crosslinking, and culture. (b) Representative images of cells encapsulated in PEMA₂₁ after being stained with calcein-AM (green, live cells) and ethidium homodimer-1 (red, dead cells) at different time points. (c) Number of total and (d) Live cells observed in cell-laden hydrogels at different time points. (e) C2C12 viability after 1, 3, 7, and 14 days in culture.

Fig. 5

(a) C2C12 spreading and alignment visualized by fluorescent staining (phalloidin/Hoechst) and confocal microscopy at different time points (1, 3, 5, and 7 days) and different types of media. The fluorescent images show F-actin (red) and nucleus (blue) for the cells encapsulated in PEMA₂₁-Gelin-S hydrogels. The first two graphs and their associated images are related to the samples conditioned in growth media. The two bottom images are taken on day 5 and 7 from the samples conditioned in the differentiation media. The percentages shown on the images are an estimation of cellular confluency inside the hydrogels. (b) Fluorescent images of alpha-actinin (in red) immunostained cells encapsulated in PEMA₂₁-Gelin-S hydrogels after 7 and 10 days of culture in differentiation media. (c) Alignment analysis of the immunostained cells in (b).

Fig. 6

Histological comparison of the Tibialis Anterior (TA) volumetric muscle loss (VML) injury site before and after and hydrogel implant treatment. (a) Photograph of the surgical site of the defect site after 7 and 21 days post-injury The white lines denote defect site boundaries, and white arrows illustrate treatment location within the different times post VML surgery. VML injury was created by an excision of approximately (8 mm diameter × 3 mm deep) of the TA muscle. Immediately after defect creation, the fascia layer was sutured, and PAMA and PAMA-Gelin-s hydrogels were injected through the incision, in the area between fascia and muscle, the skin was closed, and the animal was allowed to recover (not shown). (b) H&E staining of the histological slides after 3 weeks (c) Quantificative analysis of regenerating myofibers (%), and mean area of regenerating myofibers (μm²) at 21 days post-injury. Columns with star superscripts between one another are significantly different from one another (p < 0.05).

Fig. 7

Toluidine Blue and Masson's Trichrome Histological comparison and Blood serum analysis at 21 days post-injury. (a) Histological comparison by Toluidine Blue -stained sections of tissue retrieved from the implant region of injured TA muscle at 21 days post-injury. (b) Histological comparison by Masson's Trichrome -stained sections of tissue retrieved from implant region of injured TA muscle at 21 days post-injury and Quantification analysis of fibrosis area (%). (c) Blood serum biochemical parameters at 21 days post-injury. Columns with star superscripts between one another are significantly different from one another (p < 0.05).