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Enzymatically crosslinked gelatin-laminin hydrogels for applications in neuromuscular tissue engineering.

ABSTRACT: We report a water-soluble and non-toxic method to incorporate additional extracellular matrix proteins into gelatin hydrogels, while obviating the use of chemical crosslinkers such as glutaraldehyde. Gelatin hydrogels were fabricated using a range of gelatin concentrations (4%-10%) that corresponded to elastic moduli of approximately 1 kPa-25 kPa, respectively, a substrate stiffness relevant for multiple cell types. Microbial transglutaminase was then used to enzymatically crosslink a layer of laminin on top of gelatin hydrogels, resulting in 2-component gelatin-laminin hydrogels. Human induced pluripotent stem cell derived spinal spheroids readily adhered and rapidly extended axons on GEL-LN hydrogels. Axons displayed a more mature morphology and superior electrophysiological properties on GEL-LN hydrogels compared to the controls. Schwann cells on GEL-LN hydrogels adhered and proliferated normally, displayed a healthy morphology, and maintained the expression of Schwann cell specific markers. Lastly, skeletal muscle cells on GEL-LN hydrogels achieved long-term culture for up to 28 days without delamination, while expressing higher levels of terminal genes including myosin heavy chain, MyoD, MuSK, and M-cadherin suggesting enhanced maturation potential and myotube formation compared to the controls. Future studies will employ the superior culture outcomes of this hybrid substrate for engineering functional neuromuscular junctions and related organ on a chip applications.

SUBMITTER: Besser RR

PROVIDER: S-EPMC7141910 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Enzymatically crosslinked gelatin-laminin hydrogels for applications in neuromuscular tissue engineering.

Besser Rachel R RR Bowles Annie C AC Alassaf Ahmad A Carbonero Daniel D Claure Isabella I Jones Ellery E Reda Joseph J Wubker Laura L Batchelor Wyndham W Ziebarth Noël N Silvera Risset R Khan Aisha A Maciel Renata R Saporta Mario M Agarwal Ashutosh A

Biomaterials science 20200101 2

We report a water-soluble and non-toxic method to incorporate additional extracellular matrix proteins into gelatin hydrogels, while obviating the use of chemical crosslinkers such as glutaraldehyde. Gelatin hydrogels were fabricated using a range of gelatin concentrations (4%-10%) that corresponded to elastic moduli of approximately 1 kPa-25 kPa, respectively, a substrate stiffness relevant for multiple cell types. Microbial transglutaminase was then used to enzymatically crosslink a layer of l ...[more]

PMID: 31859298

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Project description:BackgroundTissue engineering, which involves the selection of scaffold materials, presents a new therapeutic strategy for damaged tissues or organs. Scaffold design based on blends of proteins and polysaccharides, as mimicry of the native extracellular matrix, has recently become a valuable strategy for tissue engineering.ObjectiveThis study aimed to construct composite hydrogels based on natural polymers for tissue engineering.MethodsComposite hydrogels based on blends of gelatin with a polysaccharide component (chitosan or alginate) were produced and subsequently enzyme crosslinked. The other three hydrogels, chitosan hydrogel, sodium alginate hydrogel, and microbial transglutaminase-crosslinked gelatin (mTG/GA) hydrogel were also prepared. All hydrogels were evaluated for in vitro degradation property, swelling capacity, and mechanical property. Rat adipose-derived stromal stem cells (ADSCs) were isolated and seeded on (or embedded into) the above-mentioned hydrogels. The morphological features of ADSCs were observed and recorded. The effects of the hydrogels on ADSC survival and adhesion were investigated by immunofluorescence staining. Cell proliferation was tested by thiazolyl blue tetrazolium bromide (MTT) assay.ResultsCell viability assay results showed that the five hydrogels are not cytotoxic. The mTG/GA and its composite hydrogels showed higher compressive moduli than the single-component chitosan and alginate hydrogels. MTT assay results showed that ADSCs proliferated better on the composite hydrogels than on the chitosan and alginate hydrogels. Light microscope observation and cell cytoskeleton staining showed that hydrogel strength had obvious effects on cell growth and adhesion. The ADSCs seeded on chitosan and alginate hydrogels plunged into the hydrogels and could not stretch out due to the low strength of the hydrogel, whereas cells seeded on composite hydrogels with higher elastic modulus, could spread out, and grew in size.ConclusionThe gelatin-polysaccharide composite hydrogels could serve as attractive biomaterials for tissue engineering due to their easy preparation and favorable biophysical properties.

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Enzymatically crosslinked gelatin-laminin hydrogels for applications in neuromuscular tissue engineering.

Publications

Enzymatically crosslinked gelatin-laminin hydrogels for applications in neuromuscular tissue engineering.

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