Shape memory injectable cryogel based on carboxymethyl chitosan/gelatin for minimally invasive tissue engineering: In vitro and in vivo assays

Abstract

Shape‐memory cryogels have drawn attention as an injectable system to minimize the risks associated with surgical implantation in tissue engineering. To achieve shape memory behavior with hydration as an external stimulus, it is necessary to have a porous elastic network. To achieve this, it is crucial to control the crosslinking process at the time of pore formation, especially for natural‐based polymers. In this study, a versatile method using a cryogelation method in the presence of chemical and physical crosslinkers is investigated to obtain an injectable super macroporous elastic structure based on a poly(ampholyte) (carboxymethyl chitosan) and a protein (gelatin). Mechanical, swelling, shape memorizing behavior, injectability, and in vitro and in vivo behavior of cryogels were studied. Cryogelation in a subzero temperature led to the formation of scaffolds with interconnected pores of the size of 350 μm which swelled completely after 3 min. Cryogels had crosslink density up to 22% and elastic modulus in the hydrated state up to 0.054 and 1.733 MPa at low and high strains, respectively, and low hysteresis (<30 kPa). Injectability studies confirmed the ability of the cryogels to be injected through a 16G needle. In vitro studies demonstrated good cellular penetration, cell adhesion, and high cell viability (>100%). In vivo studies using mice showed that the body's response was befitting without inflammation and any side effect for the liver and kidneys.

Biophysical Characterization and Cytocompatibility of Cellulose Cryogels Reinforced with Chitin Nanowhiskers

“…A set of unique properties-including high water retention, porosity, high pore connectivity, and consistency-make cryogels very similar to natural soft tissues [5,6]. The mechanical stability of cryogels enables their use in in vivo processes [7][8][9].…”

Cellulose nanocrystals‐reinforced dual crosslinked double network GelMA/hyaluronic acid injectable nanocomposite cryogels with improved mechanical properties for cartilage tissue regeneration

“…[45][46][47] Additionally, the preformed injectable cryogels require a highly porous structure to be able to collapse entirely and pass through a syringe (shear-thinning), and then regain their initial shape via water absorption. 48 It was observed that by increasing the HA content in the structure, pore size and porosity were increased (Figure 2A-F). The hydrophilic nature of HA contributes to the formation of larger ice crystals during the freezing process, leading to greater pore sizes after lyophilization.…”

“…These characteristics can significantly affect the physical properties of the scaffolds, cell infiltration, and nutrient‐waste exchange, which are crucial factors for successful cartilage tissue engineering 45–47 . Additionally, the preformed injectable cryogels require a highly porous structure to be able to collapse entirely and pass through a syringe (shear‐thinning), and then regain their initial shape via water absorption 48 . It was observed that by increasing the HA content in the structure, pore size and porosity were increased (Figure 2A–F).…”

Cryogel Scaffolds for Tissue-Engineering: Advances and Challenges for Effective Bone and Cartilage Regeneration

“…Another frequently used coupling reaction is known as carbodiimide coupling, reported in scheme 4 of Figure 5, based on the use of (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) along with N-hydroxysuccinimide (NHS) [33,[47][48][49][50]. These reagents are used to couple carboxylic groups of one polymer with primary amino groups or Other cross-linking protocols, largely used for the fabrication of cryogel scaffolds suitable for bone or cartilage regeneration, are based on coupling reactions with bifunctional agents, such as glutaraldehyde (GA) and divinyl sulfone (DVS) [15,44,45], reported, respectively, in scheme 2 and 3 of Figure 5.…”

“…Another frequently used coupling reaction is known as carbodiimide coupling, reported in scheme 4 of Figure 5, based on the use of (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) along with N-hydroxysuccinimide (NHS) [33,[47][48][49][50]. These reagents are used to couple carboxylic groups of one polymer with primary amino groups or hydroxyl groups of the same polymer or a different one.…”