The Use of Genipin as an Effective, Biocompatible, Anti‐Inflammatory Cross‐Linking Method for Nerve Guidance Conduits

Abstract

A number of natural polymer biomaterial‐based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross‐linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross‐linking agents. In this study, collagen‐based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross‐linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross‐linking is developed, by incorporating genipin into solution prior to freeze‐drying the NGCs. This leads to successful cross‐linking as demonstrated by higher cross‐linking degree and similar tensile strength of genipin cross‐linked conduits compared to formaldehyde cross‐linked conduits. Genipin cross‐linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross‐linked conduits, supports the viability of Schwann cells. Moreover, genipin cross‐linked conduits direct macrophages away from a pro‐inflammatory and toward a pro‐repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti‐inflammatory alternative to formaldehyde for cross‐linking clinical grade NGCs.

Biomaterial and Therapeutic Approaches for the Manipulation of Macrophage Phenotype in Peripheral and Central Nerve Repair

“…In this context, the crosslinking procedure applied to the nerve conduit can be critically relevant because it strongly affects its mechanical properties and stiffness and, consequently, the macrophage response [180]. Some of the chemical crosslinking agents commonly used are formaldehyde, hexamethylene diisocyanate, glutaraldehyde (GA), polyepoxy compounds, carbodiimides (EDAC, EDC) and genipin [180,188,189]. The effects induced by the crosslinking procedure on macrophages phenotype are described in a 2019 study by Kočí et al Here, they compared the effects of genipin and formaldehyde on collagen-based nerve guidance conduits.…”

“…Conversely, these were more expressed in the conduits crosslinked with formaldehyde. Therefore, the results suggested that genipin crosslinking can be a valuable method to direct macrophages polarization towards an anti-inflammatory phenotype [188].…”

“…Finally, self-assembling peptides (SAPs) comprise a family of biocompatible, biodegradable and easily modifiable short chain amino acid sequences, [317], capable of forming hydrogels, and have shown considerable promise for CNS injury applications [318]. SAPs have demonstrated good integration into the lesioned spinal cord and promote axonal growth [188,189] and do not induce an immune response with CD68+ macrophage numbers similar to lesioned cord controls as long as 4 weeks after injury [319,320]. Because subtyping into M1 and M2 macrophages was not explored, it is not possible to determine if SAPs may exhibit immunomodulatory functions.…”

The development of natural polymer scaffold-based therapeutics for osteochondral repair

“…Lyophilization of ECM-based biomaterials results in a porous microarchitecture. Our research group has successfully employed this approach to develop scaffolds for many indications including bone [35][36][37][38], cartilage [39][40][41], neural [42][43][44][45], respiratory [46], and cardiovascular [47][48][49][50] repair. Particular success has been achieved using lyophilized collagen scaffolds for osteochondral repair.…”

Genipin‐crosslinked collagen scaffolds inducing chondrogenesis: a mechanical and biological characterization

“…21 Furthermore, GP has showed only a 0.01% of cytotoxicity than glutaraldehyde, and its in vivo biosafety has been extensively demonstrated. 15,22 The advantage of using GP instead of other chemical crosslinkers has been demonstrated in several applications, such as heart valves, 23 pericardial patches, 24 biomaterials for cartilage and nerves regeneration, 25 and decellularized tracheal transplantation. 26 Previously, we have developed biphasic collagen/hydroxyapatite scaffolds for cartilage and bone repair, respectively.…”