Growth factor sequestration and enzyme-mediated release from genipin-crosslinked gelatin microspheres

Abstract

Controlled release of growth factors allows the efficient, localized, and temporally-optimized delivery of bioactive molecules to potentiate natural physiological processes. This concept has been applied to treatments for pathological states, including chronic degeneration, wound healing, and tissue regeneration. Peptide microspheres are particularly suited for this application because of their low cost, ease of manufacture, and interaction with natural remodeling processes active during healing. The present study characterizes gelatin microspheres for the entrapment and delivery of growth factors, with a focus on tailored protein affinity, loading capacity, and degradation-mediated release. Genipin crosslinking in PBS and CHES buffers produced average microsphere sizes ranging from 15 to 30 microns with population distributions ranging from about 15 to 60 microns. Microsphere formulations were chosen based on properties important for controlled transient and spatial delivery, including size, consistency, and stability. The microsphere charge affinity was found to be dependent on gelatin type, with type A (GelA) carriers consistently having a lower negative charge than equivalent type B (GelB) carriers. A higher degree of crosslinking, representative of primary amine consumption, resulted in a greater negative net charge. Gelatin type was found to be the strongest determinant of degradation, with GelA carriers degrading at higher rates versus similarly crosslinked GelB carriers. Growth factor release was shown to depend upon microsphere degradation by proteolytic enzymes, while microspheres in inert buffers showed long-term retention of growth factors. These studies illuminate fabrication and processing parameters that can be used to control spatial and temporal release of growth factors from gelatin-based microspheres.

Multifunctional laminarin microparticles for cell adhesion and expansion

“…PL are rich in several chemokines and GFs such as platelet derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2, -4 and -6 (BMP-2, -4, -6). Several works have suggested that PL are a valuable, non-xenogenic alternative to animal derived serum in cell culture(Bieback, 2013) (Turner, Thiele, & Stegemann, 2017) (Burnouf, Strunk, Koh, & Schallmoser, 2016) or combined with biomaterials (Santos, Sigurjonsson, Custodio, & Mano, 2018) (Oliveira, Santo, Gomes, Reis, & Mano, 2015). Physiologically, platelets are known to deliver a broad spectrum of GFs and have a main role in wound healing.…”

Angiogenesis in Tissue Engineering: As Nature Intended?

“…The release of molecules from the scaffold can be controlled by modifying the properties and composition of the material, TABLE 1 | List of common bioactive molecules used in tissue engineering vascularization and mentioned in this review. Lee et al, 2001;Gerhardt et al, 2003;Chiu and Radisic, 2010;Chow et al, 2010;Yuen et al, 2010;Shah et al, 2011;Zheng et al, 2012;Brudno et al, 2013;Nguyen et al, 2013;Alsop et al, 2014;Lai et al, 2014;Rich et al, 2014;Stamati et al, 2014;Wu et al, 2016;LaValley et al, 2017;Turner et al, 2017;Kuttappan et al, 2018;Stejskalová et al, 2019;Wang et al, 2019FGF Chow et al, 2010Tengood et al, 2011;Lai et al, 2014;Stamati et al, 2014;Kuttappan et al, 2018;Dong et al, 2019IGF Holland et al, 2005EGF Lai et al, 2014PDGF Tengood et al, 2011Brudno et al, 2013;Lai et al, 2014;Stejskalová et al, 2019;Wang et al, 2019Glycosaminoglycans Chow et al, 2010Chow et al, 2014;Wu et al, 2016 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org as well as changing the method of retaining the drug (King and Krebsbach, 2012). Boontheekul et a...…”

“…For instance, increasing the cross-linking density of gelatin can improve molecule retention and limit its diffusion (Iwanaga et al, 2003;Patel et al, 2008). Turner et al (2017) crosslinked gelatin microspheres to drive a more regulated release of VEGF or BMP2, dependent on the progressive proteolytic degradation of the scaffold. The non-specific degradation of the scaffold only partially controls the delivery time of GF, making it necessary to develop more advanced systems, which will be discussed later.…”

Injectable hydrogels delivering therapeutic agents for disease treatment and tissue engineering

“…Biomolecular species such as enzymes or metabolites also arbitrate the response of hydrogels. Turner et al (2017) [ 40 ] synthesized gelatin hydrogel microparticles (GHMs) at physiological pHs by the crosslinking reaction occurred via electrostatic interaction between anionic gelatin molecules and cationic vascular endothelial growth factors (VEGFs) or cationic bone morphogenetic protein 2 (BMP2) using genipin crosslinkers. The degradation of GHMs was derived by chromatographically purified collagenase (CLSPA collagenase), and the degradation behavior controlled the release behavior of VEGFs and BMP2s, which induce the expression of cells playing an essential role in osteoblast.…”

“…Since then, a variety of studies of injectable hydrogels to be used in tissue engineering have been actively conducted for producing better repair effect. As like the studies on the injectable hydrogels for drug delivery, recently, multi-functional and hybrid as well as stimuli-responsive injectable hydrogels, are developing for tissue repair and regeneration [ 35 , 40 , 41 , 58 ].…”