Epigallocatechin Gallate-Modified Gelatin Sponges Treated by Vacuum Heating as a Novel Scaffold for Bone Tissue Engineering

Abstract

Chemical modification of gelatin using epigallocatechin gallate (EGCG) promotes bone formation in vivo. However, further improvements are required to increase the mechanical strength and bone-forming ability of fabricated EGCG-modified gelatin sponges (EGCG-GS) for practical applications in regenerative therapy. In the present study, we investigated whether vacuum heating-induced dehydrothermal cross-linking of EGCG-GS enhances bone formation in critical-sized rat calvarial defects. The bone-forming ability of vacuum-heated EGCG-GS (vhEGCG-GS) and other sponges was evaluated by micro-computed tomography and histological staining. The degradation of sponges was assessed using protein assays, and cell morphology and proliferation were verified by scanning electron microscopy and immunostaining using osteoblastic UMR106 cells in vitro. Four weeks after the implantation of sponges, greater bone formation was detected for vhEGCG-GS than for EGCG-GS or vacuum-heated gelatin sponges (dehydrothermal cross-linked sponges without EGCG). In vitro experiments revealed that the relatively low degradability of vhEGCG-GS supports cell attachment, proliferation, and cell–cell communication on the matrix. These findings suggest that vacuum heating enhanced the bone forming ability of EGCG-GS, possibly via the dehydrothermal cross-linking of EGCG-GS, which provides a scaffold for cells, and by maintaining the pharmacological effect of EGCG.

Sustained Release of Catechin from Gelatin and Its Effect on Bone Formation in Critical Sized Defects in Rat Calvaria

“…4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) and N-methylmorpholine (NMM) were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan) and Nacalai Tesque Inc. (Kyoto, Japan), respectively. The AC-vhEGCG-GS was prepared according to previous methods 18) . Briefly, 0.07 mg of EGCG, 100 mg of gelatin, 27.5 μl of NMM, and 69.2 mg DMT-MM were mixed in 5 ml of MilliQ water at 23 o C. The dose of EGCG and gelatin determined in our previous study to result in superior bone formation was used 18,24) .…”

“…Consequently, numerous delivery systems have been developed to provide localized release of these therapeutic agents in biomedical engineering 17) . For EGCG, various techniques have been developed to retain or deliver EGCG in the affected areas; these techniques include chemical bonding to polymers 14,16,18) , layer by layer technique 19) , hydrophobic and/or hydrogen bonding 20,21) , EGCG-coating associated with Na + -mediated physical self-assembly 22) and oxidative polymerization 3) , and en-capsulation in drug carriers 23) . Although our understanding of the application of this molecule in bone regenerative therapy remains limited when compared with that in other medical fields, we previously found that gelatin sponges chemically modified with EGCG using an aqueous chemical synthesis method (hereafter designated as AC-EGCG-GS) induced superior bone formation in critical-sized bone defects (4.2 mm of diameter) in mouse calvaria 14) .…”

“…Although our understanding of the application of this molecule in bone regenerative therapy remains limited when compared with that in other medical fields, we previously found that gelatin sponges chemically modified with EGCG using an aqueous chemical synthesis method (hereafter designated as AC-EGCG-GS) induced superior bone formation in critical-sized bone defects (4.2 mm of diameter) in mouse calvaria 14) . More recently, we showed that the bone-forming ability of vacuum-heated AC-EGCG-GS (AC-vhEGCG-GS) was greater than that of vacuum-heated gelatin sponges (without EGCG) in critical-sized bone defects (9 mm of diameter) of rat calvaria 18) . Alteration of the quantity of EGCG and gelatin in AC-vhEGCG-GSs affected the bone formation results 18,24) .…”

“…More recently, we showed that the bone-forming ability of vacuum-heated AC-EGCG-GS (AC-vhEGCG-GS) was greater than that of vacuum-heated gelatin sponges (without EGCG) in critical-sized bone defects (9 mm of diameter) of rat calvaria 18) . Alteration of the quantity of EGCG and gelatin in AC-vhEGCG-GSs affected the bone formation results 18,24) . Although both AC-vhEGCG-GSs and vacuum-heated gelatin sponges successfully served as scaffolds for cells, the combination of AC-vhEGCG-GS with multipotent progenitor cells (dedifferentiated fat cells or adipose-derived stem cells) formed more bone in the congenital bone defects of rat jaws than the combination of vacuum-heated gelatin sponge with the above cells 25) .…”

Releasing Behavior of Lipopolysaccharide from Gelatin Modulates Inflammation, Cellular Senescence, and Bone Formation in Critical-Sized Bone Defects in Rat Calvaria

“…Gelatin, denatured collagen, has been widely utilized as a substrate for biomaterials and drug carriers [25][26][27]. Various groups, including our own group, have utilized this protein as a controlled release carrier for polyphenol [28], growth factors, and antibiotics [27]. A vacuum-heating technique (dehydrothermal treatment: DHT) has been utilized to promote intermolecular bonding based on esterification between the carboxyl and hydroxyl groups in polymers [29].…”