Low-Molecular-Weight Heparin-Functionalized Chitosan-Chondroitin Sulfate Hydrogels for Controlled Release of TGF-β3 and in vitro Neocartilage Formation.

You-Rong Chen,Jian Guan,Zhu-Xing Zhou,Fu-Zhen Yuan,Jia-Kuo Yu,Chao Han,Yan-Yu Yang,Bing-Bing Xu,Dong Jiang,Ji-Ying Zhang

doi:10.3389/fchem.2019.00745

Abstract

Repair of hyaline cartilage remains a huge challenge in clinic because of the avascular and aneural characteristics and the paucity of endogenous repair cells. Recently, tissue engineering technique, possessing unique capacity of repairing large tissue defects, avoiding donor complications and two-stage invasive surgical procedures, has been developed a promising therapeutic strategy for cartilage injury. In this study, we incorporated low-molecular-weight heparin (LMWH) into carboxymethyl chitosan-oxidized chondroitin sulfate (CMC-OCS) hydrogel for loading transforming growth factor-β3 (TGF-β3) as matrix of peripheral blood mesenchymal stem cells (PB-MSCs) to construct tissue-engineered cartilage. Meanwhile, three control hydrogels with or without LMWH and/or TGF-β3 were also prepared. The gelling time, microstructures, mechanical properties, degradation rate, cytotoxicity, and the release of TGF-β3 of different hydrogels were investigated. In vitro experiments evaluated the tri-lineage differentiation potential of PB-MSCs, combined with the proliferation, distribution, viability, morphology, and chondrogenic differentiation. Compared with non-LMWH-hydrogels, LMWH-hydrogels (LMWH-CMC-OCS-TGF-β3) have shorter gelling time, higher mechanical strength, slower degradation rate and more stable and lasting release of TGF-β3. After two weeks of culture in vitro, expression of cartilage-specific genes collagen type-2 (COL-2) and aggrecan (AGC), and secretion of glycosaminoglycan (GAG), and COL-2 proteins in LMWH-CMC-OCS-TGF-β3 group were significantly higher than those in other groups. COL-2 immunofluorescence staining showed that the proportion of COL-2 positive cells and immunofluorescence intensity in LMWH-CMC-OCS-TGF-β3 hydrogel were significantly higher than those in other groups. The LMWH-CMC-OCS-TGF-β3 hydrogel can slowly release TGF-β3 in a long term, and meanwhile the hydrogel can provide a biocompatible microenvironment for the growth and chondrogenic differentiation of PB-MSCs. Thus, LMWH functionalized CMC-OCS hydrogels proposed in this work will be beneficial for constructing functional scaffolds for tissue-engineered cartilage.

Highlights

Cartilage injury has brought about an increasing social and economic burden as a common joint disease (Everhart et al, 2019)
low-molecularweight heparin (LMWH)-Oxidized Chondroitin Sulfate (OCS)-CMC hydrogel was obtained by incorporating the activated LMWH into OCS-CMC hydrogel, wherein the NHS groups of LMWH can chemically linked with amino groups of CMC
Threedimensional rendering of the cell-scaffolds composites showed that PB-mesenchymal stem cells (MSCs) were evenly distributed on the surface and inside of the hydrogel (Figure 6B,e–h) with the method of centrifugal seeding which could significantly improve the distribution and proliferation of MSCs on scaffolds (Zhang Z. et al, 2015)

Summary

Introduction

Cartilage injury has brought about an increasing social and economic burden as a common joint disease (Everhart et al, 2019). As for construction of tissue-engineered cartilage, hydrogel can provide a suitable microenvironment for cartilage differentiation, and cartilagespecific extracellular matrix (ECM) regeneration (Wang et al, 2019; Zhang Y. et al, 2019). Heparin is a kind of highly sulfated anionic glycosaminoglycan existing in extracellular matrix, which can bind electropositive protein and growth factors into stable complexes by electrostatic interaction to repair and regenerate various tissues (Sun et al, 2018; Thones et al, 2019). Heparin-functionalized hydrogel scaffold can protect proteins or growth factors from degradation and maintain their biological activity in vivo, the electrostatic interaction can effectively avoid burst release and realize the sustained release of proteins or growth factors (Kim I. et al, 2018; Kim S. et al, 2018). Compared with unfractionated heparin (UFH), low-molecularweight heparin (LMWH) has superiority on the long half-life, less bleeding side effects and no need for laboratory monitoring (Ali-Hassan-Sayegh et al, 2016; Robertson and Jones, 2017)

Methods

Results

Conclusion