Comparison of chondro-inductivity between collagen and hyaluronic acid hydrogel based on chemical/physical microenvironment

https://doi.org/10.1016/j.ijbiomac.2021.05.188Get rights and content

Abstract

Achieving chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) successfully is crucial for cartilage regeneration. To date, various hydrogels with different chemical microenvironment have been used to modulate chondrogenic differentiation of BMSCs, especially collagen and hyaluronic acid hydrogel. However, the chondro-inductive ability of collagen and hyaluronic acid hydrogel has not been evaluated yet and the different chemical and physical microenvironment of these two hydrogels increase the difficulty of comparison. In this study, three different hydrogels based on collagen and hyaluronic acid (self-assembled collagen hydrogel (Col), self-assembled collagen hydrogel cross-linked with genipin (Cgp), and methacrylated hyaluronic acid hydrogel (HA)) were prepared and their chondro-inductive ability on the encapsulated BMSCs was evaluated. Col and Cgp have the same chemical composition and similar microstructure, but are different from HA, while Cgp and HA hydrogels have the same mechanical strength. It was found that chemical and physical microenvironments of the hydrogels combined to influence cell condensation. Thanks to cell condensation was more likely to occur in collagen hydrogels in the early stage, the cartilage-induced ability was in the order of Col > Cgp > HA. However, the severe shrinkage of Col and Cgp resulted in no enough space for cell proliferation within hydrogels in the later stage. In contrast, relatively stable physical microenvironment of HA helped to maintain continuous production of cartilage-related matrix in the later stage. Overall, these results revealed that the chondro-inductive ability of collagen and hyaluronic acid hydrogel with different chemical and physical microenvironment cannot be evaluated by a particular time period. However, it provided important information for optimization and design of the future hydrogels towards successful repair of articular cartilage.

Introduction

Mesenchymal stem cells (MSCs) with multi-differentiation potential have been proposed as a promising cell source for cartilage tissue engineering to regenerate injured cartilage [[1], [2], [3]]. Under some certain conditions, MSCs are able to differentiate to functional chondrocytes, which is crucial for successful cartilage regeneration [[4], [5], [6], [7]]. Nevertheless, achieving chondrogenic differentiation of MSCs successfully still remains challenge. In recent years, the cell microenvironment has been proved to regulate the behaviors of MSCs and guide their differentiation [8]. Biochemistry and biophysical cues of matrix, as the vital components of cell microenvironment, play a pivotal role in cell adhesion, spreading, migration, proliferation and chondrogenic differentiation of MSCs [9]. Suitable design of biochemical and biophysical cues of matrix is considered as an effective method to induce chondrogenic differentiation of MSCs.

Hydrogels with both high water content and 3D network structure [10], mimicking the partial physical microenvironment of cartilage extracellular matrix, provide a favorable chondro-inductive microenvironment for encapsulating MSCs [2,11]. Therefore, it is critical to choose hydrogel matrix with superior biochemistry and biophysical cues for MSCs chondrogenesis. To date, various hydrogels with different biochemical microenvironments have been used to modulate chondrogenic differentiation of MSCs, especially collagen and hyaluronic acid hydrogel. Collagen is the most abundant structural macromolecule in the cartilage ECM, it makes up more than 60% of the dry weight of cartilage, and it has tremendous adhesion sites to enhance cell–matrix interactions [12,13]. Over the years, many studies have demonstrated that collagen hydrogel is one of the most promising matrix materials for cartilage induction [6,[14], [15], [16], [17], [18]]. Yamoka et al. [16] found that collagen hydrogel could promote rapid proliferation and secretion large amount of type II collagen and proteoglycans of chondrocytes. Mimura et al. [17] implanted the collagen hydrogel/MSCs composite into an articular cartilage defect model of rabbit, MSCs could differentiate into chondrocytes and repair the defect. Previous studies [19,20] of our group also showed that collagen I hydrogel was favorable to chondrogenic differentiation of MSCs without exogenous growth factors. Hyaluronic acid is one of glycosaminoglycans, which is also an important component of the cartilage ECM and synovial fluids. Hyaluronic acid molecules with many carboxyl groups can easily interact with water molecules to form hydrogen bonds, which makes it have the function of fixing and retaining water, providing a relatively stable-moist environment for cartilage [7]. Moreover, hyaluronic acid can provide biochemical cues such as CD44 and CD168 interactions with chondrocytes via various surface receptors [21]. Recently, hyaluronic acid hydrogels have been widely applied in cartilage regeneration [[21], [22], [23]]. Toh et al. [24] showed that hyaluronic acid-tyramine hydrogels could enhance the MSCs chondrogenesis. Feng et al. [25] also demonstrated that sulfated hyaluronic acid hydrogels with enhanced growth factor retention promote MSCs chondrogenesis.

Although collagen and hyaluronic acid hydrogels both provide favorable cell microenvironment for inducing chondrogenic differentiation of MSCs, satisfactory cartilage repair effects have not been achieved yet. Thus, it is urgently needed to further optimized and design hydrogels based on collagen and hyaluronic acid. Clarification the relationship between the biochemical and biophysical cues of the two hydrogels and the chondrogenic differentiation of MSCs will be helpful. Unfortunately, it is difficult to compare the chondro-inductive ability of the two hydrogels to determine which key cues regulate the chondrogenic differentiation of BMSCs, due to the two hydrogels not only have different biochemical cues but also different biophysical cues (shown as Fig. 1). So far, a variety of biophysical cues of matrix such as mechanical strength [24,26], microstructure [27] and electrical property [28], have been proved to impact MSCs chondrogenesis. Toh et al. [24] prepared hydrogels with adjustable mechanical strength, and found that MSCs tend to chondrogenic differentiation within the lower strength hydrogels. Aliabouzar et al. [27] showed that 3D printed scaffold with square pores resulted in higher MSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. Moreover, numerous studies have also shown that negatively charged groups, such as carboxyl, hydroxyl, and sulfonic acid groups, could promote chondrogenic differentiation. Ozturk [29] and Feng [25] reported that sulfated hydrogels could promote chondrocyte proliferation mediated by the FGF signaling pathway, maintain the chondrocyte phenotype, enhance cartilage matrix deposition, and induce chondrogenic differentiation of BMSCs.

In this study, the chondro-inductive ability of collagen and hyaluronic acid hydrogel was compared under suitable design, three different hydrogels were prepared: self-assembled collagen hydrogel (Col), self-assembled collagen hydrogel cross-linked with genipin (Cgp), and methacrylated hyaluronic acid hydrogel (HA). Col and Cgp have the same chemical composition and similar microstructure, but are different from HA, while Cgp and HA hydrogels have the same mechanical strength. Then, the influence of the biochemical and biophysical cues of hydrogels on the protein adsorption, mass transfer, and chondrogenic differentiation of MSCs encapsulated in the hydrogels were investigated, and it is expected to preliminarily analyze the key cues of hydrogels matrix regulated chondrogenesis.

Section snippets

Materials

Type I collagen (Col I) was extracted from new-born calf skin, hyaluronic acid (HA) was obtained from Freda (MW: 200–400 KDa). methacrylic anhydride (MA), I2959 photoinitiator, genipin, fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from Sigma-Aldric. Bovine serum albumin (BSA), protamine (PRTM), FITC-BSA and FITC-PRTM were acquired from Solarbio. TGFβ1 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Elabscience. Mouse anti-rat collagen II primary

Zeta potential

Fig. 2a shows the zeta potential of Col (−8.6 ± 0.6 mV), Cgp (−16.0 ± 1.9 mV) and (HA -36.2 ± 0.5 mV) at pH of 7.4, three groups all presented negative electricity under physiological conditions. HA had much negative electricity at pH 7.4 since a large amount of carboxyl groups in HA molecules. The side chain of the collagen amino acid residues also contains a large number of polar groups (amino and carboxyl groups). The amino group is positively charged in acidic conditions, whereas the

Discussion

In this study, three kinds of hydrogels (i. e. Col, Cgp and HA) are chose to explore the relationship between the chemical and physical microenvironment of hydrogels and the chondrogenic differentiation of BMSCs. The results demonstrated that the cartilage-induced ability in the early stage was in the order of Col > Cgp > HA. Except for chemical cues, physical cues (e. g. microstructure, mechanical property and electrical property) of three hydrogels also significantly regulated chondrogenic

Conclusion

In this study, chemical microenvironment and some physical microenvironment (microstructure, mechanical property, electrical property, mass transfer and protein adsorption) of three hydrogels (i. e. Col, Cgp and HA) are significantly different. Chemical and physical microenvironment of hydrogels combine to influence the cell condensation which could enhance the interaction between cells and further promote chondrogenic differentiation of BMSCs. Due to the chemical microenvironment of collagen

CRediT authorship contribution statement

Jirong Yang: Conceptualization, Methodology, Writing- Original draft preparation. Zizhao Tang: Data curation. Zhaocong Luo: Visualization, Investigation. Yumei Xiao: Supervision. Xingdong Zhang: Project administration.

Declaration of competing interest

The authors indicate no potential conflicts of interest.

Acknowledgements

This study is supported by the National Natural Science Foundation of China (51873116), Shenzhen Fundamental Research Foundation (JCYJ20190812162809131) and Postdoctoral Science Foundation of China (2020M672894).

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