Hydrogel composed of type II collagen,chondroitin sulfate and hyaluronic acid for cartilage tissue engineering

Abstract

BACKGROUND:

Cartilage tissue engineering is a promising way to repair cartilage defects. Different materials have been applied in the preparation of cartilage hydrogels, but all with various disadvantages.

OBJECTIVE:

The aim of this study was to prepare cartilage hydrogel using type II collagen, chondroitin sulfate and hyaluronic acid, to explore their gelation effect and compressive strength, and to analyze the feasibility of their application in cartilage tissue engineering.

METHODS:

Type II collagen (Col II), hyaluronic acid (HA) and chondroitin sulfate (CS) were mixed in a certain proportion to prepare gel scaffolds; changes in chemical groups were detected by Fourier transform infrared. After the hydrogel was prepared, its compressive strength was measured. Umbilical cord stem cells were co-cultured with hydrogel scaffolds to observe its cytocompatibility and analyze whether stem cells had cellular activity during co-culture; histological staining was applied to observe the hydrogel loaded with stem cells.

RESULTS:

Cartilage hydrogels were successfully prepared with good compressive strength, and Fourier transform infrared analysis showed that Schiff base reaction occurred during the preparation process and tight chemical cross-linking was formed. The results of umbilical cord stem cell co-culture showed that the hydrogel had good cytocompatibility and the stem cells had good activity in the hydrogel.

CONCLUSIONS:

Cartilage hydrogels with stable structures were successfully prepared and had good compressive strength. Hydrogel scaffold could provide a suitable living environment for umbilical cord stem cells, so that they maintain normal cell morphology and activity, and has a good application potential in cartilage tissue engineering.

Keywords

Cartilage hydrogel tissue engineering scaffold

1. Introduction

Articular cartilage is composed of chondrocytes and extracellular matrix, which buffers joint pressure and reduces friction. Articular cartilage lacks innervation of nerves and has no distribution of blood vessels and lymphatic vessels. This unique physiological tissue structure allows the material exchange of articular cartilage to only be performed through the synovial fluid in the joint cavity, resulting in that the ability of cartilage to regenerate and repair is very low and difficult to heal spontaneously [1]. Relevant studies have shown that the cartilage regeneration capacity decreases significantly with age. When the range of articular cartilage defect is greater than 4 mm³, it cannot be repaired spontaneously [2]. Patients often have discomfort symptoms such as joint pain, stiffness, etc., and finally develop into arthritis, seriously affecting the quality of life of patients. Aiming at the problem of poor regeneration and repair ability of cartilage, a variety of therapeutic strategies have been proposed.

In the late 1980s, Steadman et al. proposed the technique of microfracture to repair the articular cartilage [3]. This technique surgically removes the damaged articular cartilage and exposes the subchondral bone, creates several micropores on the bone surface, allows the bone marrow stem cells in the medullary cavity to migrate and differentiate out of the medullary cavity, and forms blood clots at the cartilage defect to repair the injured cartilage. Microfracture is easy to operate and less invasive clinically, and has achieved good results in cartilage repair in a short period of time [4]. However, relevant studies have shown that the use of microfracture technology disturbs the normal joint microenvironment, destroys the structure of the calcified layer, and most of the tissues formed in cartilage defects are fibrocartilage tissue and fibrous tissue, while the articular cartilage tissue components of normal adults are mainly hyaline cartilage. Compared with hyaline cartilage, fibrocartilage tissue and fibrous tissue have less type II collagen and mucopolysaccharide content, which is worse in biomechanical properties, and cannot restore the normal function of articular cartilage. Fibrocartilage is easy to degenerate with time. Therefore, the long-term effect of microfracture technique is not reliable [5]. Goyal et al. followed the postoperative repair effect of patients treated with microfracture and found that regardless of the area of articular cartilage defects, there was a high failure rate 5 years after surgery [6]. Tissue engineering technology can promote the regeneration of original cartilage at the defect site, which brings hope for the treatment of cartilage defects.

At present, the constructed tissue-engineered cartilage is quite different from natural cartilage in tissue structure, and the extracellular matrix components of natural cartilage are mainly type II collagen (Col II), hyaluronic acid (HA) and chondroitin sulfate (CS). However, the scaffold materials such as polylactic acid (PLA) and polyglycolic acid (PGA) used in previous studies are quite different from the natural cartilage matrix components, which are prone to immune rejection after implantation in vivo, or cause a local acidic microenvironment in the tissue during degradation, and is not conducive to the proliferation and differentiation of seed cells. The scaffold components used in matrix-induced autologous chondrocyte implantation (MACI) technique are collagen I and III rather than type II collagen, so the repair tissue is mostly fibrocartilage rather than hyaline cartilage.

Therefore, how to prepare tissue-engineered cartilage materials similar to natural cartilage matrix is an important issue that urgently needs to be solved at present. In the present study, we employed collagen II, hyaluronic acid, chondroitin sulfate as raw materials to fabricate tissue-engineered cartilage hydrogels that mimic the natural cartilage matrix synthesis mechanism. However, hyaluronic acid and chondroitin sulfate do not form a strong link with type II collagen under normal conditions, so sodium periodate was used to oxidize the o-dihydroxyl groups on the surfaces of HA and CS to aldehyde groups. Therefore, they could react with the amine groups on the surface of Col II by Schiff base reaction for strong link. And the intermediates were water, which did not affect the local micro-environment. Subsequently, the colloidal properties of biomimetic cartilage hydrogel were explored, and it was co-cultured with human umbilical cord stem cells in vitro to explore its potential for application as a scaffold material in tissue engineering.

2. Materials and methods

2.1. Materials

Chondroitin sulfate, hyaluronic acid, pepsin and sodium chloride were purchased from Shanghai Shenggong (China); glacial acetic acid, concentrated hydrochloric acid and sodium hydroxide solid were purchased from Chongqing Chuandong Chemical (China); mesenchymal stem cell (MSC) special medium, Helios UltraGRO serum additive, penicillin -streptomycin antibody were purchased from HyClone (USA), 0.25% Trypsin-EDTA and collagen II were obtained from Thermo Fisher (USA).

2.2. Preparation of OCS and OHA

CS and HA powder of 4 g were separately added into 200 mL deionized water to make a solution, and then sodium periodate of 4 g (0.5 mol L⁻¹) was added into the solution as an oxidizer. This mixture was stirred overnight in the dark, and ethylene glycol was introduced for terminating oxidation reaction. The mixture was disinfected by ultraviolet irradiation and poured into sterile containers; the conductivity of mixture was adjusted to be equivalent to that of deionized water by dialysis. Next, the mixture was allowed to stand in a freezer at −20 °C for obtaining white solid. Finally, the white solid was re-crystallized from water, dried by hyophilization for oxidized chondroitin sulfate (OCS) and oxidized hyaluronic acid (OHA) (Fig. 1).

2.3. Preparation of hydrogel scaffold

Deionized water was added to OCS/OHA mixed powder with a ratio of 3 to 1, and vigorously stirred until the solute was completely dissolved to prepare a transparent solution (solution A). The pH of 500 mg Col II (10%) was neutralized by adding 1.5 mol L⁻¹ sodium hydroxide solution to prepare solution B. Subsequently, solution A was mixed with solution B, and immediate stirring was applied to make the aldehyde group of solution A reacted with the amine group of solution B (the Schiff base reaction). The semisolid mixture was transferred into a mold and solidified on standing to form hydrogel scaffold.

Fig. 1.

Oxidation process triggered by sodium periodate in chondroitin sulfate (A) and hyaluronic acid (B).

2.4. Fourier transform infrared analysis

FTIR spectroscopy was used to determine the chemical structures of OCA/OHA and hydrogel scaffold. The FTIR spectrum was recorded between 400 and 4000 cm⁻¹ by KBr FTIR spectrophotometer (Shimadzu, Japan).

2.5. Compression test

Depending on different compositions, three hydrogel scaffolds could be obtained (Col-OCS-HA, Col-CS-OHA, Col-OCS-OHA). The compressive strength measurements of these scaffolds were performed on mechanical test machine. All samples were compressed to 50% of their initial height and the compression test of each sample was then calculated on this compressive strain ratio.

2.6. Co-culture of hUMSCs with hydrogel scaffold

Human umbilical cord mesenchymal stem cells (hUMSCs) were carefully extracted from human umbilical cord jelly through a series of operations including cleaning, disinfection and seeding as previous study. Then, the cells were incubated in the special medium for hUMSCs at 37 °C in a humidified atmosphere with 5% CO₂. For following experiments, the cells were used in passage 5. Afterward, hUMSCs were added to the OCS-OHA solution, which were then mixed for 5 mins to be homogenized. The cell-gel mixture was pipetted in the cell culture plate; 5% complete medium (containing 1% antibody) was added to prevent infection, and the culture medium was changed every 3 days. At 14, 21, and 28 days of culture, the scaffold loaded with hUMSCs was fixed in 10% formalin, and paraffin sections were performed for histological analysis such as HE staining, Masson’s trichrome staining and Alcian blue staining.

2.7. Statistical analysis

Statistical analyses were performed using SPSS (version 22.0, IBM Corp., Armonk, NY, USA). All data were presented as mean ± standard deviation. Analysis of the significance of data was done either by one-way ANOVA analysis or by t-test. P < 0.05 was regarded as an indicator of statistical significance.

3. Results

3.1. Hydrogel fabrication and characterization

As shown in Fig. 2, the biomimetic cartilage gel prepared from Col II, OCS and OHA was milky white with smooth surface and without cracks or bulges. FTIR results showed peak characteristic of OCS in 1705.8 cm⁻¹, and that of OHA in 1704.8 cm⁻¹. This phenomenon may be corresponded to C=O stretching, which indicated the presence of aldehyde group. After combining OCS and OHA with Col II, characteristic peak of hydrogel was found in 1646.01 cm⁻¹, meaning the formation of C=N groups in Schiff base reaction (Fig. 3).

Fig. 2.

Frontal view (A) and side view (B) of cartilage hydrogel fabricated by COLII, OCS and OHA.

Fig. 3.

FTIR absorbance spectra showing the successful oxidation in chondroitin sulfate (A) and hyaluronic acid (B), showing the new group formed in Schiff base (C).

3.2. Compressive measurement

The compressive strength of COLII-OCS-HA was 1.92 ± 0.11 MPa, COLII-CS-OHA compressive strength was 2.84 ± 0.20 MPa, and the compressive strength of COLII-OCS-OHA was 5.26 ± 0.13 MPa. Compared with the other two groups, COLII-OCS-OHA had the best compressive performance (P < 0.05), indicating it was the most suitable for hydrogel (Fig. 4).

Fig. 4.

Compressive test of different scaffolds, the compressive strength of COLII-OCS-OHA was significantly higher than the other two.

3.3. Morphology of hUMSCs

The morphology of cells was good, and the cells could complete attachment overnight. At 1–2 days of cell culture, the cells were polygonal, short spindle and irregular, and disorganized. At 3–4 days of culture, the cells could be arranged in a ring; at 5–7 days of culture, the cells were mostly long shuttle, arranged in clusters, and the degree of fusion was high (Fig. 5).

Fig. 5.

The morphology of cells incubated in hydrogel at 3 days (A) and 7 days (B).

3.4. Histological assessment

The cells were incubated in the hydrogel, and the sections were stained as shown in Fig. 6. HE staining and Alcian blue staining showed that the hUMSCs were evenly distributed in the hydrogel. Masson’s trichrome staining showed that the type II collagen in the hydrogel had a reticular structure, and OCS and OHA were distributed in its pores, i.e., the extracellular matrix contained a large number of glycosaminoglycans. With the longer culture time, a large number of cells were still present in the hydrogel on the 28th day.

Fig. 6.

Histological analysis of the samples showed the cells survived in the hydrogel with more type II collagen and glycosaminoglycans outside the cells.

4. Discussion

In this study, Col II, OCS and OHA were selected as raw materials to prepare the hydrogel, so that the amino group in Col II could react with the aldehyde group in OCS and OHA by Schiff base. Natural materials have good biocompatibility and cytocompatibility, but the scaffold strength is weak. How to enhancing the scaffold strength is a major problem in the application of natural materials [7]. Previous crosslinking methods include chemical crosslinking, physical crosslinking and enzyme crosslinking. Compared with these crosslinking methods, the Schiff base reaction applied in this study has less stricter requirements for the external environment, and is not affected by temperature, enzyme and beam. At the same time, no toxic and harmful substance was produced. The hydrogel is used to treat cartilage defects, especially irregular cartilage defects, which can be completely filled according to the shape and size of cartilage defects. Feng et al. [8] treated articular cartilage defects in minipigs with Col II-HA-CS combined with bone marrow MSCs; at 1 month after surgery, hyaline cartilage-like tissue formation was observed in the cartilage defect area, and a small amount of chondrocyte pits appeared, indicating that this treatment could effectively repair cartilage defects. In previous studies, the use of Col II and OHA to make a hydrogel combined with bone marrow concentrate in the treatment of cartilage defects in minipigs has also achieved an ideal repair effect. These similar studies suggest that hydrogels containing Col II, CS and HA have the potential to repair cartilage defects and can promote cartilage regeneration and repair.

Stem cells are commonly used as seed cells in cartilage tissue engineering. In previous studies, bone marrow stem cells and adipose-derived stem cells were used as seed cells for cartilage defect repair [9,10]. Compared with bone marrow MSC and adipose MSC, hUMSCs have stronger proliferation ability and lower immunogenicity [11,12]. The results of Fiori et al. [13] showed that hUMSCs have more chondrogenic differentiation ability and secrete more Col II compared with bone marrow MSC, which is conducive to cartilage repair. The hUMSCs could secrete a variety of cytokines, such as chemokines, growth factors, colony-stimulating factors, tumor necrosis factor and interferon, which can promote cell proliferation, differentiation, migration and proliferation [14,15]. Rong et al. [16] used rats to establish arthritis model, and their results showed that hUMSCs had a good short-term effect on knee arthritis. Zhang et al. [17] used hUMSCs combined with acellular cartilage extracellular matrix for the repair of articular cartilage defects in goats, and their results showed that the repair effect was significantly better than that of microfracture. Sadlik et al. [18] used hUCMSCs for the treatment of cartilage defects, and at 12 months after surgery, magnetic resonance results suggested that the cartilage defects were effectively repaired. Other studies using hUMSCs to treat cartilage defects have also shown good results [19,20]. Therefore, hUMSCs are good seed cells for the treatment of cartilage defects and have excellent application potential.

5. Conclusions

In this study, cartilage hydrogels with stable structures were successfully prepared using Col II, HA and CS, and had good compressive strength. Hydrogel scaffold could provide a suitable living environment for umbilical cord stem cells, so that they maintain normal cell morphology and activity, and has a good application potential in cartilage tissue engineering, and the cartilage repair ability of this hydrogel scaffold can be further studied by animal experiments.

Footnotes

Acknowledgement

The authors would like to thank Xin Chen from the Center for Joint Surgery, Southwest Hospital, Third Military Medical University for the language support for this manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This work was financially supported by National Natural Science Foundation of China (No. 82172429).

Availability of data and materials

The datasets used in this study are available from the corresponding author upon reasonable request.

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