Bionic composite hydrogel with a hybrid covalent/noncovalent network promoting phenotypic maintenance of hyaline cartilage

Qing Wang , Xing Li , Peilei Wang , Ya Yao , Yang Xu , Yafang Chen , Yong Sun *, Qing Jiang , Yujiang Fan and Xingdong Zhang
National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China. E-mail:

Received 29th January 2020 , Accepted 11th March 2020

First published on 17th March 2020

The injectable composite hydrogel based on collagen and hyaluronic acid provided a bionic three-dimensional microenvironment and mimetic natural extracellular matrix (ECM) for the growth of cells in vivo and has been widely researched and developed for cartilage tissue engineering. Here, a novel injectable bionic hydrogel with hybrid covalent/noncovalent network derived from covalent conjugation of HA-SH and noncovalent supramolecular self-assembly of BPAA-AFF-OH short peptide was fabricated to overcome the collagen immunogenicity of animal origin and effectively maintain its biological function. Moreover, through optimizing the network structure and polymer composition, the bionic HS5FFAB5 hydrogel presented a reliable mechanical strength which depended on the highly integrated fiber structure between HA-SH and FFAB-AFF-OH molecules. The results in vitro and in vivo proved that HA-SH could provide a fundamental frame structure, while the supramolecular hydrogels could reinforce this structure via hydrogen bonds and hydrophilic/hydrophobic interactions, and endow bionic hydrogels with more abundant cell adhesion sites. The bionic composite hydrogel could improve the cell adhesion and proliferation when compared to HA-SH hydrogel, and enhanced chondrogenic related gene expression and matrix secretion by three-dimensional co-cultured in vitro and subcutaneous implantation in vivo, which further promoted phenotypic maintenance of hyaline cartilage. This bionic hydrogel with a hybrid covalent/noncovalent network is supposed to have potential application prospects in cartilage regeneration.

1. Introduction

The articular cartilage is a specific tissue which is non-vascular, has few chondrocytes, an abundant extracellular matrix and low nutrient supply, and therefore traditional clinical therapy could not effectively maintain long-term therapeutic effects on it.1–5 As essential components of the extracellular matrix (ECM) of cartilage, glycosaminoglycan and collagen (Col) were generally simulated for cartilage tissue engineering scaffolds. With the development of materials science, clinical medicine and biomedical engineering, injectable hybrid hydrogel scaffolds combined with hyaluronic acid (HA, the main component of glycosaminoglycan) and Col as cells carriers to construct bionic extracellular matrices for solving cartilage defects had received extensive attention, which could provide the necessary support for cell growth and regulate cells’ biological behaviour in the least invasive way.6–8 Bian et al. synthesized photo-cross-linkable hybrid hydrogels using methacrylated HA, chondroitin sulfate and type I Col, and studied the decoupled function of these in regulating the initial chondrogenesis, subsequent hypertrophy, and tissue mineralization by human mesenchymal stem cells.9 Wang et al. prepared a silk fibroin/collagen/hyaluronic acid (SF/COL/HA) composite scaffold via admixing, cross-linking, and lyophilizing processes and studied its physicochemical and biological properties. The optimal ratio of SF/COL/HA scaffold had a favorable effect on articular cartilage repair.10 Chen and Bian et al. prepared injectable self-crosslinking blended hydrogels by combination of collagen I and thiolated hyaluronic acid (HA-SH) and found that they could alleviate collagen I contraction in vitro and overcome the weak cell adhesive sites of hyaluronic acid.11,12 Meanwhile, the network structure of HA-SH crosslinked by disulfide bonds could avoid the introduction of toxic crosslinking agents. However, there were always some risks for pathogenic contamination and immunogenicity of animal derived collagen.

Supramolecular hydrogels have had a wide impact on tissue engineering,13 drug delivery,14 and bioelectronics fields.15 They are generally bonded via hydrogen bonds, hydrophobic/hydrophilic interactions, π–π stacking, and van der Waals interactions.16–19 They also could generally be reversibly converted between sol–gel and possess the features of injectable hydrogel.20–23 As a potential applicable scaffold in cartilage repair, gelator derived from peptides had been widely investigated owing to its advantages including a simple and efficient synthetic route, similar molecular structure to native ECM, optimal biocompatibility, and non-immunogenic properties.16–18,24 The aromatic-short peptide supramolecules consisting of natural small molecule amino acids with good biocompatibility could be specifically recognized and degraded by enzymes in vivo.25 Studies suggested that supramolecular hydrogels not only had good biocompatibility and biodegradability, but also contained a large quantity of cell adhesion sites.26–28 However, some restrictions were unavoidable, like poor mechanical compliance, lack of elasticity and rapid degradation.29 HA is abundant in human ECM and participates in many cellular activities. As a scaffold material, injectable HA hydrogels possessed many advantages such as biodegradability, minimal invisibility and easily modified functional groups. Meanwhile, HA also plays a key role of lubrication and shock absorption in joints.30 However, HA-based biomaterials inhibited cells attachment due to their hydrophilic and polyanionic properties, and thus their potential application in cartilage tissue engineering was limited. The combination of HA and supramolecular hydrogels would integrate the advantages of both to develop new injectable composite hydrogels with hybrid covalent/noncovalent network. In this case, self-crosslinked HA could provide a fundamental frame structure by chemical bonds and maintain the function of lubrication, while the supramolecular hydrogels could reinforce the structure via interactions like hydrogen bonds, hydrophilic/hydrophobic interactions and endow bionic hydrogels with more abundant cell adhesion sites. At the early stage of cartilage repair, the synergy of HA and peptides could provide a fundamental frame structure and promote cells adhesion and proliferation. After peptides were absorbed, it could provide the space for cell proliferation and secretion of ECM, which might well mimic the extracellular matrix of cartilage and biofunctions without immunogenicity.

Here, this study was aimed at preparing an injectable bionic hydrogel with sufficient fluidity, enough stability, adequate mechanical strength and good biocompatibility. The thiolated hyaluronic acid (HA-SH) was synthesised as previously reported in our lab.31–33 BPAA-AFF-OH short peptide was produced according to the principle of standard solid phase peptide synthesis (SPPS). The bionic composite hydrogel was fabricated to improve the cell adhesion, proliferation and other physiological functions. For exploring the best ratio of two components and detecting the biological property of bionic hydrogel scaffold in vitro and in vivo, five groups of hydrogels were fabricated and co-cultured with chondrocytes in vitro. Meanwhile, the phenotypic maintenance of hyaline cartilage in vivo was also investigated through a subcutaneous pocket model in rabbits.

2. Materials and methods

2.1. Materials

Hyaluronic acid (HA, Cosmetic grade, Mw = 0.3 MDa) was purchased from Bloomage Freda Biopharm Corporation (Shandong, China). 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI, 99%), N-hydroxysuccinimide (NHS, 99%), cysteamine hydrochloride (CSA·HCl, 99%) and dithiothreitol (DTT, 99%) were purchased from Best-reagent Corporation (Chengdu, China). 2-Chlorotrityl chloride resin (100–200 mesh; 1% DVB; 1.01 mmol g−1), Fmoc-L-Phe-OH and Fmoc-L-Ala-OH were purchased from Shanghai GL Biochem. (China). 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB, 99%) was purchased from Aladdin Corporation (Shanghai, China). α-Minimum Essential Medium (α-MEM, Hyclone) and Fetal bovine serum (FBS, Gibco, Australian origin) were bought from Life Technologies Corporation (USA). All the other compounds were obtained from Chengdu Best-reagent Corporation (China) and used without further purification.

2.2. The fabrication of HA-SH/peptide hybrid hydrogels

Firstly, HA (2 g) and NHS (10 mmol, 1150 mg) were added to deionized water and stirred until they formed a transparent and homogeneous solution in 24 h at room temperature. Secondly, EDCI (25 mmol, 4810 mg) in solid form was added into the mixture solution to activate the carboxyl group of hyaluronic acid for 2 h. Thirdly, the CSA·HCl (25 mmol, 2840 mg) was added to the reaction solution and stirred for 24 h and the value of pH was maintained at 4.75–5.0 during the reaction. Finally, the reaction solution was transferred into a dialysis tube with a molecular weight cut-off of 8000–14[thin space (1/6-em)]000 kDa and was dialyzed against acidic deionized water (pH = 3.5) at room temperature for 72 h with frequent changes of water. Eventually, the dialysate was freeze-dried to yield the solid HA-SH. HA-SH’s chemical structure was measured by 1H-NMR (400 MHz, Bruker AMX-400, USA) and FT-IR (NEXUS 670, NICOLET). Meanwhile, the mercapto group content of HA-SH was determined by Ellman method.34,35

The 4-biphenylacetic acid conjugating tripeptide compounds (as BPAA-tripeptides for short) was produced according to standard solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin (100–200 mesh and 1.01 mmol g−1), Fmoc-L-Phe-OH and Fmoc-L-Ala-OH. Firstly, 2-chlorotrityl chloride resin (1.0 g, 1.0 mmol) was taken into the reaction vessel and swelled in dry dichloromethane (DCM) for 10 minutes. Then the solution containing Fmoc-L-Phe-OH (581 mg, 1.5 mmol) and N,N-diisopropylethylamine (DIPEA, 420 μL, 2.4 mmol) in DCM was added and stirred for 1.5 hours. After the reaction completed, the solvent was removed and the resin was washed with DCM and quenched in a blocking solution (DCM/methanol/DIPEA = 7/2/1, v/v) for 10 minutes. The amino acid loaded resin was treated with 20% piperidine in DMF (v/v) for 30 minutes to remove the Fmoc-protecting group. Afterwards, the subsequent Fmoc-L-Phe-OH (581 mg, 1.5 mmol) in DMF was added and coupled to the amino acid loaded resin by applying O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluor-ophosphate (HBTU, 570 mg, 1.5 mmol) and DIPEA (522 μL, 3.0 mmol) as coupling reagents. The coupling of the third Fmoc-L-Ala-Oh and deprotecting of Fmoc group were repeated to elongate the peptide chain as mentioned above. Subsequently, a solution of 4-biphenylacetic acid (BPAA, 320 mg, 1.5 mmol), HBTU (570 mg, 1.5 mmol) and DIPEA (522 μL, 3.0 mmol) in DMF was added to react with the peptide chain. The resin was washed with DMF and DCM, respectively. The desired compound was then cleaved from the resin using TFA/DCM (1[thin space (1/6-em)]:[thin space (1/6-em)]99, v/v) solution. Finally, the product was precipitated in cold diethylether and analysed by 1H-NMR. The detailed spectral data was as follows: δ 8.22 (dd, J = 9.9, 7.8 Hz, 2H), 7.94 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 7.1 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.45 (t, J = 7.6 Hz, 3H), 7.39–7.10 (m, 12H), 4.50 (td, J = 9.0, 4.6 Hz, 1H), 4.44 (td, J = 8.1, 5.6 Hz, 1H), 4.24 (p, J = 7.1 Hz, 1H), 3.46 (q, J = 14.1 Hz, 2H), 2.99 (dtd, J = 22.4, 13.9, 7.0 Hz, 4H), 2.75 (dd, J = 13.9, 9.3 Hz, 2H), 1.11 (d, J = 7.0 Hz, 3H). ESI-MS m/z 576.56 [M − H]. Calcd for C34H33N3O5, 577.28. Yield: 85%.

HA-SH/peptide hybrid hydrogel was prepared as follows. Firstly, the HA-SH (15 mg) was weighed, sterilized and dissolved in 1 mL deionized water thoroughly. Similarly, 15 mg pre-lyophilized BPAA-AFF-OH was also dissolved in 1 mL deionized water and the pH adjusted to 9. Then, five hybrid hydrogels were prepared at different volume ratios: (1) BPAA-AFF-OH (FFAB), (2) BPAA-AFF-OH[thin space (1/6-em)]:[thin space (1/6-em)]HA-SH = 7[thin space (1/6-em)]:[thin space (1/6-em)]3 (HS3FFAB7), (3) BPAA-AFF-OH[thin space (1/6-em)]:[thin space (1/6-em)]HA-SH = 5[thin space (1/6-em)]:[thin space (1/6-em)]5 (HS5FFAB5), (4) BPAA-AFF-OH[thin space (1/6-em)]:[thin space (1/6-em)]HA-SH = 3[thin space (1/6-em)]:[thin space (1/6-em)]7 (HS7FFAB3), (5) HA-SH (HS). Finally, the hybrid hydrogels were adjusted to pH = 7.4 with 1 M NaOH and immediately injected into a homemade ring mould (diameter 8.5 mm, height 3.0 mm) and incubated at 37 °C until gel formation occurred.

2.3. The characterization of HA-SH/peptide hybrid hydrogels

The injection force was measured by an electro-mechanical universal testing machine (Shimadzu Autograph AGS-X, Japan) for five replicates each sample. The hybrid hydrogels were frozen and immediately lyophilized, and the morphology and interior structure were observed by scanning electron microscopy (SEM, HITACHI S-800, Japan). Transmission electron microscopy was performed using a Tecnai G2 F20 S-TWIN (FEI, USA) at an accelerating voltage of 200 kV on a carbon grid to confirm the size of the nanofibers and interactions between HA-SH and peptides. The porosity and fibre width were also calculated using Image J software. According to section 2.2, these hydrogels (diameter 8.5 mm, height 3.0 mm) were freeze-dried and weighed (Wd). And then, the freeze-dried hydrogels were immersed into 10 mL deionized water and placed in a constant temperature shaker (ZHWY-2012C, Shanghai Zhicheng, China) to shake at 90 rpm at 37 °C. At last, the hydrogels were again weighed (Ws) at certain time intervals until swollen equilibrium was reached. Every sample was measured three times. The swelling rate of the hydrogel was calculated by the following formula: swelling ratio = (WsWd)/Wd × 100%.

The mechanical properties of the hydrogels were measured by rheology testing and dynamic mechanical analysis. The Rheology measurement was performed to analyse the viscoelastic characteristics of the hydrogels at 37 °C on a TA Discovery DHR-2 rheometer with a parallel-plate geometry (40 mm) and a 1.0 mm gap. The dynamic strain was set from 0.01% to 500% with 50 points recorded. The storage modulus (G′) and loss modulus (G′′) of the hydrogels were determined by Dynamic Mechanical Analyzer (DMA, TA-Q800, USA) instrument in the multi frequency mode with a fixed frequency of 1–100 Hz at constant room temperature. The testing parameters were set with an amplitude of 40 μm, preload force of 0.002 N and force track of 105%. Every sample was tested in triplicate.

According to section 2.2, the prepared disc-shaped (diameter 8.5 mm, height 3.0 mm) hydrogels were freeze-dried and weighed (Wo), and then they were immersed in deionized water containing DTT (1 mM) and placed in a constant temperature shaker at 90 rpm at 37 °C. At certain time intervals, the hydrogels were taken out and washed in distilled water, freeze-dried and weighed again (Wr). Every sample was measured in triplicate. The disintegration behaviour of the hydrogels was expressed as percentage of weight loss and was calculated as follows: weight loss percentage = (WoWr)/Wo × 100%.

2.4. Influence of HA-SH/BPAA-AFF-OH hybrid hydrogels on chondrocyte growth

2.4.1. Chondrocytes’ isolation and culture. Articular cartilage specimens were extracted from the knees of new-born rabbits. The cartilage pieces were carefully cleaned with PBS. Thereafter, 0.25% trypsin without EDTA was applied for 30 min and then 2.5 mg mL−1 type II collagenase was employed to digest for 4 h. Finally, the isolated chondrocytes were counted and plated on Petri dishes (100 mm × 10 mm) and cultured in α-MEM (Hyclone) medium containing: 20% FBS (Gibco), 1% penicillin–streptomycin (hyclone) and 50 mg mL−1 2-phospho-L-ascorbic acid trisodium salt (phosphate-Vc, Sigma-Aldrich). After the first passage, 10% FBS was used for the two-dimensional culture of chondrocytes. The medium was replaced every 3 days, all the Petri dishes were put in a 5% CO2 container at 37 °C.
2.4.2. Fabrication of 3D chondrocytes-laden hybrid hydrogels. The BPAA-AFF-OH[thin space (1/6-em)]:[thin space (1/6-em)]HA-SH = 5[thin space (1/6-em)]:[thin space (1/6-em)]5 (HS5FFAB5) group was chosen to culture with chondrocytes in vitro due to its optimal physical properties. The 100 μL chondrocyte suspension (5 × 106 cells per mL) was added into HS5FFAB5 solution and constantly stirred with a sterile long spoon to achieve homogeneous cell distribution. Afterwards, the chondrocytes-laden solution was injected into homemade annular molds (diameter 4.7 mm, height 2.0 mm) and incubated at 37 °C. Finally, the hydrogels were separated from the mold and immersed in α-MEM medium supplemented with 10% serum, 1% penicillin–streptomycin and 0.2% Vc. The medium was replaced every two days. The wet weight and size change of hydrogels with culture time were recorded.
2.4.3. Proliferation and morphology of chondrocytes in hydrogels. After being cultured for 1, 3, 7, 14 and 21 days in vitro, the chondrocytes-HS5FFAB5 was taken out, washed twice with PBS and immersed in PBS solution containing 1 μg mL−1 of fluorescein diacetate (FDA) and 1 μg mL−1 of propidium iodide (PI). The viability and distribution of the chondrocytes in the hydrogels were observed by CLSM. The proliferation of chondrocytes was also evaluated by CCK-8 (Dojindo, Japan). Briefly, after 1, 3, 7 days, 150 μL of media containing 10% CCK-8 was added into each well for 4 h and the absorbance value was determined by using a microplate reader (Multiskan FC; Thermo) at 450 nm. The samples were subsequently dehydrated against a graded series of alcohol and dried by critical point drying for one hour to observe the surface structure by SEM. Meanwhile, the staining was performed with rhodamine–phalloidin solution (5 μg mL−1; Sigma-Aldrich) for 6 h and with DAPI (10 μg mL−1; Sigma-Aldrich) for 2 min at 25 °C to get the cytoskeleton of cells in the hydrogels by CLSM.

The quantitative determination of GAG, DNA and RNA was implemented as follows. For quantitative DNA analysis, 1 mL of 0.0125% papain in 200 mM phosphate buffer (pH = 6.5) containing 5 mM L-cysteine and 5 mM EDTA was added into 2 mL EP tube to completely digest the hydrogel, and the solution was centrifuged (12[thin space (1/6-em)]000 rpm, 4 °C) to remove debris. The DNA of the chondrocytes in the hydrogels was quantitatively analysed using Quant-iTTM PicoGreenTM dsDAN assay kit (Invitrogen) according to the manufacturer's instructions. The GAG was quantified using the above supernatant by Blyscan Glycosaminoglycan Assay (Biocolor). To analyse RNA expression, the chondrocyte loaded hydrogels were lysed with RLT lysis buffer in an RNeasy Mini kit (Qiagen) and converted to cDNA using iScriptTM cDNA synthesis Kit (Bio-rad, USA). Q-PCR analysis was carried out using SYBR Green (Roche, USA) on an C1000TM Thermal cycler machine (Bio-rad, USA). GAPDH mRNA was used as an internal control for PCR amplification. The primers (5′–3′) used in this study are listed in Table S1 (ESI). The gel pieces were embedded in optimal cutting temperature compound (OCT, Tissue-Tek) and then were sliced into a thickness of 5–10 μm. These samples were stained by Haematoxylin–Eosin (H&E), Toluidine blue (TB) and Safranin-O (SO). COL I, COL II and COL X were detected with mouse anti-rabbit primary antibody (Novus Biologicals) and the semi-quantitative results of IHC staining were calculated using Image J software.

2.5. Chondrogenic expression in vivo

All animal studies were approved by the Sichuan University Medical Ethics Committee. All animal procedures were performed in accordance with the guidelines for care and use of Laboratory Animals of Sichuan University. For in vivo study, the chondrocytes were capsulated into hydrogels (8.5 mm diameter, 3 mm height) to reach a final concentration of 5 × 106 cells per mL. The chondrocytes-laden hybrid hydrogels were implanted into the back of New Zealand white rabbits (4–6 weeks, n = 6) for assessing chondrocyte expression in vivo. The rabbits were sacrificed at 1 week and 4 weeks post-surgery and the samples were taken out and fixed overnight in 4% paraformaldehyde (Solarbio, China). The hydrogels’ degradation in vivo was evaluated by weighing the hydrogels. The tissue section staining including H&E, TB, SO, COL I, COL II and COL X was executed and quantitated using Image J software. The relative gene expression of chondrocytes was determined according to Section 2.4.3.

2.6. Statistical analysis

Results were presented as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc testing using SPSS 22.0 software and the significance level was set at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

3. Results and discussion

3.1. The synthetic and mechanical properties of HS/FFAB hydrogels

The injectable HS/FFAB hybrid hydrogel was prepared as shown in Fig. 1A. The synthesis route of thiolated hyaluronic acid (HS) has been previously reported.11,12 The substitution degree of the mercapto group was determined as 65.50% by a modified Ellman method.34,35 The 1H-NMR spectra and FT-IR spectra were used to confirm the existence of the conjugated thiol groups. The typical spectra of native and thiolated HA is shown in Fig. S1 (ESI). The new resonant peaks of HS appeared at 2.81 ppm, which represented the methylene protons of –CH2CH2SH in the spectrum of HS polymer. The new bands at 1665 cm−1, 1550 cm−1 correspond to amide I and amide II, which confirmed that thiol groups were conjugated onto HA by chemical bonding (Fig. S2, ESI). The 1H-NMR of FFAB (400 MHz, DMSO, TMS) confirmed the successful synthesis of FFAB (Fig. S3, ESI). The macroscopic pictures and injection images of the hybrid hydrogels with different component proportions are shown in Fig. 1B. The colour of these hydrogels gradually changed from transparent HS to opalescent FFAB groups, and the injection force of hydrogels gradually reduced with increasing the content of FFAB (Fig. 1C), which indicated the precursor solution was easy and convenient to operate for minimally invasive disease treatment. As shown in Fig. 1D and E, the amplitude ranged from 0.1% to 1% with a fixed frequency of 1 Hz belonging to the linear viscoelastic region of the hydrogels. Hence, the amplitude of 0.5% was chosen as the frequency sweep measurement mode in the range of 0.1–100 rad s−1 (Fig. 1F and G). The storage modulus (G′) was larger than the loss modulus (G′′) at strong frequency dependence, indicating the obvious characteristics of gelation. Compared with the other four groups of hydrogels, the mechanical properties of the HS5FFAB5 group hydrogel were the best, which might be attributed to its inner closely cross-linked network structure and the highly integrated fiber structure between HA-SH and FFAB-AFF-OH molecules. The gel time was also obtained by rheological test and tube inversion method (Table S2 and Fig. S4, ESI). These data indicated that the mass ratio of HS and FFAB was positively correlated with gelation time. Teh hybrid gel of HS5FFAB5 presented a shorter gel time, possibly due to the special internal molecular structure as shown in Fig. 2D.
image file: d0tb00253d-f1.tif
Fig. 1 (A) Schematic illustration of the bionic hydrogel with hybrid covalent/noncovalent network and in vivo and in vitro tests with a subcutaneous pocket model and chondrocytes. (B) Different proportions of HS/FFAB hybrid hydrogels. (C) The injection force of HS/FFAB hybrid hydrogels; (Mw = 0.3 MDa, 1.5 wt%, DS = 65.5%). (D–G) Mechanical properties of HS/FFAB hybrid hydrogels in frequency sweep mode and amplitude sweep mode.

3.2. The structure and properties of HS/FFAB hydrogels

The swelling test was performed as shown in Fig. 2A. The short peptide FFAB possessed a weak water absorption capacity due to its week inner shearing force, while the swelling rate of hyaluronic acid was highest, exceeding 25% in 6 h, which might be attribute to the fact that hyaluronic acid possessed more carboxyl and hydroxyl groups to retain enough water. It can be seen that swelling ratio is closely related to the ratio of hyaluronic acid after 6 h. The higher hyaluronic acid content was, the more obvious the swelling was. The 1 mM DTT was chosen to imitate the intracellular and extracellular reductive environment to study the disintegration of HS/FFAB hydrogels in vitro due to abundant disulfide bonds in the HS hydrogel, which could be effectively cleavaged in the reductive condition.36Fig. 2B indicates that all the hydrogels disintegrated smoothly under the action of DTT. The disintegration rate of the short peptide was the highest, more than 90% in 1 h, while the disintegration rate of other groups was also more than 90% except for HS5FFAB5 group hydrogel in 70% in 4 h. The CD spectra also showed that HS/FFAB hybrid hydrogels possess the characteristics both of HS and FFAB, particularly in HS5FFAB5 group hydrogel, which indicated the good combination of HS and FFAB. The abnormal disintegration behavior indicated that the optimal composition ratio (HS5FFAB5) was beneficial to enhance the steady state of the gel structure against a responsive microenvironment. The unique disintegration property could effectively alleviate the fast disintegration shortcomings of short peptide FFAB and could be conducive to long-term maintenance of short peptide biological effects to overcome the lack of active sites of pure HS hydrogels. Hence, when co-cultured with the cells, the HS/FFAB hydrogel could gradually disintegrate to provide sufficient and suitable space for the cell proliferation.
image file: d0tb00253d-f2.tif
Fig. 2 (A) Swelling ratio of HS/FFAB hybrid hydrogels. (B) Degradation behaviour of HS/FFAB hybrid hydrogels. (C) CD spectra of HS/FFAB hybrid hydrogels. (D) Different proportions of HS/FFAB hybrid hydrogels: SEM and TEM pictures. (E) Porosity of different proportions of HS/FFAB hybrid hydrogels. (F) Fibers width of different proportions of HS/FFAB hybrid hydrogels.

In further exploration of the gel structure, the pure HS hydrogel displayed interconnected porous structures. With an increasing the ratio of FFAB in the hybrid hydrogels, the micro morphology gradually changed into an irregular overlapped lamellar structure with some filaments distributed on its surface, and the indirectly measured porosity decreased with the increase of FFAB (Fig. 2E). Finer microscopic structures presented clusters of fiber evenly distributed with hyaluronic acid microspheres as shown by TEM in Fig. 2D, and the fiber width gradually reduced from 22 nm to 6 nm with increasing the proportion of FFAB (Fig. 2F). It was worth noting that the HS5FFAB5 hydrogel exhibited a unique dense porous structure, and further TEM images displayed a clearer and denser fiber network, while there were no hyaluronic acid microspheres surrounding the fiber, implying the overwhelming majority of the hyaluronic acid might be wrapped on the outside of the polypeptide fiber to enhance the interaction between HS and FFAB molecules. This unique structural composition might be the key factor that endowed it the best mechanical properties as shown in Fig. 1D–G. These results proved that gelation driving force in HS came from chemical self-crosslinking between sulfhydryl groups, while physical hydrogen bonds and hydrophilic/hydrophobic interactions promoted the gelation of FFAB molecules. Furthermore, extra enhanced hydrogen bonding might form between residual carboxyl group of HS and FFAB molecules in HS5FFAB5 hybrid hydrogels to strengthen the physical/chemical self-crosslinking density, which was likely to facilitate cell adhesion and proliferation.

3.3. The HS5FFAB5 hydrogel promoted the proliferation of chondrocytes and matrix secretion

In order to further explore the potential of optimal HS5FFAB5 hydrogel as a cell scaffold for cartilage tissue engineering, the third-passages chondrocytes were encapsulated within this hybrid hydrogel to investigate its biological properties after being cultured for 21 days in vitro. The hydrogel size evidently shrank to 70% of the original hydrogel after being cultured for 21 days (Fig. 3A–C), and the wet weight declined immediately after being cultured for 3 days and remained 0.25 times the wet weight of the 1st day after being cultured for 21 days. That was probably because that the short peptide degraded after being cultured for 3 days causing the network structure to disintegrate. In addition, the hybrid hydrogel morphology turned from semi-translucent to transparent and further to milky white, indicating that the short peptide acted as cells adhesion sites in the early stage, and gradually degraded to provide cells growth space with extended culture time while interior cells in the hybrid hydrogels secreted more and more matrix. The CLSM images, cytoskeleton and SEM pictures demonstrated that chondrocytes gradually adhered to and proliferated on the HS5FFAB5 hydrogel, which secreted a great deal of extracellular matrices with active cellular function (Fig. 3D and E). Meanwhile, the CCK-8 assays and GAG/DNA proved that the HS5FFAB5 hydrogel could effectively promote the proliferation of chondrocytes (Fig. 3F–H). Quantitative RT-PCR results indicated the expression of related genes gradually increased over time. The expression of chondrogenic related genes (Col II, Sox9 and AGG) was significantly higher than that of Col I and Col X (Fig. 3I), which meant that the HS5FFAB5 hydrogel could maintain the normal physiological function of chondrocytes, tending to produce hyaline cartilage rather than fibrous cartilage or ossification. These findings demonstrated that the HS5FFAB5 hybrid hydrogel was suitable for cartilage treatment by encapsulating chondrocytes into composite scaffolds.
image file: d0tb00253d-f3.tif
Fig. 3 (A) Macroscopy of HS5FFAB5 hydrogels cultured in vitro. (B and C) Morphological changes of HS5FFAB5 hybrid hydrogels. (D) Live/dead staining of chondrocytes cultured in HS5FFAB5 hybrid hydrogels. (E) Red cytoskeleton in low (A1–E1) and high (A4–E4) power, blue cell nucleus in low (A2–E2) and high (A5–E5) power and merge images in low (A3–E3) and high (A6–E6) power of chondrocytes as well as adhesion morphologies of chondrocytes by SEM in HS5FFAB5 hybrid hydrogel. (F–H) Proliferation of chondrocytes and quantitative analysis of cells DNA and secreted GAG in HS5FFAB5 hydrogel. (I) Q-PCR analyses of cartilage-matrix related genes, including Col I, Col II, Col X, AGG and Sox9. The data are presented as mean ± standard deviations (SD) from 3 independent experiments (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

3.4. Histological section staining analysis of HS5FFAB5 hydrogels cultured with chondrocytes in vitro

The H&E, toluidine blue (TB) and safranin-O (SO) staining as well as associated immunohistochemical staining (Col I, Col II and Col X) were detected as shown in Fig. 4A, and the corresponding pictures in low magnification are shown in Fig. S5 (ESI). Obvious cartilage lacuna formed after 14 days of culture (D4), indicating chondrocytes gradually exhibited normal morphology and proliferated during the process of culture. The TB and SO staining results showed that lots of spherical cells were found inlaid in the GAGs-rich extracellular matrix (red and blue purple). The staining range expanded and the colour gradually deepened with the prolonged culture time (E1–E5 and F1–F5). From immunohistochemical staining of Col II, it could be seen that obvious positive staining (dark brown) appeared after 14 days of culture and the staining gradually deepened in 21 days, indicating the sustained efficient secretion of Col II. In contrast, only a small amount of Col I was found in 21 days, while hardly any Col X secretion was seen over the entire incubation period. Fig. 4B–D showed the semi-quantitative results of three IHC stainings by Image J software. The secretion of Col I and Col II increased notably after 14 days and the value of Col II was significantly higher than that of Col I, whereas the secretion of Col X was relatively low. All these results showed that chondrocytes encapsulated in HS5FFAB5 hybrid hydrogel could maintain normal physiological functions and secrete specific ECM the same as hyaline cartilage.
image file: d0tb00253d-f4.tif
Fig. 4 Immunohistochemical staining for Col I, Col II, Col X (A1–A5, B1–B5, C1–C5) and photomicrographs of H&E, TB, SO staining (D1–D5, E1–E5, F1–F5) of hydrogel/chondrocytes constructs after being cultured in vitro for 1, 3, 7, 14 and 21 days. (B–D) The semi-quantitative results for Col I, Col II, Col X staining. The data are presented as mean ± standard deviations (SD) from 3 independent experiments (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

3.5. The evaluation under subcutaneous implantation in vivo

The chondrogenic capacity of the HS5FFAB5 and HA-SH hydrogels in vivo was estimated by subcutaneous implantation in New Zealand rabbits (Fig. 5A) after being cultured for 1 or 4 weeks. The Fig. 5B illustrated that the wet weight of HS5FFAB5 hydrogel declined rapidly, almost 33% and 50% at 1 week and 4 weeks, respectively. Whereas, the pure HA-SH hydrogel degraded only 5% and 12.3% at 1 week and 4 weeks, respectively. The results demonstrated that short peptide FFAB was absorbed completely after 4 weeks, which was beneficial to the proliferation of early chondrocytes by providing early adhesion sites and growth space for matrix secretion. As shown in Fig. 5C–G, the chondrogenic marker genes expression of Col I, Col II, AGG and Sox9 in the HS5FFAB5 hydrogel was significantly upregulated compared to that in the HS hydrogel and these genes expression levels increased with prolonged implantation time. Interestingly, the Col X expression in the HS5FFAB5 hydrogel group was relatively lower than that in the HS hydrogel group. The results indicated that the HS5FFAB5 hydrogel could inhibit hypertrophy of chondrocytes in comparison to the HS hydrogel and maintain the normal physiological function of chondrocytes, it was also suitable for the early repair of cartilage tissue. It has potential application prospects in the early repair of cartilage injuries.
image file: d0tb00253d-f5.tif
Fig. 5 (A) The macrograph of hydrogels. (B) Wet weight change of two groups of hydrogels. (C–G) Q-PCR analyses of cartilage-matrix related genes, including Col I, Col II, Col X, AGG and Sox9. The data are presented as mean ± standard deviations (SD) from 3 independent experiments (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

3.6. Histological section staining analysis of HS5FFAB5 hydrogels cultured with chondrocytes in vivo

Histological section analysis of subcutaneous implantation for HS5FFAB5 hydrogels encapsulating chondrocytes was consistent with the above methods in Section 3.4 and corresponding pictures in low magnification are shown in Fig. S6 (ESI). The H&E staining images revealed that the HS5FFAB5 group showed abundant cell aggregation, which filled most of the area of the hydrogel in comparison to the HS group. Meanwhile, the TB and SO staining range and colour for the HS5FFAB5 group was larger and deeper than that for the HS group, which indicated that more extracellular matrix like GAG was secreted in the HS5FFAB5 group and it was beneficial for the adhesion and proliferation of chondrocytes. When sthe ubcutaneous implantation period was extended to 4 weeks, the immunohistochemical staining (Col I, Col II and Col X) further deepened in all groups, and the staining level of the HS5FFAB5 group was obviously higher than that of the HS group. The semi-quantitative results of Col I, Col II, Col X staining are presented in Fig. 6E–G. The secretion of Col I and Col II in HS5FFAB5 hydrogels was higher than that in the HS group; especially for Col II, the expression level was significantly higher than that of HS group. However, the expression of Col X in the HS5FFAB5 group was significantly lower than that in the HS group, which was consistent with the results of Q-PCR analysis. All these results showed that the HS5FFAB5 hydrogel could promote the proliferation of chondrocytes and specific secretion of matrix, inhibit the hypertrophy trend of cartilage, and prompt the formation of hyaline cartilage.
image file: d0tb00253d-f6.tif
Fig. 6 Immunohistochemical staining for Col I, Col II, Col X (A1–D1, A2–D2, A3–D3) and photomicrographs of H&E, TB, SO staining (A4–D4, A5–D5, A6–D6) of hydrogel/chondrocytes constructs after being implanted in vivo for 1 week and 4 weeks. (E–G) The semi-quantitative results for Col I, Col II, Col X staining. The data are presented as mean ± standard deviations (SD) from 3 independent experiments (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

4. Conclusions

In this study, the combination of supramolecular peptides BPAA-AFF-OH and injectable HA-SH could effectively construct a hybrid covalent/noncovalent network by primary chemical self-crosslinking derived from reductive degradable disulfide bonds and secondary physical self-assembly with hydrogen bonding and hydrophilic/hydrophobic interactions among short peptide molecules. The optimal composition ratio of the HS5FFAB5 hybrid hydrogel presented a preferable mechanical property due to its excellent and unique microstructure. Results of research in vivo and in vitro indicated that the HS5FFAB5 hybrid hydrogel could promote the proliferation of chondrocytes and specific secretion of matrix, inhibit the hypertrophy trend of chondrocytes, and prompt the formation of hyaline cartilage. Because of its good biocompatibility and injectable properties, simple operation process as well as high security of scaffold materials, this bionic hydrogel has potential application prospects in cartilage tissue regeneration.

Conflicts of interest

There are no conflicts to declare.


This work was sponsored by National Key R&D Plan of China (Grant No. 2018YFC1105900), Sichuan Province Key R&D Program (2019YFS0007), Young Elite Scientists Sponsorship Program by CAST (2017QNRC001), and Sichuan University Innovation Spark Project (2018SCUH0089).

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Electronic supplementary information (ESI) available. See DOI: 10.1039/d0tb00253d
These authors contributed equally to this work.

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