Design and evaluation of nano-hydroxyapatite/poly(vinyl alcohol) hydrogels coated with poly(lactic-co-glycolic acid)/nano-hydroxyapatite/poly(vinyl alcohol) scaffolds for cartilage repair

The PLGA/HA/PVA hydrogel was fabricated based on our previous method using the solvent extraction, evaporation technique, and repeated freeze-thaw cycling method [27]. First, PVA (341,584, 99+% hydrolyzed, M_w 89,000–98,000, Sigma, USA) and nano-HA (677,418, nanopowder, < 200 nm particle size (BET), ≥ 97%, synthetic, Sigma, USA) were incorporated into double-distilled water. The mixture was then heated to 90 °C for 90 min in a water bath with thermostatic magnetic mixer stirring. PLGA (lactide: glycolide 50:50, ester terminated, M_w 38,000–54,000, Sigma, USA) was dissolved in dichloromethane with ultrasonic stirring, which created a primary emulsion, then the primary emulsion was added to the PVA-nano-HA mixture. The PLGA/nano-HA/PVA solution was stirred with a thermostatic magnetic mixer to evaporate the dichloromethane, and then the solution was carefully injected into a mold. Finally, the method for crosslinking is freeze-thaw [28] which mold was frozen at − 20 °C for 21 h and allowed to thaw for 3 h at room temperature. The freeze-thaw cycle was repeated five times to increase the density of crosslinking.

Based on microscopic morphology we have reported previously [27], we selected groups with PVA mass fraction of 5 wt%(E1), 10 wt%(E2), or 15 wt%(E3); PLGA of 30 wt%; and nano-HA of 5 wt% for investigation. The control group contained HA: 5 wt%, PVA: 15 wt% (CG).

The animal study was approved by the Medical Ethics Committee of Xiangya Hospital Central South University. Four-week-old white New Zealand rabbits were euthanized by air embolism. Under aseptic conditions, the articular cartilage was then collected from the rabbit hip, knee, and shoulder joints and sliced into approximately 1 × 1 × 1 mm³ sections. The cartilage fragments were washed with PBS solution three times before being digested in 0.2% collagenase type-II at 37 °C for 6 h. The supernatant was then transferred to a new tube and centrifuged at 300 g for 5 min to collect the cell pellets. The cells were cultured in DMEM medium containing 1% penicillin/streptomycin and 10% fetal bovine serum at 37 °C in a humidified incubator containing 5% CO₂. When the cells reached 80–90% confluence, they were collected and adjusted to a concentration of 1 × 10⁶/mL. The cells were observed with microscopy and adapted to grow in culture. Also, the chondrocytes were identified with hematoxylin-eosin, toluidine blue staining, and type II collagen immunohistochemistry.

The scaffold (5 × 5 × 5 mm³) was freeze-dried using a lyophilizer (VFD-1000, Biocool, China) and sterilized with ethylene oxide. A set volume of cell suspension (1 × 10⁶/100 μL) was dropped on the scaffolds (20 scaffolds per group; five for cell adhesion test, five for MTT assay, five for Western blot analysis of glycosaminoglycan and collagen type II, and five for HE, toluidine blue, and immunohistochemistry staining) in a 24-well cell culture plate. The complexes were incubated at 37 °C in a 5% CO₂ humidified incubator for 2 h, then 2 mL of fresh medium (pre-warmed to 37 °C) was carefully added to each well and incubation was continued. After the cells were inoculated with the scaffold material for 24 h, the co-cultured scaffolds were taken out, and non-adhered cells on the scaffold were eluted with PBS solution. The eluate and the culture medium of each group were collected, and the cells were counted by centrifugation. The cell adhesion rate was calculated based on the method described before [27].

MTT assay was used to evaluate cell proliferation. The growth of chondrocytes in each group was analyzed by HE, toluidine blue, and immunohistochemistry staining of collagen type II after co-culture for 3, 7, and 14 days. Total cell protein was then extracted from each group using a cell lysis solution (Cell Signaling Technology, USA). The expression of glycosaminoglycan and collagen type II were quantitatively determined by Western blotting. Western blot analysis of cell lysates was performed as described [29].

Based on the morphological characteristics and practicability of the PLGA/HA/PVA hydrogels, we chose the PLGA: 30 wt%, HA: 5 wt%, PVA: 15 wt% hydrogel as the peripheral component. For the core, the proportion of components was nano-HA: 5 wt% and PVA: 20 wt%.

A novel HA/PVA hydrogel modified with a PLGA/HA/PVA scaffold was prepared using two-step molding. PVA and nano-HA were incorporated into double-distilled water. The mixture was heated to 90 °C for 90 min in a water bath with thermostatic magnetic mixer stirring. The mixture was then injected into mold 1 (radius r), frozen for 21 h at − 20 °C, and allowed to thaw for 3 h at room temperature to form the core. The PLGA/HA/PVA solution was then injected into mold 2 (radius R), as described above, which was larger than mold 1, frozen for 21 h at − 20 °C and allowed to thaw for 3 h at room temperature to give the peripheral section. The freeze-thaw cycle was repeated five times to increase crosslinking in the transition zone between the two hydrogels (Fig. 1). Through control of the bottom surface size (r², R²) of the two molds, we prepared groups of HA/PVA hydrogel modified with PLGA/HA/PVA scaffold with different constituent ratios. Components that contained HA/PVA hydrogel (control group one, CG1) or PLGA/HA/PVA hydrogel (control group two, CG2) were used as controls. The radius of mold 2 was 10 mm, and that of mold 1 was 7 mm in experimental group one (EG1), 8 mm in experimental group two (EG2), and 9 mm in experimental group three (EG3).

A bottom-emitting image was obtained in a dark room, and the constituent ratio of each section was calculated using ImageJ software. Also, the microstructure of the composites was observed by ESEM. The mechanical properties of the composites, including compressive and tensile tests, were evaluated using a universal mechanical testing machine (DDL100, Changchun, China). The sample (20 mm × 20 mm) was cylindrical and was directly placed on the test bench for pressurization. The stress/strain rate for the test was 5 mm/min. For the compressive mechanical properties test, the compression was stopped when the compression strain reaches 60%. For the tensile mechanical properties test, we stuck the end of the material with the aluminum alloy. After 24 h, the adhesion reached the maximum strength and was tested on the machine. The stress/strain rate for the test was 5 mm/min, and the test index was the stress-strain curve and strength of the material. Three samples in each group were selected, and each sample was measured three times.

The morphology and growth features of chondrocytes were as follows. On the first day after resuscitation, the cultured cells grew on the wall of the flask with spindle-shaped morphology. On day 7, cells were spindle-shaped and whorled or parallelled along the longitudinal axis; toluidine blue staining led to cells being stained light blue and the nucleolus being stained purple-blue (Additional file 1: Figure S1); immunohistochemical staining of type II collagen (Additional file 1: Figure S1) showed yellow-brown granules in the cytoplasm of the chondrocytes, and the nucleus was stained blue. The results of staining showed that chondrocytes cultured in vitro had type II collagen expression in the cytoplasm and cell membrane. The cell growth curve (Additional file 1: Figure S1) showed that the proliferation of chondrocytes increased significantly after 3 days of culture, reached a peak on day 9, and began to decline from day 10. The second-generation chondrocytes isolated and cultured showed the best growth. As the number of passages increased, the rate of proliferation declined.

The results of cellular adhesion are shown in Fig. 2. The adhesion in the experimental groups was significantly higher than that in the control group (P < 0.05), while there were no statistical differences among the three experimental groups (P > 0.05). This result shows that the addition of PLGA significantly favored the adhesion of chondrocytes. As described above, the hydrophobic PLGA particles and nano-HA facilitated integration with surrounding cartilage and improved the porosity of the scaffolds. The cellular attachment significantly increased as a result of the combined effect of PLGA and nano-HA. Since the amount of PLGA and nano-HA were the same, there was almost no difference between the three experimental groups.

The results of cell proliferation are shown in Fig. 2. The proliferation in the groups with scaffolds was enhanced compared with the control group (P < 0.05); in addition, the proliferation of the experimental groups was higher than that for the control group (P < 0.05) and there was no statistical difference between the three experimental groups (P > 0.05). Some researchers have shown that the morphology and mechanical properties of scaffolds, such as the elastic modulus, affect how seeded cells behave [30, 31]. The results of this study showed that the presence of scaffolds helps the proliferation of cells. Furthermore, the PLGA/PVA/nano-HA hydrogels performed better than the PVA/nano-HA hydrogels. The reason for this observation might be that the elastic modulus of the PLGA/PVA/nano-HA hydrogels was more conducive to proliferation than that of the PVA/nano-HA hydrogels. The results of the tests, therefore, showed that the addition of PLGA microparticles favored the adhesion and proliferation of seeded chondrocytes.

HE staining showed numerous cells in the pores of the hydrogels (Fig. 2). Cell proliferation of the experimental groups (EG1, EG2, EG3) was found to be enhanced compared with the control group and was positively correlated with the time of co-culture. In addition, the amount of matrix secreted by chondrocytes in the experimental group was more than in the control group. Among the experimental groups (EG1, EG2, EG3), proliferation of the EG3 group was the most pronounced, and the extracellular matrix was clearly connected.

Results of toluidine blue staining were similar to those of HE staining (Additional file 1: Figure S3). The toluidine blue staining was positive after 3, 7, and 14 days of co-culture in the experimental groups (EG1, EG2, EG3) and control group (CG). Cell proliferation of the experimental groups (EG1, EG2, EG3) was observed to be more active than for the control group and was positively correlated with the time of co-culture. Among the experimental groups (EG1, EG2, EG3), the proliferation of the EG3 group was the most pronounced.

Immunohistochemical staining of type II collagen (Fig. 2) was positive in both the experimental groups (EG1, EG2, EG3) and control group (CG) after 3, 7, and 14 days of co-culture. In the experimental groups (EG1, EG2, EG3), the staining results were strongly positive. The matrix was brownish yellow, and coarse collagen fibers could be seen. The expression of type II collagen was positively correlated with the time of co-culture. Among the experimental groups, the expression of type II collagen was most pronounced in the EG3 group.

The Western blot results showed that the expression of COL2 and GAG could be detected in both the experimental and control groups (Additional file 1: Figure S2). The relative expression of COL2 and GAG increased over time (P < 0.05). After co-culturing for 3, 7, and 14 days, expression of COL2 and GAG in the experimental groups was higher than for the control group (P < 0.05), while the expression in group C was higher than for the other two groups (P < 0.05).

This finding supported the observation that the PLGA/PVA/nano-HA hydrogels performed better in improving cell proliferation and secretion of chondrogenic matrix compared with PVA/nano-HA hydrogels. The improvement shown by the hydrogel is thought to be related to the addition of PLGA microparticles, which improved the biocompatibility of the scaffolds [32, 33]. Meanwhile among the experimental group, EG3 (5% HA, 30% PLGA, 15% PVA) showed greater cell proliferation and higher expression of COL2 and GAG than the other two experimental groups. However, the pore size and porosity in EG3 were the smallest and lowest, respectively, among the experimental groups. This indicated that chondrocyte proliferation and secretion of the chondrogenic matrix was not influenced by pore size and porosity alone [34]. It is reported that the elastic modulus of a scaffold plays a role in the differentiation of stem cells [35]. Whether the elastic modulus has the same effect on chondrocytes remains a question. Based on our results, as the PVA content increased, the material became denser, and the modulus of elasticity increased, leading to better chondrocyte proliferation and secretion of the chondrogenic matrix. Therefore, we conclude that the elastic modulus, pore size, and porosity all influenced chondrocyte proliferation. More research is needed to explore the most suitable elastic modulus, pore size, and porosity for this composite.