Abstract
Recently, there has been increased attention on the treatment of cartilage repair. Overall, we constructed PHBVHHx-COL, a composite hydrogel of PHBVHHx-co-PEG and collagen, and evaluated its cartilage repair efficacy through in vitro and in vivo studies using hydrogel loaded with peripheral blood-derived mesenchymal stem cells (PBMSCs). Rheological properties and compressive mechanical properties of the hydrogels were systematically evaluated. The cytocompatibility of the hydrogels was evaluated using the Cell Counting Kit-8 test, live/dead staining, scratch test, and transwell test. The effect of chondrogenic differentiation of PBMSCs on hydrogels was evaluated using immunofluorescence staining and reverse transcription-polymerase chain reaction. Furthermore, the in vivo cartilage repair ability of the hydrogels was confirmed following in situ injections in rabbit chondral defect models. Finally, the induced polarization of the hydrogel scaffold on macrophages was explored by the expression of CD86 and CD206. In vitro experimental results confirmed that PHBVHHx-COL-gel led to better cell migration, proliferation, and chondrogenic differentiation than PHBVHHx-PEG and COL hydrogels. Hematoxylin and eosin staining indicated that the tissue of the repaired area in the PHBVHHx-COL group was nearly in fusion with the surrounding normal tissue and the reconstruction of subchondral bone was good. Safranin-O staining and COL-2 immunohistochemistry indicated that the tissue of the repaired area in the PHBVHHx-COL group had more cartilage-specific matrix secretion. The PHBVHHx-COL group exhibited more M2 macrophage infiltration and less M1 macrophage presentation than the other groups. This study demonstrated that PHBVHHx-COL scaffolds loaded with PBMSCs significantly promoted the repair of cartilage injury through immune regulation by M2 polarization and could be potential candidates for cartilage tissue engineering.
Impact Statement
In our study, for the first time, porous scaffolds of PHBVHHx were utilized for cartilage repair. We found that injectable PHBVHHx-COL gel possesses good compressive mechanical properties and biocompatibility as a tissue engineering scaffold. When loaded with PBMSCs, PHBVHHx-COL gel demonstrated a significant cartilage repair effect. After 12 weeks, the repaired cartilage tissue closely resembled normal cartilage tissue.
Keywords: PHBVHHx, peripheral blood, mesenchymal stem cells, cartilage
Introduction
Articular cartilage is hyaline cartilage, which has no blood vessels, nerves, or lymphatic vessels. Because there is no regenerative repair ability, cartilage repair remains a challenge for surgeons. 1
Polyhydroxyalkanoate (PHA), which is produced by bacteria, has been used in tissue engineering. 2,3 As a new member of the PHA family, PHBVHHx has been shown to support the growth of bone marrow-derived mesenchymal stem cells (BMSCs) and has a promising potential for medical applications. 4,5 Collagen (COL) is also a component of many different extracellular matrices, which is considered as a suitable scaffold material, especially in orthopedic tissue engineering. 6
In vitro studies show that peripheral blood-derived mesenchymal stem cells (PBMSCs) share similar cell morphology, immunophenotype, and multipotent differentiation potential with BMSCs. Additionally, PBMSCs exhibit superior adipogenic and chondrogenic differentiation abilities compared with BMSCs. In vivo experiments indicate that PBMSCs and BMSCs possess equivalent capabilities in cartilage repair. 7,8 In addition, Fu and his colleagues successfully mobilized BMSCs into the blood through the combined use of granulocyte colony stimulating factor (G-CSF) and AMD-3100, and greatly improved the acquisition efficiency of PBMSCs, providing favorable conditions for the application of stem cells. 9 Moreover, the clinical application of PBMSCs has its unique advantages, such as minimally invasive sample collection procedures, repeatability of sampling, and high recognition of patients. 10
Inflammatory reactions play an important role in the entire process of repairing tissue damage. Research has shown that during the repair process of cartilage damage, the transformation of macrophages from M0 to M2 type can promote cartilage repair. 11 Dai et al. found that M2 macrophages can secrete transforming growth factor β1 and transforming growth factors β3. These factors can promote the repair of cartilage. 12
Therefore, an ideal scaffold material for cartilage repair should have the following characteristics: good biological activity, conducive to the proliferation of MSCs, and inducing the transformation of macrophages from M0 to M2.
In this study, PHBVHHx-COL-gel, a novel composite hydrogel comprising PHBVHHx-co-PEG and COL-1, was loaded with PBMSCs and transplanted into cartilage defects. The efficacy of the hydrogels and PBMSCs in promoting cartilage injury recovery was evaluated.
Materials and Methods
PBMSCs culture and identification
Isolation and culture of PBMSCs
All the experiments were approved by the Ethics Committee of Sichuan University. First, New Zealand white rabbits were preinjected with G-CSF (intramuscular injection, 50 mg/kg/day, for 5 days); at 6 day, AMD-3100 was intramuscularly injected at a dosage of 5 mg/kg. Blood samples were obtained from arteries 1 h later, and PBMSCs were isolated using the lymphocyte separation method. Specifically, peripheral blood was diluted with an equal volume of DMEM medium containing heparin and carefully added to an equal volume of lymphocyte isolation solution (Ficoll), followed by centrifugation at room temperature at 500 g. After 30 min of centrifugation, the intermediate white film layer was collected and the process was repeated at room temperature. After centrifugation at 200 g for 10 min, the supernatant fluid was removed and the cells were resuspended in the culture medium. Then, the cells were transferred to the DMEM medium containing 15% fetal bovine serum for further culture. To ensure the activity of cells, only cells from passage 3 to passage 5 were used in this experiment.
Analysis of cell phenotype by flow cytometry
Note that 1 mL of PBMSCs suspension (cell concentration: 106/mL) was placed in a centrifuge tube, and were fluorescently labeled with the following antibodies: CD29-PE (Bioss, bs-0486R), CD34-PE (Bioss, bs-0646R), CD45-PE (Bioss, bs-0522R), CD44-PE (Bioss, bs-0521R), and CD90-PE (BD, 554898). The cells were incubated in the dark for 30 min, then washed twice with phosphate-buffered saline (PBS). The abovementioned marker proteins were detected by flow cytometry.
PBMSCs differentiation ability
PBMSCs were cultured separately in adipogenic induction medium (Cyagen Biosciences, HUXUC-90031), osteogenic induction medium (Cyagen Biosciences, HUXUC-90021), and chondrogenic induction medium (Cyagen Biosciences, HUXUC-90041). After 21 days, the ability of PBMSCs to differentiate into adipocytes, osteocytes, and chondrocytes was evaluated by oil red O staining, alizarin red staining, and alcian blue staining, respectively.
Preparation of hydrogels
The preparation of hydrogels is described in Supplementary Data S1.
Scanning electron microscopic observation of hydrogels
Before the test, the freeze-drying cylindrical hydrogels (diameter: 8 mm, thickness: 2 mm) were rapidly quenched with liquid nitrogen. Then, the cylindrical hydrogels were divided into thin slices (thickness: 1 mm). The morphology and pore size of the samples were analyzed by scanning electron microscope (SEM) (Hitachi, S-4800). All samples were coated with gold, followed by examination under SEM.
Rheological property tests of hydrogels
The test parameters are as follows: oscillation frequency, 1 Hz; temperature range: 30–50°C; change law: linear change law; data point time: 5.5 s; and number of data points: 60. Then, 100 µL of hydrogel in the sol state was added on the test bench of rheometer for measurement, and the storage modulus and loss modulus were recorded. The temperature at which the storage modulus and loss modulus are equal is the gelation temperature.
Compressive mechanical property test of hydrogels
First, the disc-shaped hydrogels (diameter: 8 mm, thickness: 2 mm) were soaked in PBS, then the excess water on the surface was removed by filter paper. Second, the hydrogels were placed on the dynamic mechanical analysis and the storage modulus of the hydrogels at room temperature was tested. The test parameters were as follows: compression frequency, 1 Hz; stress size, 2 kPa; amplitude: 40 μm; dynamic force: static force = 105:100; and each group of hydrogels was set with three parallel samples for detection.
Preparation of hydrogels loaded with PBMSCs
The hydrogel with 5% (w/w%) and the cell suspension with a concentration of 2 × 107 were mixed in a ratio of 4:1. The final cell concentration in hydrogels was 5*106. The wt% of the hydrogels was 4%. The hydrogel solidified at room temperature (cylindrical, 8 mm in diameter, 2 mm in thickness), then transferred to DMEM medium containing 15% fetal bovine serum for future research.
Effect of hydrogels on PBMSCs, in vitro
Effect of hydrogels on cell viability
The hydrogels with PBMSCs were cultured in the medium. After 1 and 7 days, the hydrogels with PBMSCs were stained with a live/dead cell staining kit, then placed under a confocal laser microscope (Leica, TCS SP8) to investigate cell viability.
Effect of hydrogels on cell proliferation
First, the hydrogel leaching solution was prepared. Four percent (w/w) of hydrogel in its sol state (1 g) was added to a 15 mL centrifuge tube, followed by the addition of 10 mL of culture medium containing 15% fetal bovine serum. The tube was then placed in a humidified incubator at 37°C with 5% CO2 for 48 h. After incubation, the sample was centrifuged at 10,000 g for 30 min, and the supernatant was collected as the leachate for further use. The cell concentration was adjusted to 5 × 104/mL; then, 100 μL per well was inoculated into 96-well plates, and the cells were cultured overnight to allow adhesion. Next, the medium was changed to leaching solution and a control group with normal culture medium was established. After 1, 3, 5, and 7 days, Cell Counting Kit (CCK)-8 reagent was added to wells in each group referring to the manufacturer’s instructions. After 2 h, the optical density of the samples was detected by a spectrophotometer (Beckman, DU-640).
Effect of hydrogels on cell migration
Scratch test
First, maker was used to draw five parallel lines with the same spacing on the bottom surface of 6-well plates. The cell concentration was adjusted to 5 × 104/mL; then,100 μL per well was inoculated into 96-well plates, and the cells were cultured overnight to allow adhesion. Next, the medium was changed to leaching solution and a control group with normal culture medium was established. After 6, 12, and 24 h, the plates were placed under the telescope and observed.
Transwell test
Note that 600 μL of leaching solution was added to a transwell chamber and incubated for 30 min. Then, 100 μL of cell suspension (containing 5 × 104 cells) was added to the upper chamber and cultured for 24 h. The cells were fixed with paraformaldehyde. The upper chamber was wiped with a sterile cotton swab for removing the cells. The bottom film of the upper chamber was collected and stained with 0.1% crystal violet staining solution. The longitudinal migration of PBMSCs from one side of the upper chamber membrane to the other was observed under a microscope.
Effect of hydrogels on cell chondrogenic differentiation
Immunofluorescence test
The hydrogels were transferred into 6-well plates. A total of 3 mL chondrogenic inducing medium (Cyagen Biosciences, HUXUC-90041) was added to each well. The medium was changed every 2 days.
After 14 and 28 days, the hydrogels were fixed in 4% paraformaldehyde. Then, anti-COL-2 primary antibody (DSHB, II-II6B3) and anti-Sox9 primary antibody (BIOSS, bs-4177R) were added into the 6-well plate together and incubated at 4°C for 24 h. Then, Alexa Fluor 555 labeled secondary antibody (Beyotime, A0460) and Alexa Fluor 647 labeled secondary antibody (Yeason, 34213ES60) were added and incubated at room temperature in dark for 4 h. At last, DAPI solution was added and incubated at room temperature in dark for 15 min. Then, the hydrogels with PBMSCs were placed under the confocal microscope for observation.
Real-time polymerase chain reaction analysis of gene expression
Total cellular RNA was extracted from PBMSCs cultured on hydrogels submerged in chondrogenic inducing medium at both 14 and 28 days, with PBMSCs grown in basal medium serving as a control. The RNA Extraction Kit (TianGen, China) was utilized according to the manufacturer’s instructions. Subsequently, total RNA was reverse transcribed into cDNA using the iScript™ cDNA Kit (Toyobo, Japan). Real-time polymerase chain reaction (PCR) reactions were conducted and monitored using the EvaGreen supermix (Bio-Rad, USA). The chondrogenic gene expression of Sox9 and COL-2 was quantified using quantitative PCR. The expression level of the GAPDH gene served as an internal control. The 2-ΔΔCT method was used to calculate the relative gene expression. The real-time PCR primer sequences are shown in Supplementary Table S1.
Reparative effect of hydrogels with PBMSCs on chondral defects
This study was approved by the Animal Ethics Committee. Forty male, 3-month-old New Zealand white rabbits were prepared, and a chondral defect (diameter: 4 mm, depth: 1 mm) was established in the femoral trochlea. Then, the rabbits were divided equally into four groups: the control group, the PHBVHHx-COL group, the PHBVHHx-PEG group, and the COL group. In the PHBVHHx-COL group, the PHBVHHx-PEG group, and the COL group, hydrogels with PBMSCs were implanted in the chondral defect. In the control group, nothing was implanted in the chondral defect. The rabbits were euthanized at 6 or 12 weeks after the operation, and the knee joints were preserved for the further research.
Macroscopic assessment
The International Cartilage Repair Society (ICRS) macroscopic evaluation criteria for cartilage repair were used for scoring. 13
Histomorphological evaluation
After the sample was fixed, decalcified, and dehydrated, it was embedded in paraffin and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E), safranin O, and type II COL immunohistochemistry referring to the manufacturer’s instructions. The sections in each group were photographed under a brightfield microscope. The staining results of the above sections were scored blindly by two researchers in related fields according to the ICRS histomorphology scoring system to evaluate the effects on the repair of cartilage in each group. 14
Effect of hydrogels on macrophage polarization
This study was approved by the Animal Ethics Committee. Forty SD rats were prepared. The rats were divided equally into four groups, the PHBVHHx-COL group, the PHBVHHx-PEG group, the COL group, and the control group. Hydrogels were injected subcutaneously into rats. In the control group, nothing was injected subcutaneously. The rats were euthanized at 7 or 14 days after the operation, and skin tissue were preserved for the further research.
After the sample was fixed, decalcified, and dehydrated, it was embedded in paraffin and sectioned at 5 μm. Rabbit antirat CD68 antibody (SAB, 41618, diluted 1:100) was used to detect CD68, while rabbit antirat CD86 (SAB, 32223, diluted 1:100) and CD206 (SAB, 3477, diluted 1:100) were used to detect CD86 and CD206. The number of cells labeled with CD68, CD86, and CD206 antibody were calculated by Image J software. Cells labeled with CD68, CD86, and CD206 antibody represent macrophages, M1 macrophages, and M2 macrophages, respectively.
Statistical analysis
SPSS 18.0 (IBM, USA) was used to analyze the data. Values are shown as the mean ± standard error. One-way analysis of variance was used for data with a homogeneous variance to identify significant differences between groups, and the rank-sum test for data with a nonhomogeneous variance. If p < 0.05, there was a statistically significant difference between groups.
Results
PBMSCs express MSC-specific markers and have the potential for multidirectional differentiation
The cultured PBMSCs showed a uniform, slender, spindle shape and a typical spiral arrangement under the microscope. The proliferation rate was fast, and there was no cell aging phenomenon such as cavitation (Fig. 1A, B).
FIG. 1.
(A, B) Morphology of P3-generation PBMSCs under an optical microscope (A, ×40; B, ×100). (C) Osteogenic differentiation of PBMSCs stained with alizarin red. (D) Adipogenic differentiation of PBMSCs stained with oil red O. (E) Chondrogenic differentiation of PBMSCs stained with alcian blue. (F–J) Flow cytometry analysis showing positive expression of CD29, CD90, and CD44 (≥95%) and negative expression of CD34 and CD45 (both ≤2%). PBMSCs, peripheral blood-derived mesenchymal stem cells.
After 21 days of culture in osteogenic induction medium, a large amount of matrix calcification with calcium nodule formation was observed by alizarin red staining (Fig. 1C). After 21 days of culture in adipogenic induction medium, a large number of round, red-stained lipid droplets were observed by oil red O staining (Fig. 1D). After 21 days of culture in chondrogenic induction medium, alcian blue staining was positive (Fig. 1E), indicating that proteoglycans were abundant in the three-dimensional (3D) cell pellets. The above experimental results confirm that PBMSCs have the potential for multidirectional differentiation.
The flow cytometry results of PBMSCs showed that the expression of the MSC surface markers CD29 (98.6%), CD44 (98.8%), and CD90 (98%) was positive and the expression of the hematopoietic cell markers CD34 (1.32%) and CD45 (1.72%) was negative (Fig. 1F). These data show that PBMSCs have MSC characteristics
Hydrogel surfaces are porous
Measurements taken using ImageJ after SEM observation revealed that PHBVHHx-PEG hydrogel and COL hydrogel exhibited relatively uniform porous structures, with a mean pore size of approximately 100 μm (SD ±8.3 μm), allowing space for matrix deposition. In contrast, the mean pore size of PHBVHHx-COL hydrogel was significantly larger, at 130 μm (SD ±7.3 μm), and its pore distribution was more uniform compared with the other two hydrogels (Fig. 2A).
FIG. 2.
(A) SEM results of the hydrogels. (B–D) Temperature-sensitive rheological results of the (B) PHBVHHx-COL,(C) PHBCHHx-PEG, and (D) COL hydrogels, showing storage modulus (G′) and loss modulus (G′′). (E) Compressive mechanical properties of hydrogels tested by dynamic mechanical analysis (DMA). The X-axis represents compression frequency (1 Hz), and the Y-axis represents the storage modulus (kPa). *, p < 0.05; **, p < 0.01; ***, p < 0.001. SEM, scanning electron microscope.
Temperature-sensitive rheological properties of hydrogels
The storage modulus (G′) of PHBVHHx-COL gel is equal to the loss modulus (G′′) at approximately 39°C. The gelation temperatures for PHBVHHx-PEG and COL are 37°C and 39°C, respectively (Fig. 2B). These hydrogels can solidify and transform into gel form after being injected into knee joint defects in their sol state.
Hydrogels have good compressive mechanical property
In biological tissue engineering, the compressive mechanical property is a very important index to measure the quality of hydrogel scaffold. As shown in Figure 2C, the storage modulus of PHBVHHx-COL-gel was 17.1 ± 1.1 kPa; the storage modulus of PHBVHHx-PEG hydrogel was 10.7 ± 0.2 kPa; and the storage modulus of COL hydrogel was 8.2 ± 0.2 kPa. The results showed that the mechanical property of PHBVHHx-PEG hydrogel was higher than COL hydrogel (p < 0.05). The mechanical property of PHBVHHx-PEG hydrogel was significantly increased after the introduction of COL, and the mechanical property of PHBVHHx-COL-gel was significantly higher than the other two hydrogels (p < 0.001).
Hydrogel have good cytocompatibility and promote PBMSC proliferation and migration
The results of confocal laser observation for PBMSCS seeded on the hydrogels showed at 1 day, there were many living cells and a few dead cells in all hydrogels, which were considered to be produced in the process of trypsin digestion. The ratio of living cells in PHBVHHx-COL was significantly higher than that in PHBVHHx-PEG and COL hydrogels. At 7 days, the ratio of living cells in PHBVHHx-COL was still significantly higher than that in PHBVHHx-PEG and COL hydrogels. The results above proved that compared with PHBVHHx-PEG and COL hydrogels, PHBVHHx-COL-gel is more suitable for cell survival (Fig. 3A, B).
FIG. 3.
(A) Confocal laser observation of live/dead-stained PBMSCs cultured on the hydrogels for 1 and 7 days (scale bar = 100 μm; green: live cells; red: dead cells). (B) Semiquantitative analysis of live PBMSCs cultured on the hydrogels for 1 and 7 days. (C) CCK-8 assay showing the effects of hydrogels on the proliferation of PBMSCs in vitro. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001. CCK-8, Cell Counting Kit-8.
CCK-8 results showed that at 1 day, compared with the control group, all hydrogels did not promote the proliferation of PBMSCs. However, at 3, 5, and 7 days, the PHBVHHx-COL and PHBVHHx-PEG hydrogel promoted the proliferation of PBMSCs compared with the control group. The effect of PHBVHHx-COL-gel on the proliferation of PBMSCs was better than those of PHBVHHx-PEG hydrogel and COL hydrogel (Fig. 3C).
Scratch test results showed that at 6 h, only PHBVHHx-COL-gel promoted the migration of PBMSCs. At 12 and 24 h, PHBVHHx-COL-gel and PHBVHHx-PEG hydrogel promoted the migration of PBMSCs, and the effect of PHBVHHx-COL-gel on the migration was better than that of PHBVHHx-PEG hydrogel. Transwell test results also showed that PHBVHHx-COL-gel and PHBVHHx-PEG hydrogel promoted the migration of PBMSCs, and the effect of PHBVHHx-COL-gel on the migration was better than that of PHBVHHx-PEG hydrogel at 24 h (Fig. 4A–C).
FIG. 4.
(A) Scratch assay results for lateral migration at 0, 6, 12, and 24 h, and the effects of hydrogels on the vertical migration of PBMSCs in vitro by transwell assay (scale bar = 10 μm). (B) Semiquantitative analysis of the scratch assay area. (C) The longitudinal migration of PBMSCs in the transwell chamber (scale bar = 100 μm). (D) Semiquantitative analysis of PBMSCs migration in the transwell assay. *p < 0.05; **p < 0.01; ***p < 0.001.
Chondrogenic differentiation of PBMSCs on hydrogels
Immunofluorometric assay
After 14 days of in vitro culture, PBMSCs on the PHBVHHx-COL gel produced more COL-2 and Sox9 compared with those on the COL hydrogel (Fig. 5A, B). Even after 28 days of in vitro culture, PBMSCs on the PHBVHHx-COL gel continued to produce higher levels of COL-2 and Sox9 than those on the COL hydrogel (Fig. 5C, D).
FIG. 5.
(A) COL-2 and SOX-9 immunofluorescence staining of PBMSCs on the hydrogels at 14 and 28 days (scale bar = 100 μm). (B, C) Average fluorescence intensity (AFI) of (B) COL-2 and (C) SOX-9 at 14 days. (D, E) AFI of (D) COL-2 and (E) SOX-9 at 28 days. (F) Gene expression results for COL-2 in PBMSCs on the hydrogels at 14 and 28 days. (G) Gene expression results for SOX-9 in PBMSCs on the hydrogels at 14 and 28 days. *p < 0.05, **p < 0.01, ***p < 0.001.
PCR results indicated that after 14 and 28 days, the gene expression of COL-2 and Sox9 in PBMSCs cultured on the PHBVHHx-COL gel and PHBVHHx-PEG hydrogel was higher than that in PBMSCs cultured on the COL hydrogel and the control group (Fig. 5E, F).
Hydrogels combined with PBMSCs promote articular cartilage regeneration
Macroscopic evaluation
The macroscopic results of cartilage regeneration at 6 and 12 weeks postsurgery are shown in Figure 6A. At 6 weeks, the cartilage defect in the PHBVHHx-COL group was completely filled, exhibiting a smooth surface, soft texture, and slight depression around the edges. In the PHBVHHx-PEG group, most of the cartilage defects were covered centrally, but there were still noticeable depressions around the edges. At 12 weeks, the appearance of the cartilage repair site in the PHBVHHx-COL group was uniform, with a color similar to that of normal cartilage and no distinct boundary between the PHBVHHx-COL group and normal cartilage. However, in the other groups, there were still obvious surface defects, with an uneven appearance and soft texture.
FIG. 6.
(A) Macroscopic results for cartilage defect repair in rabbit femoral condyles (scale bar = 4 mm). (B, C) ICRS cartilage repair macroscopic score at (B) 6 and (C)12 weeks. *p < 0.05, **p < 0.01, ***p < 0.001. ICRS, International Cartilage Repair Society.
The ICRS cartilage repair macroscopic score (Fig. 6B) indicated similar results, showing significantly better repair in the PHBVHHx-COL group compared with the other groups at both 6 and 12 weeks.
Histomorphological evaluation
Histomorphological staining illustrates the nature of the repaired tissue, showing that chondral defects are gradually filled with repair tissue over time. The results of H&E staining (Fig. 7A) were consistent with the macroscopic evaluation results. In the PHBVHHx-PEG group, cartilage defects were mostly filled with a small amount of remaining defects at 6 weeks. By 12 weeks, the cartilage surface was smooth with no obvious cracks with the surrounding normal cartilage. In the PHBVHHx-COL group, defects were almost fully repaired by hyaline cartilage-like structures, presenting a smooth surface and complete fusion with the surrounding normal cartilage tissue.
FIG. 7.
(A) hematoxylin-eosin (H&E) staining results of femoral condyle cartilage defects. The red arrow indicates the defect boundary (scale bar = 100 μm). (B) Safranin O-fast green (SO-FG) staining results (scale bar = 100 μm). (C) COL-2 immunohistochemical staining results (scale bar = 100 μm). (D, E) ICRS histomorphology score of the repaired tissue at (D) 6 and (E) 12 weeks. *p < 0.05, **p < 0.01, ***p < 0.001.
Safranin O staining (Fig. 7B) indicates polysaccharide content in cartilage, with deeper red color signifying higher polysaccharide content. In both the PHBVHHx-COL and PHBVHHx-PEG groups, the new tissue in the damaged area stained deeply, suggesting increased polysaccharide secretion and indicating similar biological functions to hyaline cartilage.
Immunohistochemical staining for COL-2 levels in the repaired tissue (Fig. 7C) revealed that at 6 weeks, new cartilage in the PHBVHHx-COL and PHBVHHx-PEG groups showed significant staining, superior to that in the other groups. However, the staining of new cartilage was lighter compared with the surrounding normal cartilage, indicating lower COL-2 content than normal cartilage. By 12 weeks, immunohistochemical staining of new cartilage in the PHBVHHx-COL and PHBVHHx-PEG groups showed no significant difference from that of the surrounding normal cartilage, with a smooth surface of the new cartilage.
The histomorphology scores were consistent with the staining results. The cartilage scores (Fig. 7D) in the PHBVHHx-COL and PHBVHHx-PEG groups were significantly superior to those in the other groups at both 6 and 12 weeks.
Immunomodulation of the hydrogel in vivo
Immunofluorescence results for subcutaneous rat implants at days 7 and 14 showed that CD86 (M1 marker) expression in the PHBVHHx-COL and PHBVHHx-PEG groups was significantly lower than in the COL and control groups (p < 0.05). Conversely, CD206 (M2 marker) expression in the PHBVHHx-COL and PHBVHHx-PEG groups was significantly higher than in the COL and control groups (p < 0.05). No significant difference was observed between the PHBVHHx-COL and PHBVHHx-PEG groups (p > 0.05) (Fig. 8A, B).
FIG. 8.
(A) Immunofluorescence staining images of macrophages in rat subcutaneous implants at 7 and 14 days. Blue dots represent cell nuclei, while green represents CD68+ cells. Red cells represent CD86+ cells (M1 macrophages) or CD206+ cells (M2 macrophages). (B, C) The proportion of (B) CD86+ (M1 macrophages) and (C) CD206+ (M2 macrophages) at 7 days. (D, E) The proportion of (D) CD86+ and (E) CD206+ at 14 days. *p < 0.05, **p < 0.01, ***p < 0.001.
Discussion
An ideal hydrogel scaffold material should be with the following characteristics: mechanical strength similar to tissue, biodegradability, and 3D structure conducive to stem cell proliferation, differentiation, and migration. 15 By this study, we found that PHBVHHx-COL injectable hydrogel has higher mechanical properties, good cytocompatibility, and promotes PBMSC proliferation and migration as a tissue engineering scaffold. PHBVHHx-COL hydrogel loaded with PBMSCs has good cartilage repair effect. After 12 weeks, the repair tissue of cartilage injury is close to the normal cartilage tissue.
PHA has drawn more attention because it comes from bacteria and has desirable biocompatibility, flexible biodegradability, and mechanical strength in vitro and in vivo. 16 In the PHA family, poor biocompatibility has limited their further applications. A few studies showed that PHB and PHBV induced prolonged acute inflammatory responses when implanted in vivo. 17,18 As a new member of the PHA family, the structure and properties of the PHBVHHx had been studied by a few studies. 4,5 The studies showed that PHBVHHx had a rougher surface, higher thermal stability, greater flexibility, and mechanical strength. In addition, PHBVHHx also promotes cell adhesion and proliferation. One study also showed that PHBVHHx promoted more BM-MSC growth and the differentiation of BM-MSCs into nerve cells. 19 In addition, two studies showed that PHBVHHx scaffolds loaded with stem cells had the similar effect on injured livers and significantly promoted the recovery of injured livers. 20,21 In this study, it was proved that PHBVHHx promotes PBMSCs proliferation and chondrogenic difference.
There are many types of COL, including COL-1, COL-2, and COL-3. 22,23 Although COL-2 is the main component of extracellular matrix of cartilage, but high price and poor physical and chemical properties limit the application. 24 In one study, researchers implanted stem cells into 2D or 3D scaffolds and found that type I COL gel could promote the transformation of cells into osteogenic or chondrogenic cells. 25 In another study, the researchers found that some special supplements were added to the pure COL hydrogel (β-phosphate bone and chondroitin sulfate cartilage) could significantly promote the differentiation of stem cells. 26,27 The results of this study showed that PHBVHHx-COL has a better chondrogenic differentiation ability than PHBVHHx-PEG hydrogel and COL hydrogel. The possible reason is that PHA itself has a strong chondrogenic ability, and the addition of COL binds to the integrin on the cell membrane, thus enhancing the chondrogenic ability of PHBVHHx.
Traditionally, bone marrow is considered as the most reliable and abundant source of MSCs. However, there are a few limitations: the first is surgical complications, including donor site pain, infection, and even sepsis. 28 In addition, the aging-related problems of BMSCs are also considered in cartilage repair. 29 Compared with BMSCs, PBMSCs have stronger ability to adipogenic and chondrogenic differentiation. 8,9 Moreover, in terms of ethics, the clinical application of PBMSCs has its unique advantages, such as minimally invasive sample collection procedures and high recognition of patients. 10
In this study, PHBVHHx-COL-gel might inhibit the transformation of macrophage M0 into M1, which was conducive to the early repair of cartilage damage. Our research results also proved that the repair site of cartilage in the PHBVHHx-COL group was repaired early, and the repair tissue was close to normal cartilage tissue.
Conclusions
This study demonstrated that PHBVHHx-COL scaffolds loaded with PBMSCs significantly promoted the repair of cartilage injury through immune regulation by M2 polarization. PHBVHHx-COL scaffolds and PBMSCs can be further studied for cartilage tissue engineering.
Authors’ Contributions
The conception and design of the study were conducted by D.X.W. and W.F., while data acquisition was carried out by P.S. and Y.H., Data analysis and interpretation were performed by P.S. and Y.H., who also drafted the article. W.F. and J.L. critically revised the article for important intellectual content. All authors gave final approval of the version to be submitted.
Disclosure Statement
Each author certifies that he or she has no commercial associations (e.g., consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the article.
Funding Information
This work was supported by the National Natural Science Foundation of China (82372490, 81972123, 82172508), Fundamental Research Funds for the Central Universities (2015SCU04A40), The Innovative Spark Project of Sichuan University (2018SCUH0034), Chengdu Science and Technology Bureau Project (2019-YF05-00090-SN), 1.3.5 Project for Disciplines of Excellence of West China Hospital Sichuan University (ZYJC21030, ZY2017301), and 1·3·5 project for disciplines of excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (2019HXFH039).
Supplementary Material
Reference
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