Stem Cell

Construction of ultrasonically treated collagen/silk fibroin composite scaffolds to induce cartilage regeneration – Scientific Reports

Time Stamp: November 16, 2023 7:00 PM
Source Node: 313262

SF purified solutions

Sericin was removed from the cocoon’s surface by placing the cocoon in a boiling NaHCO₃ solution for 60 min. The degummed reconstituted silk fibers were rinsed with deionized water and then dried and sealed. The reconstituted silk fibers were then dissolved in tri solution (CaCl₂·CH₃·CH₂·OH·H₂O, molar ratio of 1:2:8 and silk protein fiber: tri solution volume ratio of 1:10) at 72 ± 2 °C with a magnetic stirrer for 1 h. The silk protein mixture was then placed in a dialysis bag, and dialysis was carried out in running tap water for 48 h before being replaced with deionized water once per hour for 48 h. After dialysis, the mass score was 3.5 wt% of the SF solution.

Preparation of collagen

The fascia was first peeled from a bovine Achilles tendon, muscle and fat were removed, and the fascia was thoroughly ground using a slicer and tissue agitator. The protein was then agitated with 0.2 mol/L NaHCO₃ for 6 h to remove any impurities. The Achilles tendon crumbs were then washed in distilled water to adjust the pH to 7.0, followed by whisking for 1 h with 75% ethanol to defat and then washing again in distilled water to return the pH to neutral. The cleaned crumbs of 1% bovine tendon were dissolved in a solution of acetic acid (pH = 3.5, 0.5 mol/L), and the appropriate amount of 750U/mg pepsin (50:1 bovine tendon crumbs to pepsin mass ratio) was then added to the solution at 4 °C and stirred for 60 h. After centrifugation, the mixture was centrifuged and the supernatant was collected at 1200 rpm. Collagen precipitation was collected by centrifugation of the supernatant at 1200 rpm after adjusting the supernatant’s pH to 5.5 with 10 mol/L NaOH. The collagen deposits were then dissolved in a 0.5 mol/L acetate solution, followed by washing with ultrapure water and centrifuging at 1200 rpm.

Preparation of Col/SF composite scaffolds

The freshly prepared collagen and SF were mixed in a dialysis bag with a microelectric mixer attached to the handle according to the Col/SF mass ratios of 7:3, 8:2, and 9:1. The mixtures were then vibrated for 20 min using an ultrasound machine (100% vibrational power). After removing the bubbles, the mixtures were further concentrated with a sucrose solution (5 times its volume) for 6 h. The condensed material was then laid out and freeze-dried. Cross-linking of the lyophilized material was performed at 4 °C with 50 mM EDAC + 20 mM NHS dissolved in 95% ethanol for 24 h (0.9585 g EDAC and 0.23 g NHS dissolved in 100 mL of 95% ethanol). Following cross-linking, the Col/SF scaffolds were obtained by washing the scaffold three times with PBS and three times with deionized water.

Fourier-transform infrared spectroscopy (ATR-FT-IR) and SEM

We undertook the exploration of the biochemical profile of Col/SF scaffolds using ATR-FT-IR. This was performed with a Thermo Scientific Nicolet iS20 Spectrometer (Thermo Scientific, Waltham, MA, USA), equipped with a Smart iTR ATR accessory with a diamond crystal. The ATR-FT-IR absorption spectra were obtained within the range of 4000–400 cm⁻¹, utilzing 32 scans at a resolution of 4 cm⁻¹. The scaffold morphology was characterized by scanning electron microscopy (SEM). Using a scalpel, the scaffold specimens were cut into cross-sectional and longitudinal sections. The scaffold structure was observed by SEM after gold–palladium coating of the sections.

Porosity

The porosity was determined by a modified liquid displacement method. Anhydrous ethanol was added to the tube, and its volume was denoted V1. A dry scaffold sample was then added to the test tube and immersed in the solution for 5 min. Negative pressure was applied to degas the scaffold, at which point the volume of ethanol was recorded as V2. The scaffold sample was gently removed, and the remaining ethanol volume was recorded as V3. The mean values of three samples from each group were calculated.

The porosity of scaffolds was calculated using the following formula: porosity = (V1 − V3)/(V2 − V3) × 100%

$${text{Porosity}}, = ,left( {{text{V1}} – {text{V3}}} right)/left( {{text{V2}} – {text{V3}}} right) times {1}00% .$$

Water absorption

Samples of three dried scaffolds were taken from each group and impregnated with 0.01 mol/L PBS (pH = 4) for 24 h to equilibrate. The weight of the scaffold after drying its surface was designated as M0, and after drying for 12 h in the dryer as M1.

The rate of water absorption was calculated as follows: (M0 − M1)/M1 × 100%

$$left( {{text{M}}0 , – {text{ M1}}} right)/{text{M1 }} times { 1}00% .$$

Compressive testing

The scaffolds were first divided into three groups based on Col/SF ratios (7:3, 8:2, and 9:1), cut into cylinders of uniform size, and soaked in ultrapure water for 24 h before being placed in separate DMA Q800 instruments. For the stress–strain curve, the experimental module was set to DMA Strain Rate, the experimental method to Strain Ramp, the experimental camp to Compression, the experimental air bearing gas to Air, the experimental temperature to room temperature, and the experimental procedure as follows: Ramp strain − 30.000%/min to − 70.00%. The modulus of the sample was determined from the slope of the straight line fitted to each of the curves²⁰.

Cell culture

Human adipose-derived stem cells (hADSCs) were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). The cells was cultured with Dulbecco’s Modified Eagle Medium/F12 (DMEM/F12) and 10% fetal bovine serum (FBS). All cultures were maintained in a 5% CO₂ incubator at 37 °C. The medium was changed every 2 days.

Cytotoxicity assay

Three sets of Col/SF scaffolds (7:3, 8:2, and 9:1) were soaked in 75% alcohol for 48 h and then rinsed five times with a sterile 0.9% saline solution. The scaffolds were then soaked in low-sugar DMEM medium (containing 10% fetal bovine serum) in a 37 °C water bath for 24 h at a rate of 1.25 cm²/mL. Following extraction, the leachate solution was filtered through a 0.22-μm membrane. L929 cells were divided into four groups and seeded into culture dishes at a concentration of 1 × 10⁴ cells/mL with 100 μL per well and six wells per group. After incubation for 24 h, the medium was removed, 200 μL DMEM was added to the control group, while 200 μL leachate solution was added to each of the three groups of scaffolds. On days 2, 4, and 7, a culture panel was prepared with 20 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (5 mg/mL) per well for a period of 4 h. Next, 150 μL dimethylsulfoxide (DMSO) had been added to the culture solution under complete vacuum and allowed to oscillate for 10 min before the absorbance was measured at 492 nm using a multiplex enzymatic marker.

To evaluate hADSCs adhesion, the scaffolds were first transferred to a 12-well plate. Each scaffold was then inoculated with 50 μL of cell suspension containing 5 × 10⁵ hADSCs and incubated in a humidified 5% CO₂ incubator at 37 °C for 2 h. After 7 days of culture, hADSCs in the scaffold were assessed using the Calcein/PI Cell Activity and Cytotoxicity Assay kit (Beyotime, China) in line with the manufacturer’s instructions. After 30 min of incubation with a live/dead staining solution, both live (green) and dead (red) cells were observed under a confocal microscope.

Quantitative RT-PCR analysis

Gene expression of the cartilaginous markers SOX9, COL2A1, and ACAN was analyzed by quantitative PCR. Briefly, the harvested ADSCs/scaffold constructs (n = 3) were frozen with liquid nitrogen, and total RNA was extracted and purified using the Takara MiniBEST Universal RNA Extraction Kit (Takara, Japan) in line with the manufacturer’s instructions following 7 or 14 days of incubation. RNA concentrations were then quantified using a NanoDrop One spectrophotometer, and cDNA was obtained using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Japan). Quantitative RT-PCR was then performed using the Applied Biosystems 7500 Fast Real Time PCR System (Bio-Rad) using a Bio-Rad PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time). The primers used in this study are included in Table 1. The expression levels of genes were analyzed by the △△Ct method and normalized to the expression level of GAPDH.

Table 1 The sequence of primers.

Full size table

Sulfated glycosaminoglycans (sGAG) and hydroxyproline (HYP) quantification

GAG and HYP levels were measured in accordance with previously reported methods². The scaffold–cell complexes on days 7 and 14 were used for genetic and biochemical analyses (DS-DNA, GAG, and HYP levels). Quantification of DNA, GAG, and HYP was performed using SpectraMax Paradigm. After weighing with a microbalance, the scaffolds seeded with ADSCs (n = 3 in each group at each time point) were digested for 24 h in a pre-prepared papain solution (Sigma) at 60 °C overnight for the estimation of dsDNA content and GAG content. A total of 60 μL of the above-digested specimen reacted with a working solution of Hoechst 33,258 (2 μg/mL) in the dark at 37 °C for 1 h. The intensity was measured with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The readings were compared with the calf thymus DNA Standard Curve (Sigma). The total sulfated GAG content was assessed using dimethylmethylene blue assay (DMMB, Sigma). Briefly, 60 μL of the above-digested sample was mixed with DMMB reagent and allowed to react for 30 min at room temperature, and the absorbance was measured at 525 nm. The content of GAG was calculated relative to the standard curve obtained from the shark standard curve 6-sulfate (Sigma, USA). Collagen content was determined by quantifying the HYP content. Aliquots of the same digested solution were further hydrolyzed in HCl at 120 °C for 2 h, and HYP content was measured at 560 nm. HYP content was determined based on the HYP standard curve (Sigma). Normalization of GAG and HYP was performed using ds-DNA.

Animal model

All animals used in this study were adult male New Zealand rabbits that weighed between 2 and 2.5 kg. The experimental animals were provided by Beijing Vital River Laboratory Animal Technology Company. All procedures of the animal experiments were approved by the Beijing Jishuitan Hospital Animal Care and Use Committee, Beijing, China, and all of the methods were performed in accordance with the institutional guidelines for care and use of animals. The study is reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). All efforts were made to minimize the number of animals sacrificed in the experiment and their discomfort. Breeding conditions were maintained with single-cage housing, free movement inside cage, temperature of 18–23 °C, relative humidity of 50–60%, 12-h light/dark cycle, and ad libitum access to chow. 18 New Zealand rabbits were randomly divided into three groups, namely, the control group, the Col/SF scaffold group, and the Col/SF-hADSC scaffold group. For surgery, the animals were anesthetized with pentobarbital sodium and were placed in the prone position once they lost the pain reflex response. The articular cartilage was dislocated after surgical incision, exposing the articular tissue. A cylindrical cartilage defect (4 mm in diameter and 2 mm in depth) was formed by drilling corneal rings. In the Col/SF-hADSC scaffold group, hADSC-seeded scaffolds were placed in the cartilage defect area. A Col/SF scaffold was inserted in the Col/SF scaffold group. After reduction of the patella, suturing layer by layer from the joint to the skin was performed. To prevent infection, the New Zealand rabbits were given penicillin intramuscularly. The animals were sacrificed three or six months after the surgery, and further studies were carried out.

Macroscopic observations

The restored tissue was assessed in accordance with the International Society for Chondroplasty (ICRS) macroscore. Three different investigators blinded to the experimental groups graded tissue repair.

Histology and immunohistochemistry

The tissue samples were fixed in 4% paraformaldehyde for 24 h, flushed with tap water for 12 h, and then decalcified using 12.5% EDTA for 8 weeks. For the decalcified samples, a gradient ethanol series was used for dehydration. Following paraffin embedding and sectioning, 5-μm-thick sagittal sections were stained using Safranin O and Fast Green solution, Toluidine Blue staining solution, Red Picrosirius solution, and antibody against collagen II (1:200, Ab34712, Abcam, USA) to visualize type II collagen in line with the previous protocol²¹. For antigen retrieval, the sample sections were digested with pepsin (Sigma Aldridge, USA) at 37 °C for 30 min.

Statistical analysis

Statistical analyses were performed using the software GraphPad Prism 7.0. All experimental results were presented as the mean ± standard deviation. All the data were presented as mean ± standard deviation. Two-Way or One-way analysis of variance (ANOVA) test was used to compare the means among groups. While data from the same group were evaluated using Student’s t-test. Significance levels were set to at *P < 0.05, **P < 0.01, ***P < 0.001.

Ethics approval and consent to participate

All procedures of the animal experiments were approved by the Beijing Jishuitan Hospital Animal Care and Use Committee, Beijing, China and the study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).