L-polylactic acid porous microspheres enhance the mechanical properties and in vivo stability of degummed silk/silk fibroin/gelatin scaffold

All the experiments in the present study were performed according to the Declaration of Helsinki. Ethical approval was obtained from the Ethical Committee of the Jilin University First Hospital. Carboxyl-terminated PLLA (MW 5000) (Sigma, USA), Type VI Collagenase (Nordmark, Germany); SF (Beijing Shengnuodede Medical Technology Co., Ltd, China); dichloromethane (Tianjin University Kewei Company, China); anhydrous ethanol (No. 6 Tianjin Chemical Reagent Factory, China); sodium hydroxide (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd, China) Dulbecco's modified Eagle medium (DMEM)/high glucose culture medium (Hyclone, USA), fetal bovine serum (Clack, Australia), penicillin-streptomyces–amphotericin B mixed solution (Solaribo, China), Collagen type II Rabbit Polyclonal antibody (Proteintech, USA), cell counting kit-8 (CCK-8) kit (Invigentech, USA), and calcein-AM/PI double-staining kit (Solaribo, China). All other chemical reagents were obtained from commercial sources.

Carboxyl-terminated PLLA (Mw: 50 000) powder was dissolved in dichloromethane to prepare an oil phase solution with a concentration of 2.5%; NH4HCO3 was dissolved in deionized water to prepare an internal aqueous phase with a concentration of 1% NH4HCO3. Under high speed disperser stirring, the internal aqueous phase solution was added drop-wise to the prepared oil phase solution with a syringe pump. After the addition was complete, stirring was continued for 2 min to form W1/O emulsion. Quickly transfer W1/O emulsion into a stirred 0.1% PVA aqueous solution to form a double emulsion (W1/O/W2). Stir for 3 h to volatilize the organic solvent and solidify the PMs. The PMs were collected and washed with deionized water three times. Resuspend the PLLA PMs with 0.1 mol l⁻¹ NaOH solution at room temperature for 20 min, and then washed three times, pre-frozen in separate packaging, and freeze-dried to obtain porous microsphere solid powder.

Physical blending was applied for the preparation of S/SF/G/PLLA-PMs scaffold. The detailed steps refer to the patent. Here, a brief description was introduced. Degummed silk was evenly spread into a net-like structure in the mold. The G was dissolved in deionized water so that the concentration of the G solution was 30%. The PLLA PMs were blended with SF solution and G solution. The weight of PMs in 15 ml blend was 0 or 200 mg. SF concentration was 3 mg ml⁻¹, and G concentration was 7%. Fifteen milliliters of the solution was injected into a mold containing degummed silk, and freeze-dried to obtain a composite scaffold material. The composite material was immersed in 1% carbodiimide (EDC) for 8 h, washed with water, and lyophilized to finally obtain a composite tissue engineering scaffold material with or without PLLA PMs.

Gold was sprayed on the S/SF/G and S/SF/G/PLLA-PMs scaffolds, and the surface of the scaffolds was observed under an acceleration voltage of 5 kV through a scanning electron microscope (SEM; Hitachi S3400, Japan). The diameter and pore size of PLLA-PMs were obtained by analyzing SEM images of microspheres.

The porosity of S/SF/G and S/SF/G/PLLA-PMs scaffolds was measured using an ethanol infiltration method [22]. The results were calculated as following:

$$\begin{equation*}P\% = V1 - V3/V2 - V3 \times 100\% \end{equation*}$$

where V1 is the known volume of ethanol before immersing lyophilized scaffold, V2 is the total volume of ethanol and immersed scaffold after the pores are completely immersed in ethanol, and V3 is the volume of the ethanol remaining in the tube after removing the scaffold.

Dry samples of scaffolds weighed W1 were incubated in 10 ml PBS (pH 7.4) at 37 °C for 24 h. After removing the unabsorbed solution on the surface of the scaffolds with filter paper, the weight of the wet samples was recorded as W2. The swelling fold of the scaffolds was calculated as following:

$$\begin{equation*}{\text{Swelling}}\,{\text{fold}} = w2 - w1/w1.\end{equation*}$$

Structural analysis of the scaffolds was performed using FTIR spectroscopy (6800–50/NEXUS, Nicolet, America) in the attenuated total reflection mode. Samples of S/SF/G and S/SF/G/PLLA-PMs scaffolds were prepared by freeze-drying. Transmittance readings were measured by scanning at a resolution of 0.09 cm⁻¹ in the 4000–500 cm spectral region.

A universal material testing machine (Measuring range: 0–5KN, accuracy: within ±0.5% of the indicated value, speed range: 0.001–500 mm min⁻¹) was used to evaluated the Young's Modulus of the S/SF/G and S/SF/G/PLLA-P-MPs scaffolds, in triplicate for each scaffold. The scaffolds were fabricated into samples with a cuboid (10 × 10 × 5 mm) and attached to the material testing machine. A compression load was applied at a compression speed of 1 mm min⁻¹ until failure was observed. Simultaneously, Young's Modulus were obtained.

Fresh ear cartilage was harvested from the ears of 4 week old pigs under sterile conditions, digested with 0.25% trypsin plus 0.02% EDTA at 37 °C for 60 min, and then cut into pieces of about 1 mm³. The cartilage pieces were washed with PBS (supplemented with 100 U ml⁻¹ penicillin and 100 mg ml⁻¹ streptomycin) three times and digested using 0.2% collagenase VI in serum-free DMEM at 37 °C for about 8–10 h. The digested tissue was filtered through a 200 mm nylon mesh and centrifuged at 1000 rpm for 10 min to obtain chondrocytes after removal of the supernatant. The chondrocytes were re suspended in DMEM supplemented with 15% FBS, 100 U ml⁻¹ penicillin, and 100 mg ml⁻¹ streptomycin and seeded in a culture flask at a density of 5 × 105 cells ml⁻¹. The culture flask was incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. The culture medium was refreshed every 2–3 d, and chondrocytes were subcultured when they reached 80%–90% confluence. Chondrocytes at passage 2 were used for the following experiments.

Prior to use, the scaffolds were sterilized by Co60 irradiation. Before inoculating chondrocytes, the S/SF/G and S/SF/G/PLLA-PMs scaffolds were sterilized and moistened. In short, the scaffolds were made into cube samples with a size of 0.7 × 0.7 × 1.5 mm. Then, the scaffolds were immersed in ethanol for 30 min, and finally washed three times with PBS for 5 min each time. The scaffolds were immersed in DMEM/High glucose supplemented with 10% FBS, 100 units ml⁻¹ penicillin, and 100 mg ml⁻¹ streptomycin overnight. Then absorbed culture medium. Subsequently, cell suspension with the concentration of 1 × 10⁸ ml⁻¹ was added to the scaffolds, about 100 μl/scaffold. Then the scaffolds were further incubated in an atmosphere of 5% CO₂/95% humidified air at 37 °C for 5 h to attach the cells. After the cells adhered to the scaffold for 5 h, 1–2 ml of culture medium was added to immerse the scaffold–cell construct. The culture medium was refreshed every 2–3 d.

The cytotoxicity tests of S/SF/G and S/SF/G/PLLA-PMs scaffolds were performed using the CCK-8 method. Initially, the scaffolds extract was prepared by soaking a sterile scaffold in DMEM supplemented with 10% FBS at 37 °C for 72 h. After the extraction was completed, the filter was used for sterilization, and the scaffold extract was used as the culture medium for the experimental group. Additionally, DMEM containing 10% FBS was utilized as the culture medium of control groups. The chondrocytes were seeded into the wells of a 96-well culture plate at a density of 3 × 103 cells/well, and then 100 μl culture media of the control or experimental group was added (n = 6 per group). The culture plate was transferred to the incubator, and chondrocytes were incubated in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. After culturing for 1, 3, 5, and 7 d, 10 μl CCK-8 solution was added to each well and incubated for 2.5 h. An enzyme labeling instrument (Model 680, Bio-Rad, USA) was used to measure absorbance at an excitation wavelength of 450 nm. The experiments were independently repeated in quintuple [23].

CCK-8 was used to measure the proliferation of the chondrocytes seeded on the scaffolds (1 × 10⁵ cells/scaffold). After incubation for 1 and 5 d, the cell-seeded scaffolds were relocated to a new 96-well plate (1 scaffold in 1 well) with 10 μl CCK-8 solution added. After incubating with CCK-8 at 37 °C for 2.5 h, the absorbance value of the scaffolds was detected at 450 nm by using an enzyme labeling instrument. The experiments were independently repeated in triplicate.

The viability of chondrocytes in the scaffolds was evaluated by using live/dead assay. Cells were fluorescently stained by using calcein-AM for live cells and propidium iodide (PI) for dead cells. Presoaked the scaffolds (7 × 7 × 1.5 mm³) in the culture medium in a confocal special Petri dishes for 30 min, and then seeded the chondrocytes into scaffolds (2 × 10⁵ cells/scaffold). After 1, 7 d, scaffolds were washed with PBS and stained with calcein-AM and PI for 30 min at room temperature. Scaffolds were washed again with PBS, and fluorescent images were acquired using the confocal microscope.

The cell morphology and extra cellular matrix distribution on the scaffolds were further studies by SEM observation. Briefly, at day 7 and 30 of cultures, the cell/scaffold constructs were washed twice with PBS, and then fixed in 2.5% glutaraldehyde for 4 h at room temperature. After rinsing with PBS three times, the samples were sequentially dehydrated with gradient ethanol and freeze-dried, and then sprayed with gold for SEM observation.

Cell-scaffold complexes (n = 12 per group) were constructed with pig auricular chondrocytes density of 6 × 10⁷ ml⁻¹ cells, nearly 200 μl onto each scaffold followed by 5 h incubation to promote cell adhesion [24, 25]. All the samples were statically cultured in DMEM/High glucose supplemented with 10% FBS, 100 units ml⁻¹ penicillin, and 100 mg ml⁻¹ streptomycin for 4 weeks in vitro. Medium was refreshed every 2–3 d.

Cartilage specific genes were further analyzed by RT-qPCR to evaluate the in vitro cartilage formation. The total ribonucleic acid (RNA) was extracted with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and reverse transcribed into complementary DNA (cDNA) using a real-time PCR kit (TransGen Biotech, Beijing, China). RT-qPCR was performed using the SYBR Green I PCR master mix kit (TAKARA, Beijing, China). The amplification protocol was carried out as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 1 min, and 72 °C for 10 min. The PCR primers for specific mouse genes are as follows: GAPDH(F:5'-CTGCCCCTTCTGCTGAT GC-3',R:5'-TCCACGATGCCGAAGTTGTC-3'), SRY (sex determining region Y)-BOX 9 (SOX9) (5'-ACTACACAGACCAGAACTCCG-3',R:5'-TGAAGG TGGAGTAGAGCACAGAGC-3'), collagen type II (COL2A1) (F:5'-TGGGCAGAGGTATAATGATAAGGA-3', R:5'-CTTCACAGATTATGTCGTCGCAG-3'), aggrecan (ACAN)(F:5'-TGGGAAAGAAGACTTGGC TGG-3', R:5'-CTCCACCAATGTAGTATCCACGA-3').

After 4 weeks, constructs (n = 12 per group) were autologously transplanted into individual subcutaneous pockets of each pig and harvested for analysis 2, and 8 weeks after implantation.

Samples from each group were harvested after 4 weeks in vitro and 2 or 8 weeks in vivo. The specimens were fixed with 10% buffered formalin in PBS for 24 h, embedded in paraffin, sectioned into 5 mm sections, the sections were stained with Hematoxylin-Eosin (HE) staining for histological analyses, and Safranin O-Fast Green Staining to visualize the glycosaminoglycans (GAG) deposits and type II collagen expression was detected by using a rabbit anti pig type II collagen monoclonal antibody followed by horseradish peroxidase-conjugated anti-rabbit antibody. Images of the type II collagen positive area were analyzed by using ImageJ (NIH), as reported in the published study [9].

4.1.1. Macroscopic appearance

The macroscopic appearance of S/SF/G and S/SF/G/PLLA-PMs scaffolds was shown without using microscopy, we found S/SF/G/PLLA-PMs scaffolds to be pure white, while S/SF/G scaffolds were yellowish-white, and white PLLA-PMs could be observed in S/SF/G/PLLA-PMs scaffolds (figure 1).

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4.1.2. Scanning electron microscopy

Herewith, the PLLA-PMs were prepared using the reported procedure, which contained both micro- and macro-interconnected pores (figure 2(a)). PLLA-PMs exhibited dense pore distribution. The mean particle size was from 100 to 300 μm; average 245 ± 35 μm, pore size was 20–40 μm, mean 27 ± 4.8 μm. We used SEM to study the internal structure of the S/SF/G and S/SF/G/PLLA-PMs scaffolds. The SEM images were presented in figures 2(b) and (c). Both the scaffolds had porous network structures and good connectivity between the pores. The pore sizes ranging from 80 to 600 μm, average 307 ± 142 μm. However, in S/SF/G/PLLA-PMs scaffolds, part of the pore structure was occupied by PLLA-PMs.

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The porosity was measured using the reported procedure. The porosity of the S/SF/G scaffolds was 77.00 ± 1.956%, while the porosity of S/SF/G/PLLA-PMs scaffolds was decreased to 67.25 ± 3.174% due to the corporation of PLLA-PMs (figure 3(a)).

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After incubating for 24 h, the swelling ratio of S/SF/G scaffold was 267.47 ± 11.93%, while the swelling ratio of S/SF/G/PLLA-PMs scaffold was decreased to 175.57 ± 9.61% due to the corporation of PLLA-PMs(figure 3(b)).

FITR spectra showed that FITR spectra showed that both scaffolds had peaks around 1620 ∼ 1640 cm⁻¹, 1520 ∼ 1540 cm⁻¹ respectively corresponding to amide I and amide II. These regions are the major characteristic peaks of the protein-based substances that define the scaffold composition. The characteristic peaks of PLLA at 1758.68 cm⁻¹ (stretching C = O), 1185.93 cm⁻¹(stretching –C–O–), 1089.20 cm⁻¹ (tension –C–O–) were observed in S/SF/G/PLLA-PMs scaffold, indicating that the characteristic functional group has not changed during the fabricating process of the scaffold (figure 4).

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The Mechanical tests showed Young's Modulus of S/SF/G and the S/SF/G/PLLA-PMs scaffold in dry condition was 2.304 ± 0.629 and 4.409 ± 0.325 respectively (figure 5(a)). In wet condition, the Young's modulus was 0.6471 ± 0.08358 and 1.452 ± 0.1343, respectively (figure 5(b)). Young's modulus was significantly greater in the S/SF/G/PLLA-PMs scaffold than in the S/SF/G scaffold, which indicated that PLLA-PMs could remarkably improve the mechanical properties of the S/SF/G scaffolds.

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Toxicity of the S/SF/G scaffolds and S/SF/G/PLLA-PMs scaffolds were tested by the CCK-8 assay. Chondrocytes were cultured at the leach liquors of scaffolds in the medium for 1, 3, and 5 d. It was observed that most of the cells (>90%) at each leach liquor grew well. The viability of the S/SF/G and the S/SF/G/PLLA-PMs composite scaffolds was not significant compared to the control group during the process of culture, indicating no significant cytotoxicity of both composite scaffolds. Thus, the S/SF/G and the S/SF/G/PLLA-PMs composite scaffolds resulted in excellent biocompatibility (figure 6(a)).

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The cell proliferation assay has shown that after 5 d of culture, the cell proliferation rate increased significantly. In the 1, 3, and 7 d of culture, cells seeded on the S/SF/G scaffolds generally seemed to have a higher proliferation rate than that of S/SF/G/PLLA-PMs scaffolds. But at day 5 the proliferation of S/SF/G/PLLA-PMs scaffolds were higher than that of the S/SF/G scaffolds though no statistically significant difference was found (figure 6(b)). Thus, addition of PLLA PMs did not significantly reduce cell proliferation.

Fluorescent images of live/dead cells showed that chondrocytes had good viability on both the scaffolds and only a few dead cells were observed in these scaffolds (figures 7(a) and (b)). It could appear that at day 1 chondrocytes were well attached on the surface of the two scaffolds (figures 7(e) and (f)). After 7 d culture, the number of cells has increased dramatically and cells on both scaffolds were still in a dispersed state (figures 7(c) and (d)). A few cells entering the PLLA-PMs could be seen after the scaffolds were developed, but most cells were attached to the surface of both scaffolds (figures 7(g) and (h)). The magnified image and 3D image show that the cells were attached to the surface of the microspheres at different depth (figure 7(i)).

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The SEM image of the cell morphology on the scaffold was shown in figure 8. After 7 d of culture, on the S/SF/G scaffolds the cells adhered well to the surface of the scaffold and they displayed round (figure 8(a)). On the S/SF/G/PLLA-PMs scaffold, cells did not adhere to the scaffold to the full extent, they gathered and clung to each other and maintained their original shape just like they were in vivo (figure 8(b)).

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With time, the cells grew more and spread all over the internal surface of S/SF/G scaffolds (figure 8(c)); instead, on the S/SF/G/PLLA-PMs scaffolds, cells were located both inside the PLLA-PMs and along the surface of the scaffolds (figure 8(d)). Cells located on both scaffolds have strong matrix secretion capacity (figures 8(e) and (f)).

Cartilage specific genes were analyzed by RT-qPCR to evaluate the cartilage formation after 4 weeks culture in vitro. According to the current results, there was no significant difference on expression levels of cartilage specific genes ACAN, COL2A1, SOX9 between S/SF/G scaffolds and S/SF/G/PLLA-PMs scaffolds (figure 9).

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At 4 weeks, HE staining revealed that a large number of cells were distributed on the scaffolds, cells gather together, and the extracellular matrix deposits initially formed. On S/SF/G/PLLA-PMs scaffolds, the cells were mainly distributed on the surface of the PMs, and few cells enter the interior of the PMs. (figures 10(a)–(d)) Immunohistochemical results showed that both the two scaffolds showed positive aggregation of type II collagen (figures 10(e)–(h)).

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Long-term stability of cartilage construction in vivo was a key factor in determining whether the scaffold material could be used in clinic. After 4 weeks cultures in vitro, all the samples (12/12) formed ivory-white cartilage-like tissue with elasticity (figures 11(a), (b), (e) and (f)). However, it was observed that ivory-white tissue structure was distributed in stripes along the S/SF/G scaffold while not seen in the S/SF/G/PLLA-PMs scaffold. After subcutaneous implantation at 2 and 8 weeks in the mini-pig model. Both samples shrunk to vary degrees over time. They adhered with the surrounding connective tissue. The boundary between harvested samples and surrounding tissues was not very clear and their original shape and size were changed to some degree (figures 11(c), (d), (g) and (h)).

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Histology showed that compared with S/SF/G scaffolds, S/SF/G/PLLA-PMs scaffolds had a more integrated structure. Both the S/SF/G and S/SF/G/PLLA-PMs scaffolds showed inflammatory reaction, such as lymphocyte aggregation, foreign body giant cell reaction (figures 12(a), (b), (e) and (f)). Safranin O staining showed obvious extracellular matrix deposition for 2 weeks culture in vivo, and a significant decrease in extracellular matrix deposition for 8 weeks. And S/SF/G/PLLA-PMs scaffold has more obvious extracellular matrix deposition than S/SF/G scaffold (figures 12(c), (d), (g), (h), (k), (l), (o) and (p)). Immunohistochemical staining showed intense staining of cartilage-specific collagen II after 2 weeks of transplantation in vivo. The PLLA-PMs were stained darker than the rest part of the scaffold, although statistics showed no significant difference in the ratio of the staining area between the two scaffolds (figures 13(a)–(d) and (i)). At week 8 post transplantation, the type II collagen staining of the S/SF/G and S/SF/G/PLLA-PMs scaffolds reduced significantly. However, compared with S/SF/G scaffold, the S/SF/G/PLLA-PMs scaffold was stained darker, indicating that the PLLA-PMs had a better effect of maintaining long-term stability of cartilage (figures 13(e)–(i)).

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