Osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells encapsulated in Thai silk fibroin/collagen hydrogel: a pilot study in vitro

Protocols using animals were approved by the Ethics Committee for Animal Care and Use of Chulalongkorn University (certificate of approval No. 06/2560) following The Animals for Scientific Purposes Act, BE 2558 (AD 2015), which regulates the use of both vertebrates and invertebrates, and conducted under license for Animals for Scientific Purposes from the Institute for Animals for Scientific Purpose Development and National Research Council of Thailand. MSCs were isolated from femurs of 3-week-old female Wistar rats obtained from a specialist supplier (. The femurs were dissected and cleaned with phosphate-buffered saline (PBS) before being cut at the proximal and distal ends. The bone marrow was extracted using a 24-G needle fitted to a syringe containing 1 mL of α-modified Eagle’s medium (α-MEM) supplemented with 15% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The procedure was repeated until all of the bone marrow was harvested, and then the tissue was triturated by repeated aspiration through a 24-G needle. The resulting cell suspension was placed into a 75-cm² tissue culture flask containing 10 mL of medium and cultured in an incubator at 37°C, under an atmosphere of 5% CO₂ in air. The medium was changed on the fourth day and every 3 days thereafter [18].

When 80%–90% confluence was reached, the cells were released from the dish by treatment with trypsin–ethylene diamine tetraacetic acid (EDTA) and subcultured into a new set of culture dishes at 5 × 10⁴ cells/cm² on the first passage. The cells from the first passage were again subcultured before they became confluent, and the cells from the second passage were used for experiments.

Pupae-free Thai silk cocoons from Bombyx mori (Nangnoi-Srisaket 1) from the Queen Sirikit Sericulture Center (Nakhon Ratchasima province, Thailand) were used to prepare sterile aqueous SF solution by a method modified from Ratanavaraporn et al. [19]. About 40 g of the pupae-free Thai silk cocoons was added to 1 L of boiling 0.02 M sodium carbonate (Na₂CO₃) solution for 20 min. The fibers were then rinsed thoroughly with deionized water (DI) to remove sticky gum protein glue, sericin, from the fibroin filaments. The degummed silk fibers were air dried at room temperature. Then dried fibers were sterilized by autoclaving at 121°C for 15 min. The sterilized fibers were dissolved in 9.3 M LiBr at 60°C for 4 h with 4 g:16 mL silk:LiBr. The solution was dialyzed against sterile DI water for 48 h to remove the LiBr. The SF solution obtained was then centrifuged at 9,000 rpm at 4°C for 20 min to remove insoluble parts. The final concentration of regenerated aqueous SF solution was about 5.5%–6.0% wt. All equipment was sterilized before use, and the whole process was performed under sterile conditions. The sterility of aqueous SF solution was proved by microbiological attribution tests including a total plate count and total yeast and molds [10].

Hydrogels were prepared from sterile SF solution (4% wt) with or without sterile collagen solution (Nitta Gelatin) and allocated to 3 groups: group 1, hydrogels made of 4% SF solution only (SF hydrogel), group 2, hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and group 3, hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). The gelation was induced by the addition of surfactants (poloxamer-188:oleic acid at 0.07:0.93 by weight). Then each sample was mixed on a vortex mixer for 90 s before incubation at 37°C for 30–45 min.

The cells treated with trypsin and resuspended in α-MEM to obtain a cell density of 5 × 10⁶ cell/mL. Then 100 ml of the cell suspension was added and mixed with the vortexed silk solution giving a final concentration of 5 × 10⁵ cells/mL. An aliquot of 0.5 mL of the mixtures was quickly pipetted into a stainless-steel ring placed in a 24-well cell culture plate and incubated at 37°C, under an atmosphere of 5% CO₂ in air. A hydrogel formed within 45–60 min. Then the rings were removed to obtain cylindrical-shaped gels with 12 mm diameter and 5 mm thick.

All samples were then cultured in 2 mL of growth medium containing α-MEM supplemented with 15% FBS and 1% penicillin–streptomycin at 37°C under an atmosphere of 5% CO₂ in air. The medium was refreshed every 2 days. The number of cells was analyzed by DNA assay at 6 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days. The hydrogel was minced and added to 1 mL sodium dodecyl sulfate (SDS) lysis buffer and incubated at 37°C under an atmosphere of 5% CO₂ in air for 1 h to disrupt cell membranes. The obtained cell lysates were then centrifuged to separate supernatants. About 100 µL of supernatant was pipetted into a 96-well black plate, and then Hoechst 33258 reagent (20 µL Hoechst solution + 19 mL DI + 1 mL saline-sodium citrate) was added. The fluorescent intensity of the solution at 355 nm (excitation) and 460 nm (emission) was measured immediately using a microplate reading fluorometer and compared with a standard curve indicating cell numbers. The efficiency of rat MSC encapsulation was calculated at various times from 6 h.

Rat MSCs (1 × 10⁶ cells/mL) were cultured in osteogenic induction medium consisting of α-MEM supplemented with 10% FBS, 1% penicillin–streptomycin, 50 µg/mL l-ascorbic acid, 10^–8 M dexamethasone, and 10^–2 M b-glycerol phosphate and then incubated at 37°C under an atmosphere of 5% CO₂ in air. The medium was changed twice a week. The osteogenic differentiation of MSC was determined by DNA assay, alkaline phosphatase (ALP) activity assay, and calcium assay at 3 days, 7 days, 14 days, 21 days, 28 days, and 35 days.

Minced hydrogel was added to 1 mL of SDS and incubated at 37°C under an atmosphere of 5% CO₂ in air for 1 h to disrupt cell membranes and then 20 µL samples pipetted into wells of a 96-well plate. p‑Nitrophenyl phosphate (100 µL) was added to the cell lysates in each well and then together incubated at 37°C under an atmosphere of 5% CO₂ for 15 min. The reaction was stopped with 80 µL of 0.02 N NaOH, and p-nitrophenol, which is a product of hydrolysis of p-nitrophenol phosphate catalyzed by ALP, was measured using a microplate reading spectrophotometer at 405 nm [20].

Minced hydrogel was added to 1 mL SDS and incubated at 37°C under an atmosphere of 5% CO₂ in air for 1 h to disrupt cell membranes and then 100 µL samples pipetted into wells of a 48-well plate. 1 M HCl (100 µL) was added to the cell lysates and the mixture incubated at 37°C under an atmosphere of 5% CO₂ in air for 4 h to extract calcium. Aliquots (10 ml) of the cell lysate extracts were pipetted into wells of a 48-well plate. Ethanolamine buffer (1 mL of 0.88 M) and o-cresolphthalein complex substrate (OCPC) (100 ml of 0.63 M) were mixed together with the sample in each well of the plates. The reaction between OCPC and calcium yielded calcium-o-cresolphthalein complex measured using a microplate reading spectrophotometer at 570 nm [21].

After rat MSCs had been cultured in hydrogels for 6 weeks, hydrogels were cut into slices and dehydrated in a graded series of ethanol concentrations before drying with a critical point dryer. The dried specimens were sputtered with gold using a fine coater. The specimens were then examined using digital scanning electron microscopy (SEM; JSM-6610LV; Jeol). To determine the elemental composition of the specimens, the energy dispersive spectroscopy (EDS; 10–30 kV X-Max^N, Oxford Instruments) spectrum was plotted and analyzed.

All experiments were performed in triplicate. The results are reported as mean ± standard deviation (SD). Hydrogels were compared using a one-way analysis of variance (ANOVA) with a post hoc least significant difference (LSD) test. The level of significance was set at P < 0.05.

The results of the DNA assay at 6 h revealed that the encapsulation efficiency of rat MSCs in SF hydrogels was 18.20 ± 3.21 (×10⁴ cells), in SF/0.05C hydrogels was 14.03 ± 0.06 (×10⁴ cells), and in SF/0.1C hydrogels was 10.74 ± 3.24 (×10⁴ cells). Rat MSCs proliferated on all hydrogels in the first 24 h, and then cell numbers declined before reaching plateau phase at 5 days. Subsequently, the number of cells encapsulated was maintained during days 5–14 of culture. The MSC cultured on SF/0.1C hydrogel exhibited the lowest encapsulation efficiency and cell numbers during day 3–5, but when the cultured cells reach plateau phase during days 7–14, there were no significant differences in cell numbers among the various hydrogels (Figure 1).

Figure 1

At 21 days, the cell proliferation in SF/0.1C hydrogels was significantly greater than in SF hydrogels. We found were no significant differences observed between the 3 groups of hydrogels at other times (Figure 2).

Figure 2

After 1 week of culture, ALP was found increased in MSCs cultured in all hydrogels. Then the ALP level in SF/0.1C hydrogel decreased, whereas in SF and SF/0.05C hydrogels, the activity continued increasing to the highest level at 3 weeks (Figure 3).

Figure 3

Calcium level in SF and SF/0.05C hydrogels was significantly higher in SF/0.1C hydrogel at 21 days, but no differences were seen in other time points (Figure 4).

Figure 4

SEM analysis revealed random distribution of osteoblast-like cells within the pores of scaffold in all hydrogels. Nevertheless, the culture in SF/0.1C hydrogel showed most abundant fibril-like matrix, whereas the SF hydrogel exhibited least matrix amount (Figure 5). EDS analysis showed the presented amount of calcium level in the following order: SF/0.05C > SF > SF/0.1C. The Ca/P ratios of SF, SF/0.05C, and SF/0.1C were 4.28 ± 2.38, 2.91 ± 1.09, and 3.61 ± 1.75, respectively (Table 1).

Figure 5