Osteogenic differentiation of encapsulated cells in dexamethasone–loaded phospholipid–induced silk fibroin hydrogels

doi:10.12336/biomatertransl.2022.03.005

Biomaterials Translational ›› 2022, Vol. 3 ›› Issue (3): 213-220.doi: 10.12336/biomatertransl.2022.03.005

• RESEARCH ARTICLE • Previous Articles Next Articles

Osteogenic differentiation of encapsulated cells in dexamethasone–loaded phospholipid–induced silk fibroin hydrogels

Chavee Laomeephol¹^,², Helena Ferreira³^,⁴, Sorada Kanokpanont²^,⁵^,⁶, Jittima Amie Luckanagul¹^,⁷, Nuno M Neves³^,⁴, Siriporn Damrongsakkul²^,⁵^,⁶^,^*()

1 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
2 Biomaterial Engineering for Medical and Health Research Unit, Chulalongkorn University, Bangkok, Thailand
3 3B’s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal
4 ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal
5 Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand
6 Biomedical Engineering Research Center, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand
7 Center of Excellence in Plant–produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand

Received:2022-08-02 Revised:2022-09-06 Accepted:2022-09-16 Online:2022-09-28 Published:2022-09-28
Contact: Siriporn Damrongsakkul E-mail:Siriporn.D@chula.ac.th
About author:Siriporn Damrongsakkul, Siriporn.D@chula.ac.th.

Abstract

Abstract:

The tissue engineering triad comprises the combination of cells, scaffolds and biological factors. Therefore, we prepared cell– and drug–loaded hydrogels using in situ silk fibroin (SF) hydrogels induced by dimyristoyl glycerophosphoglycerol (DMPG). DMPG is reported to induce rapid hydrogel formation by SF, facilitating cell encapsulation in the hydrogel matrix while maintaining high cell viability and proliferative capacity. In addition, DMPG can be used for liposome formulations in entrapping drug molecules. Dexamethasone (Dex) was loaded into the DMPG–induced SF hydrogels together with human osteoblast–like SaOS–2 cells, then the osteogenic differentiation of the entrapped cells was evaluated in vitro and compared to cells cultured under standard conditions. Calcium production by cells cultured in DMPG/Dex–SF hydrogels with Dex–depleted osteogenic medium was equivalent to that of cells cultured in conventional osteogenic medium containing Dex. The extended–release of the entrapped Dex by the hydrogels was able to provide a sufficient drug amount for osteogenic induction. The controlled release of Dex was also advantageous for cell viability even though its dose in the hydrogels was far higher than that in osteogenic medium. The results confirmed the possibility of using DMPG–induced SF hydrogels to enable dual cell and drug encapsulation to fulfil the practical applications of tissue–engineered constructs.

Key words: DMPG, osteogenic differentiation, silk fibroin, three–dimensional cell culture, tissue engineering

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Figures/Tables 4

Figure 1. (A) Elastic modulus of the 5, 10 and 15 mM DMPG–3% (w/v) SF hydrogels determined by unconfined compression (mean ± SD, n = 10). *P ≤ 0.05 (one–way analysis of variance followed by Bonferroni post–hoc tests). (B–D) Compressive stress–strain plots of 5 (B), 10 (C), and 15 (D) mM DMPG–3% (w/v) SF hydrogels. DMPG: dimyristoyl glycerophosphoglycerol; SF: silk fibroin.

Figure 2. (A) Diffusivity of fetal bovine serum from the DMPG–SF hydrogels, determined from the protein released into the supernatants using Bradford protein assay. (B) Release profile of dexamethasone from 5, 10 and 15 mM DMPG/dexamethasone–SF hydrogels (containing 0.625, 1.25 and 1.875 mM dexamethasone, respectively) in 0.01 M phosphate–buffered saline (pH 7.4). Data are expressed as mean ± SD, and were analysed by one–way analysis of variance followed by Bonferroni post–hoc tests. The experiment was repeated three times. DMPG: dimyristoyl glycerophosphoglycerol; SF: silk fibroin.

Figure 3. The number of SaOS–2 cells entrapped within DMPG–SF hydrogels cultured under different conditions. Group 1: cell–loaded DMPG–SF hydrogels cultured in proliferation medium; Group 2: cell–loaded DMPG–SF hydrogels cultured in osteogenic medium; Group 3: cell–loaded DMPG/Dex–SF hydrogels cultured in proliferation medium; Group 4: cell–loaded DMPG/Dex–SF hydrogels cultured in Dex–depleted osteogenic medium. Cell number was determined using Hoechst 33258 DNA quantification assay. Data are expressed as mean ± SD, and were analysed by one–way analysis of variance followed by Bonferroni post–hoc tests. The experiment was repeated three times. Dex: dexamethasone; DMPG: dimyristoyl glycerophosphoglycerol; SF: silk fibroin.

Figure 4. Osteogenic differentiation of encapsulated SaOS–2 cells in DMPG– or DMPG/Dex–SF hydrogels. Group 1: cell–loaded DMPG–SF hydrogels cultured in proliferation medium; Group 2: cell–loaded DMPG–SF hydrogels cultured in osteogenic medium; Group 3: cell–loaded DMPG/Dex–SF hydrogels cultured in proliferation medium; Group 4: cell–loaded DMPG/Dex–SF hydrogels cultured in Dex–depleted osteogenic medium. (A) Alkaline phosphatase activity of the encapsulated cells, represented as the concentration of nitrophenol standard normalised to the number of cells. (B) Calcium content deposited in the hydrogels. Data are expressed as mean ± SD. The experiment was repeated three times. *P ≤ 0.05 (one–way analysis of variance followed by Bonferroni post–hoc tests). (C) Appearance of the SaOS–2 cells encapsulated in hydrogels and cultured under different conditions at each time point. The opacity of Groups 2 and 4 hydrogels can be seen, implying high calcium deposition in the samples. (D) Microscopic analysis of alizarin red staining of calcium deposition within the cell–entrapped hydrogels cultured for 7 days. The orange–red stain indicates calcium mineralisation. Scale bars: 50 μm. DMPG: dimyristoyl glycerophosphoglycerol; Dex: dexamethasone; SF: silk fibroin.

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Osteogenic differentiation of encapsulated cells in dexamethasone–loaded phospholipid–induced silk fibroin hydrogels

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