1. bookVolume 12 (2018): Edition 6 (December 2018)
Asian Biomedicine's Cover Image
Asian Biomedicine
Détails du magazine
License
Format
Magazine
eISSN
1875-855X
Première parution
01 Jun 2007
Périodicité
6 fois par an
Langues
Anglais
Accès libre

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

Jirun Apinun,
Jirun Apinun,
Sittisak Honsawek,
Sittisak Honsawek,
Somsak Kuptniratsaikul,
Somsak Kuptniratsaikul,
Jutarat Jamkratoke et
Jutarat Jamkratoke et
Sorada Kanokpanont
Sorada Kanokpanont
Publié en ligne: 24 Oct 2019
Volume & Edition: Volume 12 (2018) - Edition 6 (December 2018)
Pages: 273 - 279
Asian Biomedicine's Cover Image
Asian Biomedicine
Détails du magazine
License
Format
Magazine
eISSN
1875-855X
Première parution
01 Jun 2007
Périodicité
6 fois par an
Langues
Anglais

Hydrogels are cross-linked hydrophilic polymer networks that can contain a high amount of water. Their hydrated network architecture provides a place for cells to adhere, proliferate, and differentiate. They are generally nontoxic and biocompatible. In tissue engineering, hydrogels can be used to encapsulate living cells as a cell delivery system and as a scaffold for tissue regeneration. Moreover, hydrogels can be delivered to a target site in a minimally invasive manner. Nevertheless, the gelling process must be benign for cell survival and to avoid damage to loaded cells [1].

Silk fibroin (SF), the structural protein in natural silkworm fibers, is a naturally derived polymer. Attributes of silk include good biocompatibility and slow biodegradability. In tissue engineering, silk has been widely studied in the design of various forms of scaffolds for regeneration of various tissues [2, 3]. SF can be processed into a hydrogel using time controlled chemical and physical methods, and makes it appropriate for the development of injectable in situ-forming hydrogels for bone tissue engineering [4, 5, 6]. Gelation of SF can be induced by surfactant and vortexing [7, 8, 9]. To apply in situ-forming SF hydrogel as a cell carrier in bone defect treatment, a suitable processing time is essential. SF gelation can be induced in 45–60 min by adding surfactants composed of a mixture of oleic acid and poloxamer-188 (poloxamer-188:oleic acid at 0.07:0.93 by weight), which is expected to be appropriate to prepare hydrogels to encapsulate cells, delivery to target sites, and set the gels in situ [10].

Collagen is a major protein in the extracellular matrix and plays an important role in supporting and strengthening the structural integrity of connective tissue. Due to its biocompatibility, relative lack of immunogenicity, nontoxicity, and promotion of cell adhesion, it is a promising biomaterial for tissue engineering.

Scaffolds made of combined SF and collagen have been developed to attain benefits of bioactivity from collagen and mechanical properties from the porous structure of SF. Various designs of SF/collagen scaffold have been explored for potential tissue engineering of various tissues [11, 12, 13, 14, 15, 16]. Moreover, enhanced biomineralization has been demonstrated in coexistence with collagen and SF-derived polypeptide [17]. Therefore, hydrogels developed from a combination of collagen and SF could be expected to be beneficial in bone tissue engineering.

The aim of the present study was to evaluate the biocompatibility and osteogenic potential of rat mesenchymal stem cells (MSCs) encapsulated in injectable surfactant-induced Thai SF hydrogel supplemented with various concentrations of collagen compared with Thai SF hydrogel without added of collagen in cell culture in vitro.

Materials and methods
Cell isolation

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.

Scaffold preparation and cell encapsulation

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.

Cell viability and proliferation

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.

Osteogenic differentiation

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.

ALP activity assay

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].

Calcium assay

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].

Scanning electron microscopy and energy dispersive spectroscopy

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-MaxN, Oxford Instruments) spectrum was plotted and analyzed.

Statistical evaluation

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.

Results
Cell viability and proliferation

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

Growth kinetics of rat mesenchymal stem cells cultured in hydrogels. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). Error bars are standard deviations of triplicate experiments.

Osteogenic differentiation

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

DNA assay of rat mesenchymal stem cells cultured in hydrogels in osteogenic induction medium. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). *P < 0.05 (one-way analysis of variance with a post hoc least significant difference test). Error bars are standard deviations of triplicate experiments.

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

Alkaline phosphatase (ALP) activity of rat mesenchymal stem cells cultured on hydrogels in osteogenic induction medium. Units indicate mM p-nitrophenol produced per min per cell. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). Error bars are standard deviations of triplicate experiments.

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

Calcium content of rat mesenchymal stem cells cultured on hydrogels in osteogenic induction medium. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). *P < 0.05 (one-way analysis of variance with a post hoc least significant difference test). Error bars are standard deviations of triplicate experiments.

SEM and EDS

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

SEM images of rat MSCs cultured in hydrogels under different magnifications. SF (hydrogel made of 4% silk fibroin (SF) solution only, SF/0.05C (hydrogel made of 4% SF solution + 0.05% collagen solution), and SF/0.1C (hydrogel made of 4% SF solution + 0.1% collagen solution).

Elemental analysis result by EDS

HydrogelsC (% wt)O (% wt)Ca (% wt)P (% wt)Ca/P
SF45.03 ± 5.5637.61 ± 3.7513.99 ± 1.684.03 ± 1.814.28 ± 2.38
SF/0.05C39.36 ± 2.9633.99 ± 10.5619.27 ± 7.137.38 ± 3.372.91 ± 1.09
SF/0.1C45.92 ± 10.0038.96 ± 3.1110.86 ± 6.044.27 ± 4.373.61 ± 1.75
Discussion

Development of an injectable hydrogel made of Thai SF and collagen theoretically provides benefits from bioactivity of collagen and mechanical properties with porous structure from SF, which are suitable for the use in bone tissue engineering. Inducing an appropriate gelation time for this hydrogel for proper cell loading and delivery without toxicity or damage to loaded cells is essential. In the present study, we controlled the gelation time of SF solution to be 45–60 min by adding a surfactant composed of a mixture of oleic acid and poloxamer-188 and vortexing [10]. Combining these two methods created shorter gelation time using less surfactant and a shorter vortexing time. This probably resulted in a more benign milieu for survival and differentiation of the loaded cells than using each gel induction method separately.

The viability and proliferation of encapsulated rat MSCs demonstrated in the present study verified the biocompatibility of SF and combined SF/collagen hydrogel scaffolds. Even though the SF hydrogel with collagen seems to have less encapsulation efficiency for rat MSCs, the growth at the plateau phase of all hydrogels was comparable. We observed that cell viability declined during days 1–5. Cell numbers then plateaued and were maintained until day 14 of culture. This indicated a balance between apoptotic cell death and proliferation similar as found in culture of human MSCs in a chitosan– hyaluronan-based hydrogel in vitro [22]. The cell density at plateau phase was approximately 20%–30% of loaded cells. The viability of rat MSCs in SF and SF/collagen hydrogels presented in the present study was lower than that reported for other hydrogels [23, 24, 25]. Factors contributing to loss of viability of rat MSCs in SF and SF/collagen hydrogels may include cell damage from surfactants, limited cell distribution in the hydrogel, inadequate mass transport, or partial degradation of the hydrogel.

The highest ALP activity and calcium content detected in rat MSCs cultured in SF and SF/0.05C hydrogels was at 3 weeks significantly demonstrated the potential of the gels to allow induction of osteogenic differentiation of encapsulated rat MSCs in induction medium even with the apparent initial cell loss and the low proliferation. The rat MSC growth kinetics in the induction medium revealed the same characteristics as those in the growth medium. The cell numbers decreased in the first week before maintaining their level in the second week. From the third week, there was greater proliferation of rat MSCs in SF/0.1C than in SF and SF/0.05C hydrogels. By contrast, SF/0.1C showed less ALP activity and calcium content, indicating less osteogenic differentiation. According to these findings, SF/0.1C appeared to express a greater promotional effect on proliferation than differentiation than the SF and SF/0.05C hydrogels. This finding does not support the proposal of enhanced biomineralization with combined SF and collagen in hydrogels. We postulate that in blended SF and collagen hydrogels, not only the biological activity of collagen results in varied osteogenic differentiation in each hydrogel, but also the physicochemical properties of the hydrogels, such as density, stiffness, hydrophobicity, gel stability, and water absorption, are altered due to the addition of collagen, and must also be taken into account. Further studies on pre- and postencapsulated rat MSCs in hydrogels are warranted.

Comparable distributions of osteoblast-like cells within the pores of the scaffold were observed by SEM of each hydrogel group. However, a greater amount of matrix was noted in SF hydrogels supplemented with collagen. This finding indicates the beneficial effect of collagen when inducing matrix formation.

Calcium content analyzed by EDS was highest in the SF/0.05C hydrogel, followed by that in the SF hydrogel, and was lowest in SF/0.1C. This observation appeared to be correlated with the results of ALP activity and calcium assay, which does not meet the expectation of facilitated mineralization in combined SF and collagen hydrogel. Compared with the Ca/P ratio of hydroxyapatite and calcium phosphate (1.67–2.0) [17], the Ca/P ratio found in this study was around 2.9–4.3, suggesting that other forms of calcium compounds presented in the hydrogels during the osteogenic stimulus.

Conclusion

The injectable Thai SF and SF/collagen hydrogel scaffolds induced by surfactant demonstrated biocompatibility in cell encapsulation and the potential for allowing osteogenic differentiation of rat MSCs. Collagen combined in the hydrogel yielded a positive effect on proliferation and matrix formation; however, benefits on osteogenic differentiation and biomineralization were not demonstrated as expected. We conclude that these hydrogels could be used as scaffolds to promote bone regeneration in situ. However, further study of their physicochemical properties, biodegradation, and more information regarding their ability to allow or promote osteogenic differentiation is suggested to obtain a better understanding to optimize this system as a scaffold and cell carrier in bone tissue engineering.

Figure 1

Growth kinetics of rat mesenchymal stem cells cultured in hydrogels. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). Error bars are standard deviations of triplicate experiments.
Growth kinetics of rat mesenchymal stem cells cultured in hydrogels. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). Error bars are standard deviations of triplicate experiments.

Figure 2

DNA assay of rat mesenchymal stem cells cultured in hydrogels in osteogenic induction medium. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). *P < 0.05 (one-way analysis of variance with a post hoc least significant difference test). Error bars are standard deviations of triplicate experiments.
DNA assay of rat mesenchymal stem cells cultured in hydrogels in osteogenic induction medium. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). *P < 0.05 (one-way analysis of variance with a post hoc least significant difference test). Error bars are standard deviations of triplicate experiments.

Figure 3

Alkaline phosphatase (ALP) activity of rat mesenchymal stem cells cultured on hydrogels in osteogenic induction medium. Units indicate mM p-nitrophenol produced per min per cell. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). Error bars are standard deviations of triplicate experiments.
Alkaline phosphatase (ALP) activity of rat mesenchymal stem cells cultured on hydrogels in osteogenic induction medium. Units indicate mM p-nitrophenol produced per min per cell. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). Error bars are standard deviations of triplicate experiments.

Figure 4

Calcium content of rat mesenchymal stem cells cultured on hydrogels in osteogenic induction medium. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). *P < 0.05 (one-way analysis of variance with a post hoc least significant difference test). Error bars are standard deviations of triplicate experiments.
Calcium content of rat mesenchymal stem cells cultured on hydrogels in osteogenic induction medium. Squares indicate hydrogels made of 4% silk fibroin (SF) solution only (SF hydrogel), circles indicate hydrogels made of 4% SF solution + 0.05% collagen solution (SF/0.05C hydrogel), and triangles indicate hydrogels made of 4% SF solution + 0.1% collagen solution (SF/0.1C hydrogel). *P < 0.05 (one-way analysis of variance with a post hoc least significant difference test). Error bars are standard deviations of triplicate experiments.

Figure 5

SEM images of rat MSCs cultured in hydrogels under different magnifications. SF (hydrogel made of 4% silk fibroin (SF) solution only, SF/0.05C (hydrogel made of 4% SF solution + 0.05% collagen solution), and SF/0.1C (hydrogel made of 4% SF solution + 0.1% collagen solution).
SEM images of rat MSCs cultured in hydrogels under different magnifications. SF (hydrogel made of 4% silk fibroin (SF) solution only, SF/0.05C (hydrogel made of 4% SF solution + 0.05% collagen solution), and SF/0.1C (hydrogel made of 4% SF solution + 0.1% collagen solution).

Elemental analysis result by EDS

HydrogelsC (% wt)O (% wt)Ca (% wt)P (% wt)Ca/P
SF45.03 ± 5.5637.61 ± 3.7513.99 ± 1.684.03 ± 1.814.28 ± 2.38
SF/0.05C39.36 ± 2.9633.99 ± 10.5619.27 ± 7.137.38 ± 3.372.91 ± 1.09
SF/0.1C45.92 ± 10.0038.96 ± 3.1110.86 ± 6.044.27 ± 4.373.61 ± 1.75

Nicodemus GD, Bryant SJ. Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng Part B Rev. 2008; 14:149–65.NicodemusGDBryantSJCell encapsulation in biodegradable hydrogels for tissue engineering applicationsTissue Eng Part B Rev2008141496510.1089/ten.teb.2007.0332Search in Google Scholar

Thitiwuthikiat P, Ii M, Saito T, Asahi M, Kanokpanont S, Tabata Y. A vascular patch prepared from Thai silk fibroin and gelatin hydrogel incorporating simvastatin-micelles to recruit endothelial progenitor cells. Tissue Eng Part A. 2015; 21:1309–19.ThitiwuthikiatPIiMSaitoTAsahiMKanokpanontSTabataYA vascular patch prepared from Thai silk fibroin and gelatin hydrogel incorporating simvastatin-micelles to recruit endothelial progenitor cellsTissue Eng Part A20152113091910.1089/ten.tea.2014.0237Search in Google Scholar

Vorrapakdee R, Kanokpanont S, Ratanavaraporn J, Waikakul S, Charoenlap C, Damrongsakkul S. Modification of human cancellous bone using Thai silk fibroin and gelatin for enhanced osteoconductive potential. J Mater Sci Mater Med. 2013; 24:735–44.VorrapakdeeRKanokpanontSRatanavarapornJWaikakulSCharoenlapCDamrongsakkulSModification of human cancellous bone using Thai silk fibroin and gelatin for enhanced osteoconductive potentialJ Mater Sci Mater Med2013247354410.1007/s10856-012-4830-0Search in Google Scholar

Floren M, Migliaresi C, Motta A. Processing techniques and applications of silk hydrogels in bioengineering. J Funct Biomater. 2016; 7:pii:E26. doi: 10.3390/jfb7030026FlorenMMigliaresiCMottaAProcessing techniques and applications of silk hydrogels in bioengineeringJ Funct Biomater20167piiE26doi10.3390/jfb7030026Ouvrir le DOISearch in Google Scholar

Wang HY, Zhang YQ. Processing silk hydrogel and its applications in biomedical materials. Biotechnol Prog. 2015; 31:630–40.WangHYZhangYQProcessing silk hydrogel and its applications in biomedical materialsBiotechnol Prog2015316304010.1002/btpr.2058Search in Google Scholar

Rockwood DN, Preda RC, Yücel T, Wang X, Lovett ML, Kaplan DL. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc. 2011; 6:1612–31.RockwoodDNPredaRCYücelTWangXLovettMLKaplanDLMaterials fabrication from Bombyx mori silk fibroinNat Protoc2011616123110.1038/nprot.2011.379Search in Google Scholar

Kang GD, Nahm JH, Park JS, Moon JY, Cho CS, Yeo JH. Effects of poloxamer on the gelation of silk fibroin. Macromol Rapid Commun. 2000; 21:788–91.KangGDNahmJHParkJSMoonJYChoCSYeoJHEffects of poloxamer on the gelation of silk fibroinMacromol Rapid Commun2000217889110.1002/1521-3927(20000701)21:11<788::AID-MARC788>3.0.CO;2-XSearch in Google Scholar

Wu X, Hou J, Li M, Wang J, Kaplan DL, Lu S. Sodium dodecyl sulfate-induced rapid gelation of silk fibroin. Acta Biomater. 2012; 8:2185–92.WuXHouJLiMWangJKaplanDLLuSSodium dodecyl sulfate-induced rapid gelation of silk fibroinActa Biomater2012821859210.1016/j.actbio.2012.03.007Search in Google Scholar

Yücel T, Cebe P, Kaplan DL. Vortex-induced injectable silk fibroin hydrogels. Biophys J. 2009; 97:2044–50.YücelTCebePKaplanDLVortex-induced injectable silk fibroin hydrogelsBiophys J20099720445010.1016/j.bpj.2009.07.028Search in Google Scholar

Apinun J, Yamdech R, Damrongsakkul S, Jamkratoke J, Kanokpanont S. Viability of rat’s bone marrow-derived mesenchymal stem cells in a surfactant-induced Thai silk fibroin hydrogel. In: Biomedical Engineering International Conference: BMEiCON 2017: Proceedings of the 10th Biomedical Engineering International Conference; 2017 31 Aug – 2 Sept; Hokkaido, Japan.ApinunJYamdechRDamrongsakkulSJamkratokeJKanokpanontSViability of rat’s bone marrow-derived mesenchymal stem cells in a surfactant-induced Thai silk fibroin hydrogelInBiomedical Engineering International Conference: BMEiCON 2017: Proceedings of the 10th Biomedical Engineering International Conference201731 Aug – 2 SeptHokkaido, Japan10.1109/BMEiCON.2017.8229144Search in Google Scholar

Ghezzi CE, Marelli B, Donelli I, Alessandrino A, Freddi G, Nazhat SN. The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen-silk fibroin construct. Biomaterials. 2014; 35:6236–47.GhezziCEMarelliBDonelliIAlessandrinoAFreddiGNazhatSNThe role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen-silk fibroin constructBiomaterials20143562364710.1016/j.biomaterials.2014.04.040Search in Google Scholar

Ghezzi CE, Marelli B, Donelli I, Alessandrino A, Freddi G, Nazhat SN. Multilayered dense collagen-silk fibroin hybrid: a platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineages. J Tissue Eng Regen Med. 2015; 11:2046–59.GhezziCEMarelliBDonelliIAlessandrinoAFreddiGNazhatSNMultilayered dense collagen-silk fibroin hybrid: a platform for mesenchymal stem cell differentiation towards chondrogenic and osteogenic lineagesJ Tissue Eng Regen Med20151120465910.1002/term.2100Search in Google Scholar

Long K, Liu Y, Li W, Wang L, Liu S, Wang Y, Ren L. Improving the mechanical properties of collagen-based membranes using silk fibroin for corneal tissue engineering. J Biomed Mater Res A. 2015; 103:1159–68.LongKLiuYLiWWangLLiuSWangYRenLImproving the mechanical properties of collagen-based membranes using silk fibroin for corneal tissue engineeringJ Biomed Mater Res A201510311596810.1002/jbm.a.35268Search in Google Scholar

Shen Y, Redmond SL, Papadimitriou JM, Teh BM, Yan S, Wang Y, et al. The biocompatibility of silk fibroin and acellular collagen scaffolds for tissue engineering in the ear. Biomed Mater. 2014; 9:015015. doi: 10.1088/1748-6041/9/1/015015ShenYRedmondSLPapadimitriouJMTehBMYanSWangYet alThe biocompatibility of silk fibroin and acellular collagen scaffolds for tissue engineering in the earBiomed Mater20149015015doi10.1088/1748-6041/9/1/015015Ouvrir le DOISearch in Google Scholar

Wang G, Hu X, Lin W, Dong C, Wu H. Electrospun PLGA-silk fibroin-collagen nanofibrous scaffolds for nerve tissue engineering. In Vitro Cell Dev Biol Anim. 2011; 47:234–40.WangGHuXLinWDongCWuHElectrospun PLGA-silk fibroin-collagen nanofibrous scaffolds for nerve tissue engineeringIn Vitro Cell Dev Biol Anim2011472344010.1007/s11626-010-9381-4Search in Google Scholar

Wei G, Li C, Fu Q, Xu Y, Li H. Preparation of PCL/silk fibroin/collagen electrospun fiber for urethral reconstruction. Int Urol Nephrol. 2015; 47:95–9.WeiGLiCFuQXuYLiHPreparation of PCL/silk fibroin/collagen electrospun fiber for urethral reconstructionInt Urol Nephrol20154795910.1007/s11255-014-0854-3Search in Google Scholar

Marelli B, Ghezzi CE, Alessandrino A, Barralet JE, Freddi G, Nazhat SN. Silk fibroin derived polypeptide-induced biomineralization of collagen. Biomaterials. 2012; 33:102–8.MarelliBGhezziCEAlessandrinoABarraletJEFreddiGNazhatSNSilk fibroin derived polypeptide-induced biomineralization of collagenBiomaterials201233102810.1016/j.biomaterials.2011.09.039Search in Google Scholar

Vachiraroj N, Ratanavaraporn J, Damrongsakkul S, Pichyangkura R, Banaprasert T, Kanokpanont S. A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes. Int J Biol Macromol. 2009; 45:470–7.VachirarojNRatanavarapornJDamrongsakkulSPichyangkuraRBanaprasertTKanokpanontSA comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutesInt J Biol Macromol200945470710.1016/j.ijbiomac.2009.07.010Search in Google Scholar

Ratanavaraporn J, Kanokpanont S, Damrongsakkul S. The development of injectable gelatin/silk fibroin microspheres for the dual delivery of curcumin and piperine. J Mater Sci Mater Med. 2014; 25:401–10.RatanavarapornJKanokpanontSDamrongsakkulSThe development of injectable gelatin/silk fibroin microspheres for the dual delivery of curcumin and piperineJ Mater Sci Mater Med2014254011010.1007/s10856-013-5082-3Search in Google Scholar

Ginis I, Weinreb M, Abramov N, Shinar D, Merchav S, Schwartz A, et al. Bone progenitors produced by direct osteogenic differentiation of the unprocessed bone marrow demonstrate high osteogenic potential in vitro and in vivo. Biores Open Access. 2012; 1:69–78.GinisIWeinrebMAbramovNShinarDMerchavSSchwartzAet alBone progenitors produced by direct osteogenic differentiation of the unprocessed bone marrow demonstrate high osteogenic potential in vitro and in vivoBiores Open Access20121697810.1089/biores.2012.9904Search in Google Scholar

Takahashi Y, Yamamoto M, Tabata Y. Osteogenic differentiation of mesenchymal stem cells in biodegradable sponges composed of gelatin and β-tricalcium phosphate. Biomaterials. 2005; 26:3587–96.TakahashiYYamamotoMTabataYOsteogenic differentiation of mesenchymal stem cells in biodegradable sponges composed of gelatin and β-tricalcium phosphateBiomaterials20052635879610.1016/j.biomaterials.2004.09.046Search in Google Scholar

Lindborg BA, Brekke JH, Scott CM, Chai YW, Ulrich C, Sandquist L, et al. A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells. Tissue Eng Part A. 2015; 21:1952–62.LindborgBABrekkeJHScottCMChaiYWUlrichCSandquistLet alA chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cellsTissue Eng Part A20152119526210.1089/ten.tea.2014.0335Search in Google Scholar

Moshaverinia A, Chen C, Akiyama K, Xu X, Chee WW, Schricker SR, Shi S. Encapsulated dental-derived mesenchymal stem cells in an injectable and biodegradable scaffold for applications in bone tissue engineering. J Biomed Mater Res A. 2013; 101:3285–94.MoshaveriniaAChenCAkiyamaKXuXCheeWWSchrickerSRShiSEncapsulated dental-derived mesenchymal stem cells in an injectable and biodegradable scaffold for applications in bone tissue engineeringJ Biomed Mater Res A201310132859410.1002/jbm.a.34546Search in Google Scholar

Raucci MG, Alvarez-Perez MA, Demitri C, Sannino A, Ambrosio L. Proliferation and osteoblastic differentiation of hMSCs on cellulose-based hydrogels. J Appl Biomater Funct Mater. 2012; 10:302–7.RaucciMGAlvarez-PerezMADemitriCSanninoAAmbrosioLProliferation and osteoblastic differentiation of hMSCs on cellulose-based hydrogelsJ Appl Biomater Funct Mater201210302710.5301/JABFM.2012.10366Search in Google Scholar

Sánchez-Ferrero A, Mata Á, Mateos-Timoneda MA, Rodríguez-Cabello JC, Alonso M, Planell J, Engel E. Development of tailored and self-mineralizing citric acid-crosslinked hydrogels for in situ bone regeneration. Biomaterials. 2015; 68:42–53.Sánchez-FerreroAMataÁMateos-TimonedaMARodríguez-CabelloJCAlonsoMPlanellJEngelEDevelopment of tailored and self-mineralizing citric acid-crosslinked hydrogels for in situ bone regenerationBiomaterials201568425310.1016/j.biomaterials.2015.07.062Search in Google Scholar

Articles recommandés par Trend MD

Planifiez votre conférence à distance avec Sciendo