Hydrogel is one of the promising platforms as a matrix that provides a cell niche to control cell fate due to their similarity to natural extracellular matrix (ECM). Especially, the physical properties of the hydrogel, such as mechanical properties, topography and scale, are important parameters to control cell fate and fabricate mature artificial tissue for 2D and 3D hydrogel cell culture platforms, because cells can sense the external microenvironment and be able to respond to and convert this information. in biochemical signals for their viability, proliferation, differentiation and development. However, in 3D hydrogel, key physical parameters, elastic modulus and swelling ratio, are generally coupled, so this can hinder accurate cell analysis based on a single variable.
In addition, ECM is a multi-level matrix that is composed of fibrous proteins such as collagen, fibronectin, and elastin, etc., and polymeric molecules such as proteoglycan, so the hydrogel alone cannot provide the physical and structural properties that ECM has. Furthermore, independent diffusion control could be realized by introducing microchannels into the hydrogel structure while maintaining the elastic modulus. The second was 2) to provide an ECM mimetic microenvironment, a “nanofiber composite,” composed of nanofibers and polymer hydrogel to control cell fate by engineering the nanofibers and utilizing the structural strength of the nanofiber composite.
Heterogeneous nanofibers can impart multifunctionality to the nanofiber composition, such as electrical conductivity and topological change.
Introduction to Matrix for Tissue Engineering
Hydrogels as the role of extracellular matrix (ECM)
Nanofibers as the role of ECM
Comprehensive examination of mechanical and diffusional effects on cell behavior
- Materials and Method
- Synthesis of methacrylic dextran(‘MDex’)
- Fabrication of MGel-MDex hydrogels
- Mechanical characterization of hydrogels
- Diffusional characterization of hydrogels
- Cell culture
- Results and Discussion
- Synthesis of methacrylic dextran(‘MDex’)
- Physical properties of MDex-linked hydrogel
- Cell behavior in MDex-linked hydrogels
- Hepatocarcinoma cells
- Fibrobalsts
- Mesenchymal stem cells
- Conclusion
- Reference
- Introduction
- Materials and Method
- Generation of induced hepatic progenitor cells (iHEP)
- Fabrication of iHEP-laden hydrogels
- Immunocytochemistry
- Quantitative real-time PCR (qRT-PCR)
- Development of murine acute liver injury model
- Implantation of iHEP-laden hydrogel
- Histological and blood analysis
- Results and Discussion
- Mechanical properties of hydrogels controlled by polymeric crosslinker
- Microchannel architecture of hydrogels controlled by photolithography·
- Viability and proliferation
- Albumin
- CYP1A2
- In vivo evaluation of iHEP-laden hydrogels
- Conclusion
- Reference
Introduction to Matrix for Tissue Engineering Figure 1-1. Synergistic control of mechanics and microarchitecture of 3D bioactive hydrogel platform to promote the regenerative potential of engineered liver tissue. a) Schematic illustrations of the total experimental process to produce engineered hepatic tissue. Relative distribution for iHEPs in each hydrogel condition over time (a) without and (b) with. c, d) The diffusion rates (kP) defined by the trend line in (a) and (b) with Eq.
Enhanced mechanical and electrical properties of heteroscaled hydrogels infused with
Materials and Method
- Synthesis of methacrylic gelatin (MGel)
- Synthesis of dispersible-hybrid nanofibers (dhNF)
- Fabrication of dhNF-infused hydrogels
- Scanning electron microscopy
- Impedance
- Electrical stimulation
Results and Discussion
- Fabrication of dispersible hybrid nanofibers
- Mechanical properties
- Electrical conductivity
- Cell viability and proliferation in 3D culture
- Effect of electrical stimulation
Conclusion
Mechanotopography-driven design of dispersible nanofiber-laden hydrogel as a 3D
Materials and Method
- Preparation of GO and rGO laden dispersible nanofibers
- Physicomechanical characterization of nanofibers
- Fibroblast encapsulation in nanofiber-infused hydrogels
- In vitro characterization - immunocytochemistry
- Statistical analysis
15 wt% MGel and up to 6 mg ml-1 graphene-based materials (GO and rGO) were dissolved in 2,2,2-trifluoroethanol/deionized (DI) water (7:3 volume ratio) and mixed by sonication for 10 min . Cell morphology in nanofiber hydrogel composite was also observed by SEM after 4% paraformaldehyde fixation and after DI washing. The force-distance curve for obtaining adhesion force of the short nanofiber was measured when the cantilever approached and retracted from the nanofiber in contact mode.
The negative value in the force plot was the attractive force between the cantilever and the sample that pulled the cantilever away from the sample. Twenty short nanofibers were randomly selected for each sample and their average value was reported. The surface adhesion force of the short nanofiber was obtained from the force-distance curve using a Dimension AFM (DI-3100, Veeco).
The force was calculated based on the results of the displacement and spring constant of the cantilever. Nanofiber was dried in vacuo and placed between two metal probes of an impedance analyzer (4294A, Agilent). The fibroblasts were dispersed in a pre-gel solution (1 × 10 cells mL – 1) and photocross-linked to encapsulate in a hydrogel.
Cell-laden hydrogels were immersed in cell culture medium and continuously cultured at 37 °C under 5% CO2. To evaluate the effect of soluble profibrotic factor, the culture medium was supplemented with TGF-β1 (recombinant human, Peprotech) at 2 ng mL-1, being replenished every 2 days [42, 44]. After permeabilization with 0.1% Triton X-100 in PBS, samples were blocked with 2% bovine serum albumin for 2 h at room temperature.
To identify alpha-smooth muscle actin (α-SMA) expression, primary antibody containing mouse anti-α-SMA antibody (1:500, Thermo Fisher) was added to the samples for 3 h at room temperature followed by incubation in the secondary antibody containing anti-mouse IgG for 1 hour at room temperature. Mean and standard deviation values from multiple independent experiments were reported in this study (n = 10 for material characterization, n = 6 for cell viability and proliferation, and n = 30 for image quantification).
Results and Discussion
- Fabrication and Characterization of nanofibers with controlled mechanotopography
- Fabrication and characterization of nanofiber-laden hydrogel
- Viability and proliferation
- Myofibroblast differentiation
However, there was a small but significant increase in moduli for rGO-Gel NF compared to GO-Gel NF at the same graphene concentration. In addition, the adhesion strength of rGO-Gel NF was significantly greater than that of GO-Gel NF at the same graphene content. However, the moduli of rGO-Gel NF hydrogel were significantly improved by the increasing presence of rGO (Fig. 4d).
The increase in moduli by rGO-Gel NF was correlated with the amount of rGO present in the nanofibers; On the other hand, the swelling ratios decreased to a smaller extent, 25.5% on average for Gel NF hydrogels and GO-Gel NF hydrogels, when increasing the nanofiber concentration to 2% (Fig. 4f). Even more remarkable, for rGO-Gel NF hydrogels, the swelling ratio decreased by only 39%, only 4% higher than MGel hydrogels, while the moduli were 36% larger than MGel hydrogels (Fig. 4g).
The proliferation rates in Gel NF hydrogels were higher than those in MGel hydrogels at respective mechanical stiffnesses. The proliferation rates in GO-Gel NF hydrogels were slightly lower than those in Gel NF hydrogels (Fig. 5b, e & h). In addition, the spreading rates in rGO-Gel NF showed an even further decrease compared to those in GO-Gel NF hydrogels (Fig. 5b, f & h).
This phenomenon was more frequently observed in nanofiber-loaded hydrogels than MGel hydrogels with increasing MGel concentration, again due to the limited spatial accessibility. a, b) Fluorescent images of fibroblasts encapsulated in MGel hydrogels in the presence of TGF- at different MGel concentrations, Gel NF hydrogels, GO-Gel NF hydrogels and rGO-Gel NF hydrogels at different nanofiber concentrations (scale: 100μm ). It was evident that the cell spreading became more extensive with the presence of nanofibers and the incorporation of GO or rGO into the nanofibers (Gel NF. < GO-Gel NF < rGO-Gel NF). The cell area in Gel NF hydrogels was lower than those in MGel hydrogels, combined with higher cell aspect ratio and α-SMA expression, showing greater degree of myofibroblast differentiation (Fig. 8c & e).
Interestingly, the difference in cell aspect ratio and cell area between Gel NF, GO-Gel NF and rGO-Gel-NF hydrogels decreased, especially at higher nanofiber concentrations, where α-SMA expression was the highest (Fig. 8d & f). Overall, the nanofiber-loaded hydrogels with controllable mechanotopographic properties could be used as a highly efficient 3D cell culture platform for the in-depth study of tissue fibrosis. a, b) Immunocytochemistry images of -smooth muscle actin (red) and actin (green) from fibroblasts in MGel hydrogels in the presence of TGF- at different MGel concentrations, Gel NF hydrogels, GO-Gel NF hydrogels and rGO - Gel NF hydrogels in different nanofiber concentrations.
Conclusion
울산에서 9년, 미국에서 1년을 보낸 후 박사학위를 받았습니다. 공부와 연구에 전념하며 보낸 20대이지만, 소중한 사람들을 만나 풍성하고 뜻깊게 보낸 시간이었습니다. 제가 새로운 출발을 할 수 있도록 도와주신 분들께 김선태 연구원으로서 감사의 말씀을 전하고 싶습니다.
우선 차채녕 교수님을 지도교수로 모실 수 있었던 것은 축복이었습니다. 교수님께서는 선생님이 아니라 선배연구원이라고 하시지만, 교수님께서 저에게 주신 가르침이 저를 연구원으로 만들었습니다. 이제 의사로서 새로운 출발을 앞두고 있는 김선태 선생님의 교훈을 기억하며 다시 훌륭한 연구자로 뵙겠습니다.
그리고 WFIRM의 객원연구원으로 지내며 새로운 환경에서 무명의 시간임에도 불구하고 물질적으로나 정신적으로 많은 지원을 해주시고 잘 적응하고 연구할 수 있도록 도와주신 이상진 교수님에게도 감사의 말씀을 전하고 싶습니다. 나의 약점을 깨닫고, 이를 보완할 수 있는 동기를 부여하고, 독립적인 연구자가 될 수 있도록 지도함으로써 나는 다음 단계로 성장하여 박사학위를 받을 수 있었습니다. 올해를 의미있게 만들어주셔서 감사합니다.
또한 WFIRM의 James Yu 교수님께도 감사의 말씀을 전하고 싶습니다. 연구지도를 받지는 못했지만, 넓고 깊은 경험을 바탕으로 저의 고민과 진로에 대해 마치 자기 일처럼 조언과 격려를 해주셔서 큰 도움이 되었습니다. 그리고 박사학위 논문의 심사위원으로 시간을 내어 자기 세탁물처럼 조언해주신 강현욱 교수님, 김지윤 교수님, 박태은 교수님, 이창영 교수님에게도 감사의 말씀을 전하고 싶습니다. . 재학생.
진짜 형처럼 챙겨주시고 조언도 해주셔서 너무 감사했어요. 또한, 저와 함께 일해주신 부산대학교 연구원들과 WFIRM, 특히 Dr. 오랜 시간 동안 저를 형처럼, 아버지처럼 보살펴주시고, 박사학위 공부를 더욱 풍성하게 해주신 정진오 교수님, 장지수 교수님에게도 감사드립니다.
