4.4.2. Cells Embedded and Cultured in Hydrogel Scaffold

A certain concentration of P(OEGMA-co-MEO2MA) microgel solution was prepared and mixed well with an equal volume of the uniform cell suspension to form a mixed solution, in which the cell concentration was about 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/mL and the microgel concentration was 3.0 wt%. To a 48-well culture plate, 0.5 mL of the above cell/gel mixture was added and placed in a cell culture incubator. With increasing temperature, the solution gelled gradually. After the morphology of the gel in the culture plate was fixed, 0.5 mL of the culture solution was added to each well and recorded as day 0. The culture solution was changed regularly to ensure a nutritious environment for cell growth. The whole process is shown in Scheme 2. A certain concentration of P(OEGMA-co-MEO2MA) microgel solution was prepared and mixed well with an equal volume of the uniform cell suspension to form a mixed solution, in which the cell concentration was about 1 x 105 cells/mL and the microgel concentration was 3.0 wt%. To a 48-well culture plate, 0.5 mL of the above cell/gel mixture was added and placed in a cell culture incubator. With increasing temperature, the solution gelled gradually. After the morphology of the gel in the culture plate was fixed, 0.5 mL of the culture solution was added to each well and recorded as day 0. The culture solution was changed regularly to ensure a nutritious environment for cell growth. The whole process is shown in Scheme 2.

**Scheme 2.** The preparation of the P(OEGMA-co-MEO2MA) thermosensitive hydrogel scaffold. **Scheme 2.** The preparation of the P(OEGMA-co-MEO2MA) thermosensitive hydrogel scaffold.

### 4.4.3. Cell Viability (MTT Assay)

4.4.3. Cell Viability (MTT Assay) MTT assay was used to detect the viability of cells in the scaffolds. From day 0 of cell culture, three wells were selected each day and 100.0 μL of MTT solution was added to MTT assay was used to detect the viability of cells in the scaffolds. From day 0 of cell culture, three wells were selected each day and 100.0 µL of MTT solution was added to the

the wells. After continuing to incubate for 4 h, the gel pieces were removed, and 10.0 mL of DMSO was added. Then, the mixture was shaken for 10 min in the dark to dissolve the

From day 0 of cell culture, three wells were selected out after culturing for certain days, and one drop of AO/EB double staining solution was added to the selected wells. After further incubation for 1h, the cells were removed, and the morphology and growth state of the cells in the scaffold were observed by an inverted fluorescence microscope.

**Author Contributions:** Conceptualization, Y.L.; methodology, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.- L.Y.; validation, Y.L., Y.-N.L. and Y.-L.Y.; formal analysis, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.-L.Y.; investigation, P.Z., W.-F.Y. and C.-Y.Z.; resources, Y.L.; writing—original draft preparation, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.-L.Y.; writing—review and editing, Y.L., Y.-N.L. and Y.-L.Y.; visualization, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.-L.Y.; supervision, Y.L.; project administration, Y.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript. **Funding:** This research was funded by the Natural Science Foundation of Hunan Province (No. 2021JJ30597), Scientific Research Projects of Health Commission of Hunan Province (No. 202113022002), and Hengyang Guided Science and Technology Project (No. 2020jh042809).

4.4.4. Cell Activity and Morphology (AO/EB Staining)

**Institutional Review Board Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

1. Khademhosseini, A.; Langer, R. A decade of progress in tissue engineering. *Nat. Protoc.* **2016**, *11*, 1775–1781.

2. Esdaille, C.J.; Washington, K.S.; Laurencin, C.T. Regenerative engineering: A review of recent advances and future directions.

**Informed Consent Statement:** Not applicable. **Data Availability Statement:** Not applicable.

**References** 

https://doi.org/10.1038/nprot.2016.123.

*Regen. Med.* **2021**, *16*, 495–512. https://doi.org/10.2217/rme-2021-0016.

Vis spectrophotometer, and the MTT curve of cells was plotted.

wells. After continuing to incubate for 4 h, the gel pieces were removed, and 10.0 mL of DMSO was added. Then, the mixture was shaken for 10 min in the dark to dissolve the formed formazan. The absorbance of the solution at 490 nm was measured using a UV-Vis spectrophotometer, and the MTT curve of cells was plotted.

4.4.4. Cell Activity and Morphology (AO/EB Staining)

From day 0 of cell culture, three wells were selected out after culturing for certain days, and one drop of AO/EB double staining solution was added to the selected wells. After further incubation for 1h, the cells were removed, and the morphology and growth state of the cells in the scaffold were observed by an inverted fluorescence microscope.

**Author Contributions:** Conceptualization, Y.L.; methodology, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.- L.Y.; validation, Y.L., Y.-N.L. and Y.-L.Y.; formal analysis, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.-L.Y.; investigation, P.Z., W.-F.Y. and C.-Y.Z.; resources, Y.L.; writing—original draft preparation, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.-L.Y.; writing—review and editing, Y.L., Y.-N.L. and Y.-L.Y.; visualization, Y.-N.L., P.Z., W.-F.Y., C.-Y.Z. and Y.-L.Y.; supervision, Y.L.; project administration, Y.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Foundation of Hunan Province (No. 2021JJ30597), Scientific Research Projects of Health Commission of Hunan Province (No. 202113022002), and Hengyang Guided Science and Technology Project (No. 2020jh042809).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

