## 4 Discussion  
PLA has been extensively studied as a candidate for drug delivery and tissue engineering due to its biocompatibility, biodegradability and good mechanical properties; however, hydrophilicity modification is necessary to decrease its hydrophobicity for in vitro and in vivo studies. Sodium hydroxide (NaOH) is a common chemical used in every lab. Its hydroxyl (-OH) groups can be introduced through hydrolysis, which cleaves the ester bonds. In addition, the introduced -OH groups can be used to bind bioactive molecules such as collagen [53], fibronectin [54], and arginine-glycine-aspartic acid (RGD) [55] to regulate cell adhesion.  
In our early exploration of surface modification, we have tried other coating strategies, including coating of pure NaOH (10 N) and collagen. However, may due to the thickness of the filament or the high temperature processing, PLA has a certain degree of resistance to NaOH erosion, that results in a poor cell adhesion. In contrast, cells were flourishing on fibers that formed by collagen crosslinking. While collagen fibers forming networks in- and out-side of 3D scaffold, there is no optional way to precisely control the distribution and direction of these networks, nor of their pore size. The affection of collagen fibers on cell growth superseded the one brought by 3D scaffolds.  
In this study, we used FBS as the ground and top layer and incorporated with NaOH to generate a sandwich coating (FBS+NaOH+FBS) on the PLA surface. Fetal bovine serum is a widely used serum supplement for in vitro cell culture. In the pilot study of FBS coating, cells successfully attached on scaffold surface, which indicates an enhancement both in hydrophilicity and cell compatibility, simultaneously, FBS did not introduce a morphology change. Following with a second layer treatment of NaOH, there is an attenuation in element deposition as well as in hydrophilicity. This phenomenon indicated a chemical erosion caused by NaOH, but chemical bond cleavage may also associate with remained free chemical bonds that are available for further deposition. The increased FBS deposition in third layer coating confirmed this hypothesis (Supplementary Information, Figures S2 and S3). The increase of hydrophilicity and cell attachments are also indirectly supported an additional deposition of chemicals on eroded chemical bonds (Supplementary Information, Figures S1 and S4). Due to the prominent enhancement on hydrophilicity and bio-friendliness, we utilized the three-layer coating strategy in this study. This deposition-erosion-deposition strategy may be also appropriate for other biomaterials' surface modification.  
With the developing of 3D printing, there are several techniques used to fabricate 3D structure. The one used in this study is fused-deposition modeling (FDM). This method of printing deposits melted thermoplastic in thin layers and laydown at the designed location associated with CAD  
Figure 10: (**A**) Picrosirius Red Stain for bottom layer of 3D squares after 21 days incubation. The red color represents type III collagen (black arrows) and yellow color represents type I collagen (yellow arrows). Cells incubated in monolayer set as control (fare left figures). (**B**) Alizarin Red stain for calcium deposition after 21 days incubation (scale bar is 100 \(\upmu\)m).  
model. Therefore, FDM can be used to fabricate complicated 3D structure. However, due to the high extruding temperature required by biomaterials, such as the extruding temperature for PLA is 225 \({}^{\circ}\)C, no bio-factors nor cells can be printed with biomaterials. This is the biggest limitation of FDM during tissue engineering.