Thanks for the great report. This is a bug but at the same time is not a bug either, let me explain.

You edited the image externally with the layers baked in. You reloaded the image correctly in Blender, but Pixel Art Studio does not watch for that: this is where the bug is, and I'm going to fix that (it's going to detect when the texture was reloaded).

But then, by editing the texture externally and reloading in Blender, it's then up to you to manage layers in PIxel Art Studio manually, as Pixel Art Studio is not communicating with Aseprite about any layer information (as you are editing the baked PNG, a raw PNG image with no layer information, it's not like a Photoshop PSD file or an *.aseprite file that has layers information).

So I'm going to fix the bug of Pixel Art Studio not detecting that a texture was reloaded, but then it's up to you to manage duplicate data (for example, in your example, the black pixel stroke is going to appear baked in the PNG you just reloaded AND repeated in the layer you had with it; likewise the pixel art grid will still be in its separate layer BUT ALSO baked in the texture already, duplicated).

Yep, that exactly happened to me, I was actually using Aseprite to edit the textures that I was painting with Pixel Art Studio and brought it back to Blender to keep editing it there. I will check the update now, thank you :)