Alginate solution

A 1.5% (w/v) alginate solution (Sigma-Aldrich, Missouri, USA) was initially prepared for inner encapsulation. Initially, 9 log CFU/mL of the respective LAB strain was mixed with 20 mL of alginate solution at a 1:5 (v/v) ratio and was then ready for the further microencapsulation step^{14 (link)}. For the extrusion method, the alginate mixtures were added dropwise through a 3 mL-syringe into 100 mL of CaCl₂ (1 mol/L) (Merck KGaA, Darmstadt, Germany) and left for 30 min for gelation to achieve the alginate beads^{39 (link),49 (link)}. For the emulsion method, the alginate beads were settled by adding the alginate mixtures to 100 mL of soybean oil (Sigma-Aldrich, Missouri, USA) containing 0.2% (v/v) of Tween 80 while stirring with a magnetic stirrer. Next, 100 mL of CaCl₂ (1 mol/L) was added into the mixture to solidify the alginate beads, which were then harvested by centrifugation at 350g for 10 min at 4 °C^{40 (link)}. Alginate beads obtained from the extrusion and emulsion methods were rinsed with and then kept in 0.1% (w/v) peptone water (Becton, Dickinson and Company, Maryland, USA) at 4 °C^{14 (link)}. For the spray drying method, alginate mixtures were atomized through the spray dryer (Mini Spray Dryer B-290, Buchi, Flawil, Switzerland) with an inlet temperature of 130 °C, and the alginate beads were then collected from the collecting vessel^{23 (link)}.

The procedure of animal surgery was approved by the Institutional Review Board of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2017-129). We used 32-month-old female nude mice (BALB/c) in this experiment, and each group included ten mice. The calvarial defect (3.5mm) operation was performed in the left parietal bones of mice with a dental drill.
The hDPSCs transfected by NC, miR-20a-5p, and anti-miR-20a-5p were resuspended in 1.2% alginate solution (Sigma) at a density of 5×10⁶ cells per ml in a conical tube, and the suspension was dropped into 1 ml of pre-warmed CaCl₂ to form the spherical alginate scaffolds which were approximately 3.5mm in diameter. The prepared alginate scaffold with cells was placed in the defect area. After 8 weeks, the skull samples were harvested and fixed in 4% polyoxymethylene for further experiments.

A suspension containing the equivalent concentration of 10¹¹ CFU/mL of γ-irradiated B. ovis was resuspended in 2 mL of 1% alginate solution (Sigma-Aldrich; product number 448869 with 96.1% deacetylation, and 35 cps of viscosity) and dripped with a 33G needle into 10 mL of polymerization solution (0.5 mM CaCl₂). After dripping, the microcapsules were homogenized for 15 minutes and washed twice in MOPS buffer (3-[Nmorpholino] propanesulfonic). Microcapsules containing 10¹⁰ CFU/mL of γ-irradiated B. ovis were homogenized for 30 minutes in a solution of 1% chitosan (pH 4.55, dissolved in 1% acetic acid) and washed once in MOPS buffer solution for 5 minutes. For the preparation of empty alginate-chitosan capsules, the procedure was similar, but without incorporation of bacteria into the alginate.

Bipolymeric NCs were prepared in two steps according to a previously reported method with some modifications [34 ]. The first step, emulsification, was used to prepare oil in water NE. For the oily phase, 30 mL ASH extract was mixed with 19 mL castor oil (Acros Organics, USA) and 1 g Span 20 (Acros Organics, USA) using a homogenizer (GLH 850, Omni Inc., USA) at 5000 rpm for 15 min. The oily phase was added gradually to a 100 mL solution of Tween 80 (1% w/v, Acros Organics, USA) using a homogenizer at 10,000 rpm for 45 min and then sonicated using a probe sonicator (Model LC 60/H, Elma, Germany) at 40% amplitude for 15 min with 5 s on/off and temperature adjusted to 15 °C.
In the second step, ionic gelation, a 100-mL alginate solution (0.03 or 0.06% w/v, Sigma-Aldrich, USA) was added gradually to ASH extract NE while stirring for 30 min. A 20-mL calcium chloride solution (0.06% w/v, Acros Organics, USA) was gradually added and stirred for 30 min. Finally, a 20-mL chitosan solution (0.03% or 0.06% w/v, Sigma-Aldrich, USA) in 1% acetic acid (pH 4–4.5, Acros Organics, USA) was gradually added with stirring for 30 min. The obtained NC suspension was sonicated to unify the particle size using a probe sonicator at 40% amplitude for 5 min with 5 s on/off and temperature adjusted to 15 °C. The preparation was stored at 4 °C. All samples were prepared using deionized distilled water.

Alginate solution of 0.5% w/v (from sodium alginate powder with medium viscosity, Sigma-Aldrich, St. Louis, MO, USA) and chitosan solution of 0.5% w/v (from 85% deacetylated chitosan flakes with medium molecular weight, Sigma-Aldrich, St. Louis, MO, USA) were prepared as reported [41 (link)]. The pH of the Alginate solution was adjusted to 6, 8, 10, and 11.5, while the pH of the chitosan solution was maintained at around 5.5 for the electrofabrication of chitosan membranes. Fluorescein isothiocyanate-labeled dextran (F-dextran) with molecular weights of 4 kDa and 1× phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals used in this study can be obtained from major suppliers.