Programmable Electro-Assembly of Collagen: Constructing Porous Janus Films with Customized Dual Signals for Immunomodulation and Tissue Regeneration in Periodontitis Treatment

Abstract

Currently available guided bone regeneration (GBR) films lack active immunomodulation and sufficient osteogenic ability- in the treatment of periodontitis, leading to unsatisfactory treatment outcomes. Challenges remain in developing simple, rapid, and programmable manufacturing methods for constructing bioactive GBR films with tailored biofunctional compositions and microstructures. Herein, the controlled electroassembly of collagen under the salt effect is reported, which enables the construction of porous films with precisely tunable porous structures (i.e., porosity and pore size). In particular, bioactive salt species such as the anti-inflammatory drug diclofenac sodium (DS) can induce and customize porous structures while enabling the loading of bioactive salts and their gradual release. Sequential electro-assembly under pre-programmed salt conditions enables the manufacture of a Janus composite film with a dense and DS-containing porous layer capable of multiple functions in periodontitis treatment, which provides mechanical support, guides fibrous tissue growth, and acts as a barrier preventing its penetration into bone defects. The DS-containing porous layer delivers dual bio-signals through its morphology and the released DS, inhibiting inflammation and promoting osteogenesis. Overall, this study demonstrates the potential of electrofabrication as a customized manufacturing platform for the programmable assembly of collagen for tailored functions to adapt to specific needs in regenerative medicine.

Keywords: Janus porous structure; collagen; electro‐assembly; immunomodulatory activity; periodontitis treatment.

Scheme 1

a) Illustration of how soluble salts modulate the electro‐assembly of collagen and the potential mechanism. Anions dissociated from soluble salts screen the collagen's positive charge in solution through electrostatic interactions, resulting in the slow migration of collagen molecules to the cathode under the applied electric field, then assemble into a loosely organized (i.e., high water content) hydrogel network at the cathode surface due to the locally high pH generated by the cathode reaction. b) Sequential two‐step electro‐assembly of collagen using the solutions with pre‐programmable ionic environments (i.e., salt species, concentration), allows customization of a collagen‐based Janus porous film with specific porous microstructures (i.e., porosity and pore size) and bioactive salt composition (i.e., anti‐inflammatory drug, DS) that endow the bio‐functions of inflammatory regulation. c) The coupling of specific microstructures and anti‐inflammatory composition of Janus film can provide asymmetric cellular responses (preventing fibroblast infiltration but promoting osteoblast growth, and differentiation) and immunomodulatory functions, thereby inhibiting inflammation and promoting periodontal tissue regeneration.

Figure 1

Salt effect on electro‐assembly of collagen and pore generation. a) Illustration of the electro‐assembly of collagen in the absence and presence of soluble NaCl. b) The collagen film (designated as “E‐Col”) electro‐assembled under an environment absence of salts is thin and robust, while the film (designated as “E‐Col/Cl⁻”) electro‐assembled under an environment presence of sodium chloride is thicker and softer (note: electro‐assembly conditions is 6.67 mA/cm² for 1000 s). c) SEM images show that the porosity of the film increases with the concentration of soluble Cl⁻ salt in the electrolyte. d) Surface pore size distribution histograms of films prepared with different concentrations of Cl⁻. (n = 100). e) The zeta potential of dissolved collagen molecules under various environments with different concentrations of Cl⁻, SO₄ ²⁻, and Cit³⁻ salt species, indicating the charge of collagen molecular gradually decreases with increasing soluble salt concentration. (n = 3). f) Quantitative determination of collagen film growth rate in the presence of varying amounts of different salts. (n = 5). g) The addition of salts with high anion valences allows a wider range of tuning of pore size and porosity of the electro‐assembled collagen films after freeze drying.

Figure 2

The electrofabrication of porous collagen films using DS. a) Optical cross‐sectional images of the collagen films fabricated using varying concentration of DS (note: electro‐assembly conditions is 6.67 mA cm⁻² for 1000 s), indicating the thickness of hydrogel films (designated as “E‐Col/DS”) is increasing with the concentration of DS. b) The SEM images of the surfaces of E‐Col/DS hydrogel films after freeze drying, revealing that the film's porosity and surface porous size are increasing with the increasing concentration of DS. b) Histogram of pore size distribution on the surface of E‐Col/DS film prepared with different concentrations of DS. c) Standard calibration curve for DS. The release kinetics of d) cumulative amount curves and e) cumulative released percentage per unit area of E‐Col/DS films fabricated using varying concentrations of DS within 7 days. (n = 4).

Figure 3

Electrofabrication of Janus porous collagen/DS films and its characteristics. a) Illustration of electrofabrication of Janus films (designated as “E‐Janus Col/DS”) using a two electro‐assembly steps method. b) Optical image of the freeze‐dried E‐Janus Col/DS film and its SEM images show the macroscopic and microscopic morphological details of E‐Janus Col/DS films. One surface is dense and without pores, while the other surface is loose and porous. Besides, the crosssection of Janus film demonstrates the dramatic transition from the porous layer to the dense layer [note: the up‐interface (i.e., porous face side) and low‐interface (i.e., dense face side) of the Janus film are marked by red and green dashed lines respectively]. c) ATR‐FTIR and d) XRD spectra further respectively proves the asymmetric chemical and crystalline structures of Janus film. e) Representative stress–strain curve of wet E‐Janus Col/DS film, indicating its higher tensile stress than the commercial Bio‐Gide film. f) Optical images show the Janus film can be twisted or knotted and allowed to return to its original shape. g) Photographs taken after liquid drops were contacted with the dense surface or porous surface of the Janus film. h) Dynamic water contact angle measurements for the two surfaces of the Janus Col/DS film demonstrate that the porous face promotes liquid spreading and exhibits higher hydrophilicity compared to the dense face. i) The optical images of the dense face and porous face of the E‐Janus Col/DS film adhere to the surface of wet pigskin.

Figure 4

The Janus porous structure of the E‐Janus Col/DS film provides anisotropic guidance for fibroblasts and its porous surface facilitates the adhesion and proliferation of osteoblasts. a) The experimental approach involves a cell crown insert sealed with a film (either with the dense face up or the porous face up), positioned on a well plate to isolate upper and lower volumes for assessing anisotropic cell guidance. b–d) 3D fluorescence images demonstrate that when L929 fibroblast cells were seeded onto the Janus Col/DS film, they did not penetrate the dense layer but instead grew into the porous layer (n = 5). e) Confocal and SEM images of MC3T3‐E1 osteogenic precursor cells (marked in pseudo‐purple color) depict cell adhesion in the porous layer of the Janus Col/DS film. f) Comparison of the proliferation of osteogenic precursor cells (MC3T3‐E1) on different films suggests that the Janus porous structure promotes cell proliferation (n = 4). ^* p < 0.05, ^*** p <0.001. All data are presented as mean ± SD. One‐way ANOVA was used for comparison.

Figure 5

In Vitro anti‐inflammatory, osteogenic, and anti‐bacterial functions of E‐Janus Col/DS film. a) Illustration of the LPS activated macrophages (i.e., RAW264.7) cultured on the DS‐loaded porous surface of E‐Janus Col/DS film. b) Representative flow cytometry results of CD 86+ (M1 markers) and CD206+ (M2 markers) expression of macrophages incubated on TCP (i.e., blank group), the non‐porous surfaces of E‐Col films, the porous surfaces of E‐Col/Cl⁻ and E‐Janus Col/DS films for 1 day and c) the quantitative percentages of M1 and M2 polarization of RAW 264.7 in different groups (n = 4). d) The pro‐inflammatory and anti‐inflammatory related genes expression of RAW264.7 after 24 h culturing on the films surface using qPCR, which indicating the loaded DS and porous surface structure of E‐Janus Col/DS film are both important for decreasing the inflammatory (n = 4). e) Illustration of RAW264.7 and MC3T3‐E1 cells co‐cultured on the porous surface of the film. f) ALP and Alizarin Red S staining of cells seeded on the surface of different films’ surfaces for 14 days and 21 days, respectively, and g) the quantitative ALP activity results indicating that the MC3T3‐E1 cells present increasing osteogenic differentiation on the porous surface of E‐Janus Col/DS film (n = 4). h) Antibacterial activity of E‐Janus Col/DS film against Staphylococcus aureus. i) Inhibition zone experiment of different samples. Quantification of j) inhibition zone diameter and k) killing efficiency rate (n = 5). ^* p < 0.05, ^** p < 0.01, ^*** p <0.001. All data are presented as mean ± SD. One‐way ANOVA was used for comparison.

Figure 6

Tissue responses and degradation to different films 1, 4, and 12 weeks after subcutaneous implantation in rats. a) Optical images, H&E staining images with different magnifications for 1 week subcutaneous implantation [Note: black dotted line indicates the area of implanted materials, M; black triangle marks the dense layer of Janus film; red star marks the tissue ingrowth into the Janus porous film]. The H&E staining images with different magnifications for the different film for b) 4 weeks and c) 12 weeks after subcutaneous implantation. (n = 4).

Figure 7

In vivo evaluation of E‐Janus Col/DS film for periodontitis treatment. a) Schematic illustrates the induction of the periodontitis in rats and the procedure of surgery for film implantation. b) Relative mRNA expression levels of the corresponding proinflammatory genes (IL 1β, IL 6, and IFN γ) in the periodontal tissue at 4 and 8 weeks after surgery. c) Micro‐CT images of rat maxillas at 4 and 8 weeks after implantation of different film materials. All specimens were normalized and Micro‐CT images calibrated to enable quantitative comparison, and the distance between the alveolar bone crest (ABC) and the cementum junction (CEJ) marked with the red dashed line. d) Quantification of AB recovery determined by measuring the distance between the ABC and CEJ and the bone mineral density using 3D reconstruction volume after 4 and 8 weeks after surgery. (n = 4). ^* p < 0.05, ^** p < 0.01, ^*** p <0.001. All data are presented as mean ± SD. One‐way ANOVA was used for comparison.

Figure 8

Histological observation of periodontal tissue. Representative H&E staining and Masson's trichrome (MT) staining images comparing the periodontal tissue regeneration in different groups after repair period of 4 and 8 weeks. (AB, host alveolar bone; DE: dentine; PDL: periodontal ligament; FT, fibrous; The distance between the cementum junction (CEJ) and the alveolar bone crest (ABC) marked with the green dashed line). (n = 4), scale bar: 500 and 100 µm for low‐power (2x) and high‐power (10x) objectives, respectively).