Radiation cross-linked collagen scaffolds facilitate root coverage and keratinized gingival regeneration

Abstract

The study aimed to develop a radiation cross-linked collagen scaffold (RCS) and assess its potential for root coverage and keratinized gingival regeneration, addressing the prevalent issue of gingival recession and limitations of traditional treatments. RCS was prepared through electron beam irradiation and cross-linking followed by freeze-drying. Its properties were evaluated, including Fourier transform infrared analysis, swelling behavior, microscopic observation, porosity measurement, compression modulus and structural stability. In a rat gingival recession model with 96 rats divided into four groups, the root coverage index and gingival health indices were measured, and histological analyses were conducted. The cross-linked network structure of RCS provided excellent mechanical properties and stability. In the rat model, RCS effectively promoted gingival regeneration, with the RCS group achieving a root coverage index of 87.7 ± 2.7 %, which was 54.13 %, 42.83 % and 8.41 % higher than that of the sham operation group, non-crosslinked group and chemical crosslinked group respectively. Histological analysis showed that RCS promoted anti-inflammatory macrophage polarization, enhanced collagen deposition and gingival lamina propria fiber density and increased angiogenesis. Additionally, RCS exhibited good biosafety, as blood indices and organ coefficients remained normal. In conclusion, RCS effectively promotes gingival regeneration and is a promising keratinized gingiva substitute for gingival recession, offering a new option for oral tissue repair.

Keywords: Collagen; Gingiva; Immunomodulation; Macrophages; Regeneration.

Graphical abstract

Fig. 1

Preparation and characterization of collagen scaffolds for root coverage. (a) Freeze-drying; (b) Fourier transform infrared spectroscopy; (c) liquid absorption capacity; (d) Pore size, surface and pore microstructure; (e) Swelling and stability; (f) Compressive properties.

Fig. 2

Appearance and tissue healing assessment of root coverage and keratinized gingival regeneration. (a) Representative gross appearance of the gingiva in SD Rats; (b) Representative results of Hematoxylin and Eosin staining on day 28; (c) Gingival index; (d) Root coverage index (mean ± standard deviation, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 3

Inflammatory response and macrophage analysis. (a) Expression of iNOS in gingival tissue. (b) Expression of CD163 in gingival tissue. (c) Quantitative analysis of the positive expression area of iNOS. (d) Quantitative analysis of the positive expression area of CD163 (bar = 20 μm; mean ± standard deviation, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 4

Distribution of collagen fibers and vascularization in gingival tissue and their characteristics in different groups.(a) Representative Masson staining showing the distribution and arrangement of collagen fiber. (b) Sirius red staining depicting the newly formed collagen fiber bundles and their organization. (c) Immunohistochemistry of collagen- I, confirming the preponderance of collagen fibers. (d) Collagen volume fraction of gingival tissue, demonstrating the variation in fiber density over time and across different groups. (e) Immunohistochemistry of CD31 for vascularization characterization, revealing the differences in angiogenesis and the relationship between collagen fibers and blood vessels. (f) Vascularization characteristics, showing distinct blood vessel distribution patterns (bar = 50 μm, mean ± standard deviation, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001).

Fig. 5

Blood routine examination, blood biochemistry, organ coefficients and H&E staining results on day 28. (a) Biochemical parameters such as ALT, AST, BUN and CRE levels among all groups showed no significant change (n = 6). (b) Blood routine parameters such as RBC, WBC, HGB and PLT levels among all groups showed no significant change (n = 6). (c) Organ coefficients parameters including heart, liver, spleen and kidney levels among all the groups showed no significant change (n = 6). (d) The histochemical analysis of the heart, liver, spleen, and kidney revealed no apparent differences compared to the SHAM control(bar = 50 μm; mean ± standard deviation).