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. 2021 Dec 26:15:68-81.
doi: 10.1016/j.bioactmat.2021.12.019. eCollection 2022 Sep.

Polyphosphate-crosslinked collagen scaffolds for hemostasis and alveolar bone regeneration after tooth extraction

Jun-Ting Gu  1 , Kai Jiao  1 , Jing Li  1 , Jian-Fei Yan  1 , Kai-Yan Wang  1 , Fu Wang  1 , Yan Liu  1 , Franklin R Tay  2 , Ji-Hua Chen  1 , Li-Na Niu  1
Affiliations

Polyphosphate-crosslinked collagen scaffolds for hemostasis and alveolar bone regeneration after tooth extraction

Jun-Ting Gu et al. Bioact Mater. .

Abstract

Post-extraction bleeding and alveolar bone resorption are the two frequently encountered complications after tooth extraction that result in poor healing and rehabilitation difficulties. The present study covalently bonded polyphosphate onto a collagen scaffold (P-CS) by crosslinking. The P-CS demonstrated improved hemostatic property in a healthy rat model and an anticoagulant-treated rat model. This improvement is attributed to the increase in hydrophilicity, increased thrombin generation, platelet activation and stimulation of the intrinsic coagulation pathway. In addition, the P-CS promoted the in-situ bone regeneration and alveolar ridge preservation in a rat alveolar bone defect model. The promotion is attributed to enhanced osteogenic differentiation of bone marrow stromal cells. Osteogenesis was improved by both polyphosphate and blood clots. Taken together, P-CS possesses favorable hemostasis and alveolar ridge preservation capability. It may be used as an effective treatment option for post-extraction bleeding and alveolar bone loss.

Statement of significance: Collagen scaffold is commonly used for the treatment of post-extraction bleeding and alveolar bone loss after tooth extraction. However, its application is hampered by insufficient hemostatic and osteoinductive property. Crosslinking polyphosphate with collagen produces a modified collagen scaffold that possesses improved hemostatic performance and augmented bone regeneration potential.

Keywords: Alveolar ridge preservation; Blood clotting; Osteogenesis; Polyphosphate.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Characterization of polyphosphate-crosslinked collagen scaffold (P-CS). A. Schematic of the synthesis of P-CS. B. Crystal violet staining of pristine collagen scaffold (CS) and P-CS. Bar: 5 mm. C. SEM-EDAX spectrum of P-CS. Bar: 5 μm. D. XPS spectrum of P 2p region of P-CS. E. ATR-FTIR spectra of ammonium polyphosphate (APP), CS and P-CS. The parts of the spectra outlined in the square inset were magnified for clarity. The spectra of CS and P-CS was normalized along the collagen amide I peak (∼1640 cm−1). Peaks associated with amide A (∼3300 cm−1), amide I and amide II (∼1545 cm−1) are characteristic of collagen.
Fig. 2
Fig. 2
Physical properties of P-CS. A. Images and quantitative analysis of water contact angle on pristine collagen scaffolds (CS) and polyphosphate-crosslinked collagen scaffolds (P-CS) at the time a water droplet contacted the specimens. B. Time for CS and P-CS to absorb a water droplet. C. Water absorption ratio, D. volume expansion ratio, E. Moduli of elasticity (Young's moduli) of the different scaffolds. F. Observation and G. estimation of the percentage degradation of CS and P-CS after enzymatic hydrolysis. Data represent means and standard deviations; ***: p < 0.001 (Student's t-test; n = 6).
Fig. 3
Fig. 3
Hemostasis of P-CS in vitro. A. Dynamic blood clotting index of pristine collagen scaffolds (CS) and polyphosphate-crosslinked collagen scaffolds (P-CS) after clotting for 5, 15 or 30 min. B. Thrombin generation of CS and P-CS at different time points. C. Representative SEM images of platelets and fibrin on different specimens. Platelet morphology was outlined in yellow (yellow arrows). Images in yellow rectangles were magnified to show the difference in fibrin formation on CS or P-CS. Red arrows: fibrin strands. Bar: 5 μm. D. The diameter of fibrin formed on CS and P-CS. E. P-selectin secretion by whole blood after treated with different scaffolds. F. Influence of CS and P-CS on the prothrombin time (PT) and activated partial thromboplastin time (APTT). Data represent means and standard deviations; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001 (Student's t-test, n = 6).
Fig. 4
Fig. 4
Hemostasis of P-CS in vivo. Bleeding time and blood loss of pristine collagen scaffolds (CS) and polyphosphate-crosslinked collagen scaffolds (P-CS) in A. the rat hepatic trauma model. B. the femoral artery injury model. C. the tooth extraction model. Data represent means and standard deviations; ns: no significant difference; *: p < 0.05; **: p < 0.01 (Student's t-test, n = 6).
Fig. 5
Fig. 5
In-situ bone regeneration in the rat alveolar bone defect model. A. Sagittal view of the rat maxilla on the 7th and 21st day after surgery. Ctrl: control specimens in which defects were not treated with any graft material. CS: pristine collagen scaffolds. P-CS: polyphosphate-crosslinked collagen scaffolds. Bar: 2 mm. B. Ratio of bone volume/total volume. C. Bone mineral density. For (B) and (C), parameters were calculated based on 3D reconstruction of micro-CT scans. D. Buccal ridge height; E. palatal ridge height; F. alveolar ridge width reduction at 21 days post-surgery. Data represent means and standard deviations; ns: no significant difference; *: p < 0.05; **: p < 0.01; ***: p < 0.001 (one-way ANOVA, n = 6).
Fig. 6
Fig. 6
Histological changes in the alveolar bone defect on the 21st day after surgery. Ctrl: control specimens in which defects were not treated with any graft material. CS: pristine collagen scaffolds. P-CS: polyphosphate-crosslinked collagen scaffolds. A. Representative images of the bone defect specimens that were stained with Goldner's trichrome. Black bar: 1 mm. B. Statistical analysis of the Goldner's trichrome staining results (positive areas/total areas). C. Representative images of the bone defect specimens that were stained with von Kossa stain. White bar: 1 mm. D. Statistical analysis of the von Kossa staining results. E. Representative images of the bone defect specimens that were stained with Masson's trichrome. Black bar: 1 mm. F. Statistical analysis of the Masson's trichrome staining results. Data represent means and standard deviations; **: p < 0.01; ***: p < 0.001 (one-way ANOVA, n = 6).
Fig. 7
Fig. 7
Interaction between blood clots and BMSCs. CS: pristine collagen scaffolds. P-CS: polyphosphate-crosslinked collagen scaffolds. A. Schematic of transwell migration assay. B. Images (bar: 1 mm) and C. quantitative analysis of cell migration in various groups. D. Schematic of co-culture of BMSCs with different scaffolds. Cell morphology after incubation for 12 h was detected by E. immunofluorescence (bar: 50 μm) and G. SEM (bar: 10 μm). The morphology of the BMSCs was outlined in red. The parts of the images outlined by the red squares were magnified. Blue arrows showed contacts of the filopodia with the scaffold matrix. F. Cell spreading areas of the BMSCs in the 4 groups were quantitatively analyzed. H. Normalized cytoplasmic gene expression of osteogenesis markers after osteogenic induction of the BMSCs for 14 days. For (C), (F) and (H), data represent means and standard deviations. Groups in each chart that labeled with different lowercase letters are significantly different (p < 0.05; two-way ANOVA, n = 6).
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