A photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel promotes bone regeneration.

OBJECTIVES: In order to evaluate the effect of methacrylated hyaluronic acid (HAMA) hydrogels containing the recombinant human amelogenin (rhAm) in vitro and in vivo. BACKGROUND: The ultimate goal in treating periodontal disease is to control inflammation and achieve regeneration of periodontal tissues. In recent years, methacrylated hyaluronic acid (HAMA) containing recombinant human amyloid protein (rhAm) has been widely used as a new type of biomaterial in tissue engineering and regenerative medicine. However, there is a lack of comprehensive research on the periodontal regeneration effects of this hydrogel. This experiment aims to explore the application of photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel for periodontal tissue regeneration and provide valuable insights into its potential use in this field. MATERIALS AND METHODS: The effects of rhAm-HAMA hydrogel on the proliferation of human periodontal ligament cells (hPDLCs) were assessed using the CCK-8 kit. The osteogenic differentiation of hPDLCs was evaluated through ALP staining and real-time PCR. Calvarial parietal defects were created in 4-week-old Sprague Dawley rats and implanted with deproteinized bovine bone matrix in different treatment groups. The animals were euthanized after 4 and 8 weeks of healing. The bone volume of the defect was observed by micro-CT and histological analysis. RESULTS: Stimulating hPDLCs with rhAm-HAMA hydrogel did not significantly affect their proliferation (p > .05). ALP staining and real-time PCR results demonstrated that the rhAm-HAMA group exhibited a significant upregulation of osteoclastic gene expression (p < .05). Micro-CT results revealed a significant increase in mineralized tissue volume fraction (MTV/TV%), trabecular bone number (Tb.N), and mineralized tissue density (MTD) of the bone defect area in the rhAm-HAMA group compared to the other groups (p < .05). The results of hematoxylin and eosin staining and Masson staining at 8 weeks post-surgery further supported the results of the micro-CT. CONCLUSIONS: The results of this study indicate that rhAm-HAMA hydrogel could effectively promote the osteogenic differentiation of hPDLCs and stabilize bone substitutes in the defects that enhance the bone regeneration in vivo.

[Comparison of a new thermosensitive rhAm carrier versus traditional PGA carrier for in vitro antibacterial activity and biocompatibility].

OBJECTIVE: To compare a new thermosensitive recombinant human amelogenin (rhAm) carrier and traditional propylene glycol alginate (PGA) carrier for their characteristics, antibacterial activity, and biocompatibility with human periodontal membrane fibroblasts. METHODS: PGA-rhAm was prepared by mixing 3.3% PGA and rhAm, and CS-ßGP-rhAm was prepared by mixing 2% chitosan (CS) with rhAm and then with 60% ß-sodium glycerophosphate solution (ßGP) as the crosslinking agent. The biophysical properties of the prepared carriers were characterized, and their antibacterial activity was assessed by observing Staphylococcus aureus growth. The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts (hPDLFs) using CCK8 assay and scratch test, and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting; osteogenic differentiation of the cells was detected using alkaline phosphatase staining. RESULTS: PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s. CS-ßGP-rhAm had a solidification capacity of 6 min at 37 ℃ with a pH value close to that of the oral cavity and a swelling rate of about 90%. CS-ß GP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks. CS-ßGPrhAm more effectively inhibited the growth of S. aureus than rhAm-loaded PGA. While PGA did not obviously affect the proliferation of hPDLFs, both CS-ßGP and CS-ßGP-rhAm significantly promoted the cell proliferation(P < 0.001). Scratch test showed that after rhAm loading, both CS-ßGP and PGA promoted cell migration (P < 0.01). CS-ßGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67, RUNX2, collagen I, and ß-catenin (P < 0.05); PGA-rhAm only enhanced RUNX2 (P < 0.05) and OCN (P < 0.01) mRNA expressions without significant effects on the protein expressions. Alkaline phosphatase staining results showed that CS-ßGP, but not PGA, promoted osteogenic differentiation of hPDLFs. CONCLUSION: CS-ßGP carrier is capable of sustained release of rhAm, inhibiting the growth of S. aureus, and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.

Self-assembly and mineralization of full-length human amelogenin and its functional fragments in vitro.

OBJECTIVES: To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) in vitro and its role in hydroxyapatite (HA) crystal formation. METHODS: The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified in vitro. The protein solution of 100 µg‧mL-1, pH=8, was prepared in Tris-HCl and incubated at room temperature for 1-15 min. Their self-assembly behaviors were observed and compared under a transmission electron microscope (TEM). The full-length AM was added to artificial saliva and incubated for 3 days. A scanning electron microscope (SEM) was used in observing the morphology of the induced new crystals. Then, TARP and LRAP were added. The resulting solution was incubated for 3 days and then observed again. RESULTS: When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor. CONCLUSIONS: The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals in vivo, providing a theoretical basis for the role of the fragments in the growth and maturation of HA crystals.

Self-assembly and mineralization of full-length human amelogenin and its functional fragments / 华西口腔医学杂志

OBJECTIVES@#To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) @*METHODS@#The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified @*RESULTS@#When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor.@*CONCLUSIONS@#The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals