Biosynthetic polyphosphate enhances osteogenesis of human periodontal ligament stem cells and promotes periodontal bone regeneration in a murine periodontal bone defect model

Jiaqi Chen¹Dongying Lei¹Xinyi Liu¹Zipeng Chen²Jiaying Li¹Liang Huang¹Huifen Liu¹Xuebin Yang³Wei Wei⁴Sijing Xie^1*

• ¹Nanjing Stomatological Hospital, School of Medicine, Nanjing University, Nanjing, China
• ²Nanjing University School of Chemistry and Chemical Engineering, Nanjing, China
• ³University of Leeds School of Molecular and Cellular Biology, Leeds, United Kingdom
• ⁴Nanjing University, Nanjing, China

Periodontal bone regeneration remains a significant challenge in clinical dentistry due to the complex structure of periodontal tissues and their limited intrinsic regenerative capacity, driving the need for innovative biomaterial-based solutions. Although polyphosphates (Poly(P)) demonstrate promising regenerative potential, conventional chemical synthesis methods face limitations, including high production costs and concerns over product impurity. This study introduces an eco-friendly biosynthesis strategy that utilizes a genetically accessible environmental bacterium overexpressing polyphosphate kinase (PPK1) to produce high-purity polyphosphates (Bio-Poly P) from phosphate sources derived from wastewater. Structural characterization confirmed that Bio-Poly P's physicochemical properties are comparable to those of chemically synthesized polyphosphates. In vitro studies revealed that Bio-Poly P at 1.25 and 2.5 mg/ml did not reduce the human periodontal ligament stem cells (hPDLSCs) proliferation compared to the control after 24, 48, and 72 hours of culture. After 7 days, qRT-PCR results showed that Bio-Poly P at 0.25, 1.25, and 2.5 mg/ml significantly enhanced the gene expression of hPDLSCs for COLIA1 compared with the control (P<0.0001). Only the 1.25 mg/ml group significantly upregulated the OCN (P<0.0001) and OPN (P<0.01) expressions. After 14 days of culture, all three test groups significantly upregulated the gene expression of all osteogenic markers (RUNX2, OCN, OPN, and COL1A1) compared to the control. Alkaline phosphatase (ALP) staining and biochemical quantitative assay confirmed that all three test groups significantly enhanced the hPDLSCs ALP activities compared with the control group. Alizarin Red staining showed that the group with 1.25 mg/ml enhanced calcium accumulation, indicating extracellular matrix mineralization compared to untreated controls. After 4 weeks in vivo implantation in a murine periodontal bone defect model, micro-CT analysis showed that Bio-Poly P significantly enhanced the bone mineral density, total bone volume/tissue volume ratio and trabecular thickness compared with the bone defect alone group, which showed a significant alveolar bone loss. It is also comparable with the positive control group (Bio-Oss®) (P>0.05). These results confirmed that Bio-Poly P combines biosafety with effective osteoinductive properties promoting substantial bone regeneration both in vitro and in vivo, which indicates that Bio-Poly P could be used as a cost-effective and sustainable alternative for periodontal regeneration.