A polysaccharide-based composite hydrogel sustained delivery P24 peptide in PLGA microsphere for osteogenesis potential in vitro and in vivo

Background

Demonstrated the great potential of facilitating bone regeneration, bone morphogenetic protein-2 (BMP-2) derived synthetic oligopeptide (S[PO4] KIPKASSVPTELSAISTLYLDDD, P24) has exhibited good osteoinductive and ectopic bone formation features. However, the defect of alveolar is always with irregular shapes and the bone formation is a long process, hence the application of P24 requires sustained release at certain concentration in irregular defects throughout the process of bone regeneration.

Aim/Hypothesis

We hypothesized that this P24-loaded hydrogel system could modulate osteogenic differentiation and hydrogel mineralization in vitro. After implanted in a rat critical size cranial defect, the composite hydrogels would be hypothesized to enhance bone regeneration.

Materials and Methods

PLGA-P24 microspheres were prepared with water-in-oil-in-water double emulsion. 10% nano hydroxyapatite (n-HA) and 5% PLGA-P24 microspheres were dispersed in 4% aldehyde dextran solution. Then composite hydrogel (CD n-HA PLGA-P24) was prepared via adding 0.5 ml mixture solution into 0.5 ml 4% hydroxypropyl chitosan solution. Consequently, we created a novel P24 sustained delivery system incorporated the Poly (lactic-co-glycolic acid) (PLGA) microspheres loaded P24 and n-HA microparticles into composite hydrogels. Then composite hydrogels were constructed, characterized, and tested for preosteoblastic (MC3T3-E1) in vitro. The osteoinductivity ability of composite hydrogel was tested by establishing an ectopic osteogenic model of the rat dorsal muscle pocket. After establishing the rat calvarial defect model, the bone defect was implanted the composite hydrogel with P24 microspheres to observe the ability of this composite system to repair the bone defect in vivo.

Results

The P24 peptides showed a sustained drug release profile over 60 days and markedly enhanced the efficiency in promoting the proliferation of cells and their expression of alkaline phosphatase (ALP). Additionally, the results of osteogenic gene expression and mineralization jointly confirmed that composite hydrogels could evoke a better cell osteodifferentiation viability in vitro. Through HE staining, Masson's trichrome staining and immunohistochemical staining, the results all above showed that the P24 microspheres composite hydrogel had a good osteoinductivity effect of ectopic osteogenic model in vivo. From the results of Micro CT, HE staining, Masson's trichrome staining and immunohistochemical staining in rat calvarial defect model, we knew that the composite hydrogel with P24-loaded microspheres composite hydrogel has a good ability to promote bone regeneration and is a potential bone tissue engineering material.

Conclusion and Clinical Implications

In this study, the osteogenic capability in vitro and regeneration capacity in vivo of a novel P24 sustained delivery system were investigated. The composite hydrogels enhanced the viability and osteogenic differentiation in vitro. In addition, the hydrogels had osteoinduction potential and promoted bone regeneration in vivo of rat ectopic bone formation and critical-sized calvarial defect model. In summary, the P24-hydrogel system construct could be a promising approach for bone regeneration.