Background: A nanohydroxyapatite-coated chitosan scaffold has been developed in recent years, but the effect of this composite scaffold on the viability and differentiation of periodontal ligament stem cells (PDLSCs) and bone repair is still unknown. This study explored the behavior of PDLSCs on a new nanohydroxyapatite-coated genipin-chitosan conjunction scaffold (HGCCS) in vitro as compared with an uncoated genipin-chitosan framework, and evaluated the effect of PDLSC-seeded HGCCS on bone repair in vivo. Methods: Human PDLSCs were cultured and identified, seeded on a HGCCS and on a genipinchitosan framework, and assessed by scanning electron microscopy, confocal laser scanning microscopy, MTT, alkaline phosphatase activity, and quantitative real-time polymerase chain reaction at different time intervals. Moreover, PDLSC-seeded scaffolds were used in a rat calvarial defect model, and new bone formation was assessed by hematoxylin and eosin staining at 12 weeks postoperatively. Results: PDLSCs were clonogenic and positive for STRO-1. They had the capacity to undergo osteogenic and adipogenic differentiation in vitro. When seeded on HGCCS, PDLSCs exhibited significantly greater viability, alkaline phosphatase activity, and upregulated the bone-related markers, bone sialoprotein, osteopontin, and osteocalcin to a greater extent compared with PDLSCs seeded on the genipin-chitosan framework. The use of PDLSC-seeded HGCCS promoted calvarial bone repair. Conclusion: This study demonstrates the potential of HGCCS combined with PDLSCs as a promising tool for bone regeneration.

Increasing evidence has revealed that the surface characteristics of biomaterials, such as chemical composition, stiffness, and topography, especially nanotopography, significantly influence cell growth and differentiation. In this study, we examined the effect of surface biomimetic apatite nanostructure of a new hydroxyapatite-coated genipin-chitosan conjugation scaffold (HGCCS) on cell shape, cytoskeleton organization, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Cell shape and cytoskeleton organization showed significant differences between cells cultured on genipin-cross-linked chitosan framework and those cultured on HGCCS with surface apatite network-like nanostructure after 7 days of incubation in the osteogenic medium. The result of specific alkaline phosphatase activity as an indicator of osteogenic differentiation showed that the alkaline phosphatase activity of rat bone marrow-derived mesenchymal stem cells was higher on HGCCS. Based on quantitative real-time polymerase chain reaction, HGCCS induced highest mRNA expression of osteogenic differentiation makers, runt-related transcription factor 2 by 7 days, osteopontin by 7 days, and osteocalcin by 14 days, respectively. The enhanced ability of cells on HGCCS to produce mineralized extracellular matrix and nodules was also assessed on day 14 with Alizarin red staining. The results of this study suggest that the surface biomimetic apatite nanostructure of HGCCS is a critical signal cue to promoting osteogenic differentiation in vitro. These findings open a new research avenue to controlling stem cell lineage commitment and provide a promising scaffold for bone tissue engineering.

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Biomaterial surfaces and their nanostructures can significantly influence cell growth and viability. Thus, manipulating surface characteristics of scaffolds can be a potential strategy to control cell functions for stem cell tissue engineering. In this study, in order to construct a hydroxyapatite (HAp) coated genipin-chitosan conjugation scaffold (HGCCS) with a well-defined HAp nanostructured surface, we have developed a simple and controllable approach that allows construction of a two-level, three-dimensional (3D) networked structure to provide sufficient calcium source and achieve desired mechanical function and mass transport (permeability and diffusion) properties. Using a nontoxic cross-linker (genipin) and a nanocrystallon induced biomimetic mineralization method, we first assembled a layer of HAp network-like nanostructure on a 3D porous chitosan-based framework. X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM) analysis confirm that the continuous network-like nanostructure on the channel surface of the HGCCS is composed of crystalline HAp. Compressive testing demonstrated that the strength of the HGCCS is apparently enhanced because of the strong cross-linking of genipin and the resulting reinforcement of the HAp nanonetwork. The fluorescence properties of genipin-chitosan conjugation for convenient monitoring of the 3D porous scaffold biodegradability and cell localization in the scaffold was specifically explored using confocal laser scanning microscopy (CLSM). Furthermore, through scanning electron microscope (SEM) observation and immunofluorescence measurements of F-actin, we found that the HAp network-like nanostructure on the surface of the HGCCS can influence the morphology and integrin-mediated cytoskeleton organization of rat bone marrow-derived mesenchymal stem cells (BMSCs). Based on cell proliferation assays, rat BMSCs tend to have higher viability on HGCCS in vitro. The results of this study suggest that the fluorescent two-level 3D nanostructured chitosan-HAp scaffold will be a promising scaffold for bone tissue engineering application.

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As a biocompatible and bioactive natural tissue engineering scaffold, porcine acellular dermal matrix (PADM) has limitations for the application in tissue regeneration due to its low strength and rapid biodegradation. Here, purified PADM was modified by a nontoxic cross-linker (genipin) to enhance its mechanical properties and improve its resistance to enzymatic degradation. In vitro testing results demonstrated that the stiffness of the genipin cross-linked PADM was improved and biodegradation rate was decreased. Results of cell proliferation assays showed that the cross-linking reaction by genipin did not undermine the cytocompatibility of PADM. Furthermore, genipin cross-linking imparted an observable fluorescence allowing visualization of the scaffold's three-dimensional (3D) porous structure and cell distribution by confocal laser scanning microscopy (CLSM). Immunostaining of the cell nuclei and cytoskeleton indicated that MC3T3-E1 preosteoblasts were tightly adhered to and uniformly distributed onto the cross-linked PADM scaffold. Results of this study suggest that the 3D porous genipin cross-linked PADM with intrinsic fluorescence may have broader applications for tissue engineering scaffolds where higher mechanical stiffness is needed.

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