Chitosan/Hyaluronic Acid/MicroRNA-21 Nanoparticle-Coated Smooth Titanium Surfaces Promote the Functionality of Human Gingival Fibroblasts.

Purpose: Forming a compact biological seal between the gingiva and the implant interface around the percutaneous parts of an implant is one of the key issues in preventing peri-implantitis. Methods: In this study, since microRNA-21 (miR-21) has been approved to promote fibroblast proliferation and collagen formation in skin fibrosis, we prepared miR-21-loaded chitosan (CS)/tripolyphosphate (TPP)/hyaluronic acid (HA) nanoparticles (CTH NPs) and cross-linked them to smooth Ti surfaces with 0.2% gel solution for reverse transfection, after which isolated human gingival fibroblasts were cultured on the miR-21-functionalized Ti substrates. Results: An optimal CS:TPP:HA ratio (1:0.15:0.1) and N/P ratio (20:1) were chosen to produce appropriate nanoparticles. Finally, the CTH/miR-21 nanoparticle-coated smooth Ti surfaces demonstrated increased fibroblast adhesion, proliferation and expression of extracellular matrix-related genes along with similar cytotoxicity and cell spreading on the miR-21-functionalized Ti surface and the unmodified smooth Ti surface. Conclusion: The chitosan-based nanoparticles might be an efficient nonviral miRNA vector to form a stable biological seal in percutaneous areas of Ti for clinical use.

In situ controlled release of stromal cell-derived factor-1α and antimiR-138 for on-demand cranial bone regeneration.

Bone regeneration involves complex physiological processes, which is generally regulated and controlled by multiple bioactive molecules. In situ controlled release of combined bioactive factors in a spatiotemporal sequence for adapting the demand of bone regeneration is a desired strategy. In this study, nanoparticle/hydrogel composite system was constructed by incorporating stromal cell derived factor-1α (SDF-1α) and chitosan/tripolyphosphate/hyaluronic acid/antimiRNA-138 nanoparticles (CTH/antimiR-138 NPs) in chitosan/ß-sodium glycerol phosphate (CS/GP) hydrogel for rat critical-size calvarial bone regeneration. The fast release of SDF-1α promoted the migration of mesenchymal stem cells (MSCs) for 6 d, while the sustained release of antimiR-138 from the nanoparticle/hydrogel compound enhanced the osteogenic differentiation of MSCs over 21 d. 8 weeks after surgery, calvarial specimens were evaluated by microcomputed tomography (µ-CT), histological analysis and immunohistochemistry. Comparing with blank group and hydrogel group, hydrogels incorporated with SDF-1α and/or CTH/antimiR-138 NPs significantly enhanced bone regeneration (p<0.05). In addition, the expression of collagen type-1 (COL-1), osteopontin (OPN) and osteocalcin (OCN) proteins were enhanced in the combined drug group (incorporated both SDF-1α and CTH/antimiR-138 NPs) in comparison to the hydrogel group. Our research indicated the in situ formation of NPs/hydrogel composite could provide temporal sequence-release of SDF-1α and CTH/antimiR-138 NPs for on-demand MSCs homing and cranial bone regeneration.

Platelet-rich plasma gel composited with nondegradable porous polyurethane scaffolds as a potential auricular cartilage alternative.

Total auricular reconstruction is still a challenge, and autologous cartilage transplant is the main therapy so far. Tissue engineering provides a promising method for auricular cartilage reconstruction. However, although degradable framework demonstrated excellent initial cosmetic details, it is difficult to maintain the auricular contour over time and the metabolites tended to be harmful to human body. In this study, biocompatible and safe nondegradable elastic polyurethane was used to make porous scaffold in specific details by rapid prototyping technology. Platelet-rich plasma contains fibrin and abundant autologous growth factors, which was used as cell carriers for in vitro expanded cells. When crosslinking polyurethane framework, platelet-rich plasma and cells together, we successfully made polyurethane/platelet-rich plasma/cell composites, and implanted them into dorsal subcutaneous space of nude mice. The results showed that this method resulted in more even cell distribution and higher cell density, promoted chondrocyte proliferation, induced higher level expressions of aggrecan and type II collagen gene, increased content of newly developed glycosaminoglycans, and produced high-quality cartilaginous tissue. This kind of cartilage tissue engineering approach may be a potential promising alternative for external ear reconstruction.

In vitro studies on human periodontal ligament stem cell sheets enhanced by enamel matrix derivative.

Numerous preclinical and clinical studies have focused on the periodontal regenerative functions of enamel matrix derivative (EMD), a heat-treated preparation derived from enamel matrix proteins (EMPs) of developing porcine teeth. In this study, periodontal ligament (PDL) stem cells (PDLSCs) were isolated, and the effects of EMD on the extracorporeal induction process and the characteristics of PDLSC sheets were investigated for their potential as a more effective stem-cell therapy. EMD-enhanced cell sheets could be induced by complete medium supplemented with 50 µg/mL vitamin C and 100 µg/mL EMD. The EMD-enhanced cell sheets appeared thicker and more compact than the normal PDLSC sheets, demonstrated more layers of cells (3-7 layers), secreted richer extracellular matrix (ECM), showed varying degrees of increases in mRNA expression of periodontal tissue-specific genes (COL I, POSTN), calcification-related genes (RUNX2, OPN, OCN) and a cementum tissue-specific gene (CAP), and possessed a better mineralization ability in terms of osteogenic differentiation in vitro. These EMD-enhanced cell sheets may represent a potential option for stem-cell therapy for PDL regeneration.

The use of platelet-rich fibrin combined with periodontal ligament and jaw bone mesenchymal stem cell sheets for periodontal tissue engineering.

Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration.

Microarc-oxidized titanium surfaces functionalized with microRNA-21-loaded chitosan/hyaluronic acid nanoparticles promote the osteogenic differentiation of human bone marrow mesenchymal stem cells.

Dental implants have been widely used for the replacement of missing teeth in the clinic, but further improvements are needed to meet the clinical demands for faster and tighter osseointegration. In this study, we fabricated safe and biocompatible chitosan (CS)/hyaluronic acid (HA) nanoparticles to deliver microRNA-21 (miR-21) and thereby accelerate osteogenesis in human bone marrow mesenchymal stem cells (hBMMSCs). The CS/HA/miR-21 nanoparticles were cross-linked with 0.2% gel solution onto microarc oxidation (MAO)-treated titanium (Ti) surfaces to fabricate the miR-21-functionalized MAO Ti surface, resulting in the development of a novel coating for reverse transfection. To characterize the CS/HA/miR-21 nanoparticles, their particle size, zeta potential, surface morphology, and gel retardation ability were sequentially investigated. Their biological effects, such as cell viability, cytotoxicity, and expression of osteogenic genes by hBMMSCs on the miR-21-functionalized MAO Ti surfaces, were evaluated. Finally, we explored appropriate CS/HA/miR-21 nanoparticles with a CS/HA ratio of 4:1 and N/P ratio 20:1 for transfection, which presented good spherical morphology, an average diameter of 160.4±10.75 nm, and a positive zeta potential. The miR-21-functionalized MAO Ti surfaces demonstrated cell viability, cytotoxicity, and cell spreading comparable to those exhibited by naked MAO Ti surfaces and led to significantly higher expression of osteogenic genes. This novel miR-21-functionalized Ti implant may be used in the clinic to allow more effective and robust osseointegration.

A new method of wound treatment: targeted therapy of skin wounds with reactive oxygen species-responsive nanoparticles containing SDF-1α.

OBJECTIVE: To accelerate wound healing through promoting vascularization by using reactive oxygen species (ROS)-responsive nanoparticles loaded with stromal cell-derived factor-1α(SDF-1α). METHODS: The ROS-reactive nanomaterial poly-(1,4-phenyleneacetone dimethylene thioketal) was synthesized, and its physical and chemical properties were characterized. ROS-responsive nanoparticles containing SDF-1α were prepared through a multiple emulsion solvent evaporation method. The loading capacity, stability, activity of the encapsulated protein, toxicity, and in vivo distribution of these nanoparticles were determined. These nanoparticles were administered by intravenous infusion to mice with full-thickness skin defects to study their effects on the directed chemotaxis of bone marrow mesenchymal stem cells, wound vascularization, and wound healing. RESULTS: The synthesized ROS-reactive organic polymer poly-(1,4-phenyleneacetone dimethylene thioketal) possessed a molecular weight of approximately 11.5 kDa with a dispersity of 1.97. ROS-responsive nanoparticles containing SDF-1α were prepared with an average diameter of 110 nm and a drug loading capacity of 1.8%. The encapsulation process showed minimal effects on the activity of SDF-1α, and it could be effectively released from the nanoparticles in the presence of ROS. Encapsulated SDF-1α could exist for a long time in blood. In mice with full-thickness skin defects, SDF-1α was effectively released and targeted to the wounds, thus promoting the chemotaxis of bone marrow mesenchymal stem cells toward the wound and its periphery, inducing wound vascularization, and accelerating wound healing.

MAPs/bFGF-PLGA microsphere composite-coated titanium surfaces promote increased adhesion and proliferation of fibroblasts.

Infection and epithelial downgrowth are two major problems with maxillofacial transcutaneous implants, and both are mainly due to lack of stable closure of soft tissues at transcutaneous sites. Fibroblasts have been shown to play a key role in the formation of biological seals. In this work, titanium (Ti) model surfaces were coated with mussel adhesive proteins (MAPs) utilizing its unique adhesion ability on diverse inorganic and organic surfaces in wet environments. Prepared basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic acid) (PLGA) microspheres can be easily synthesized and combined onto MAPs-coated Ti surfaces, due to the negative surface charges of microspheres in aqueous solution, which is in contrast to the positive charges of MAPs. Titanium model surfaces were divided into three groups. Group A: MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces. Group B: MAPs-coated Ti surfaces. Group C: uncoated Ti surfaces. The effects of coated Ti surfaces on adhesion of fibroblasts, cytoskeletal organization, proliferation, and extracellular matrix (ECM)-related gene expressions were examined. The results revealed increased adhesion (P < 0.05), enhanced actin cytoskeletal organization, and up-regulated ECM-related gene expressions in groups A and B compared with group C. Increased proliferation of fibroblasts during five days of incubation was observed in group A compared with groups B and C (P < 0.05). Collectively, the results from this in vitro study demonstrated that MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces had the ability to increase fibroblast functionality. In addition, MAPs/bFGF-PLGA microsphere composite-coated Ti surfaces should be studied further as a method of promoting formation of stable biological seals around transcutaneous sites.