Local Application of Semaphorin 3A Combined with Adipose-Derived Stem Cell Sheet and Anorganic Bovine Bone Granules Enhances Bone Regeneration in Type 2 Diabetes Mellitus Rats.

Bone tissue regeneration is considered to be the optimal solution for bone loss. However, diabetic patients have a greater risk of poor bone healing or bone grafting failure than nondiabetics. The purpose of this study was to investigate the influence of the complexes of an adipose-derived stem cell sheet (ASC sheet) and Bio-Oss® bone granules on bone healing in type 2 diabetes mellitus (T2DM) rats with the addition of semaphorin 3A (Sema3A). The rat ASC sheets showed stronger osteogenic ability than ASCs in vitro, as indicated by the extracellular matrix mineralization and the expression of osteogenesis-related genes at mRNA level. An ASC sheet combined with Bio-Oss® bone granules promoted bone formation in T2DM rats as indicated by microcomputed tomography (micro-CT) and histological analysis. In addition, Sema3A promoted the osteogenic differentiation of ASC sheets in vitro and local injection of Sema3A promoted T2DM rats' calvarial bone regeneration based on ASC sheet and Bio-Oss® bone granule complex treatment. In conclusion, the local injection of Sema3A and the complexes of ASC sheet and Bio-Oss® bone granules could promote osseous healing and are potentially useful to improve bone healing for T2DM patients.

Semaphorin 3A-modified adipose-derived stem cell sheet may improve osseointegration in a type 2 diabetes mellitus rat model.

Although titanium (Ti) implants are considered to be an optimal choice for the replacement of missing teeth, it remains difficult to obtain sufficient osseointegration in patients with type 2 diabetes mellitus (T2DM). The present study aimed to investigate whether adipose-derived stem cells (ASCs) may be used to improve Ti implant osseointegration in T2DM conditions with the addition of semaphorin 3A (Sema3A), a recently identified osteoprotective protein. Cell morphology was observed using a scanning electron microscope. Cell proliferation was determined using Cell Counting Kit­8. Osteogenic differentiation was confirmed by the staining of alkaline phosphatase, collagen secretion and calcium deposition. An in vivo evaluation was performed in the T2DM rat model, which was induced by a high­fat diet and a low­dose streptozotocin intraperitoneal injection. A Sema3A­modified ASC sheet was wrapped around the Ti implant, which was subsequently inserted into the tibia. The rats were then exposed to Sema3A stimulation. The morphology and proliferation ability of ASCs remained unchanged; however, their osteogenic differentiation ability was increased. Micro­computed tomography scanning and histological observations confirmed that formation of new bone was improved with the use of the Sema3A-modified ASCs sheet. The present study indicated that the Sema3A­modified ASCs sheet may be used to improve osseointegration under T2DM conditions.

Biofunctionalization of titanium implant with chitosan/siRNA complex through loading-controllable and time-saving cathodic electrodeposition.

Although titanium (Ti) implants are already being broadly used, further improvement is still mandatory for improving its clinical performance. To this end, small interfering RNA (siRNA) biofunctionalization may present a novel strategy. In this study, siRNA biofunctionalization was realized on a titania nanotube array (TNT) on Ti by the cathodic electrodeposition (CED) of a chitosan (CS)/siRNA complex. The siRNA deposition amount was linearly correlated to the current density and the siRNA release profile was sensitive to the pH value of the surrounding solution. To confirm the activity of the cathodically electrodeposited siRNA, the siRNA targeting green fluorescent protein (siGFP) was introduced by CED, and the primary rat mesenchymal stem cells (rMSCs) encoding a GFP expression motif were seeded. High siRNA delivery efficiency and sustained target gene down-regulation were observed, which were much better than the conventional CS/siRNA nanoparticle delivery system and were related to the enhanced siRNA access into the cytoplasm. Simultaneously, good cytocompatibility was observed from the CS/siRNA biofunctionalization, as indicated by the good cell adhesion and viability. Finally, the siRNA targeting casein kinase-2 interacting protein-1 (siCkip-1), reported to have osteogenic potential, was introduced by CED to assess the potential of CS/siRNA biofunctionalization for application in bone implants. The CS/siCkip-1 biofunctionalization magnificently enhanced the osteogenic differentiation of rMSCs in terms of improved osteogenesis related gene expression, collagen secretion and calcium deposition, showing high promise for application in bone implants. The study demonstrates a new and convenient way to functionalize the biomedical metal implants with CS/siRNA for surface activation via local siRNA delivery.

Incorporation of osteogenic and angiogenic small interfering RNAs into chitosan sponge for bone tissue engineering.

Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs), ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1) and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1), which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and angiogenic siRNAs may be used as a scaffold for bone regeneration. The dual siRNA concept may also be useful in the biofunctionalization of other materials.

Immobilization of chitosan film containing semaphorin 3A onto a microarc oxidized titanium implant surface via silane reaction to improve MG63 osteogenic differentiation.

Improving osseointegration of extensively used titanium (Ti) implants still remains a main theme in implantology. Recently, grafting biomolecules onto a Ti surface has attracted more attention due to their direct participation in the osseointegration process around the implant. Semaphorin 3A (Sema3A) is a new proven osteoprotection molecule and is considered to be a promising therapeutic agent in bone diseases, but how to immobilize the protein onto a Ti surface to acquire a long-term effect is poorly defined. In our study, we tried to use chitosan to wrap Sema3A (CS/Sema) and connect to the microarc oxidized Ti surface via silane glutaraldehyde coupling. The microarc oxidization could formulate porous topography on a Ti surface, and the covalently bonded coating was homogeneously covered on the ridges between the pores without significant influence on the original topography. A burst release of Sema3A was observed in the first few days in phosphate-buffered saline and could be maintained for >2 weeks. Coating in phosphate-buffered saline containing lysozyme was similar, but the release rate was much more rapid. The coating did not significantly affect cellular adhesion, viability, or cytoskeleton arrangement, but the osteogenic-related gene expression was dramatically increased and calcium deposition was also abundantly detected. In conclusion, covalent bonding of CS/Sema could strongly improve osteogenic differentiation of osteoblasts and might be applied for Ti implant surface biofunctionalization.