Bone Mesenchymal Stem Cells Delivered By Silk Hydrogel For Enhanced Bone Regeneration

[Objective]Stem-cell-based bone engineering is considered to be a promising method for bone regeneration.It is well recognized that the seeded stem cells play a crucial role in bone engineering.However,the low survival rate of stem cells after implanted in vivo remains a challenge in bone engineering.In order to acquire bone regeneration with a long time in vivo preservation of the seeded cells'viability and function,this study seeks to use silk hydrogel to quantitatively encapsulate rat bone mesenchymal stem cells,then the cell containing silk hydrogel was dropped onto porous silk scaffold for rat calvarial defect repairmen.[Materials and Methods]1.Using SEM and FI-TR to evaluate the characterization of the porous silk scaffold.Evaluate the permeation condition of the silk hydrogel/scaffold complex.2.Rat bone mesenchymal stem cells were mingled with sonicated silk solutions at the concentrations of 1.0×10~5/mL,1.0×10~6/mL and 1.0×10~7/mL before gelation.The distribution of encapsulated stem cells was observed under microscope.MTT experiment was carried to evaluate the proliferation of encapsulated stem cells.The osteogenic potential of different experiment groups was evaluated using alkaline phosphatase(ALP)activity assay,calcium deposition assay and RT-PCR experiment.3.The different groups of stem cells encapsulated silk hydrogel were dropped to porous silk scaffolds and applied on rat calvarial defect.Micro CT and Van Gibson staining were used to evaluate the bone regeneration result.Dil-staining cell tracing experiment was included to observe the preservation of cell viability and function after implanted in vivo for 8 weeks.[Results]1.The porous silk scaffold possesses an interconnected structure.Both small molecules and proteins could permeate through the silk hydrogel/scaffold complex in about 20 minutes.2.The encapsulated stem cells were homogenously distributed in the silk hydrogel.MTT shows stable cell viability after incubated in vitro for ten days.The hydrogel with 1.0×10~7/mL stem cells exhibited the highest ALP expression,most calcium deposition and strongest osteogenic-related genes expression.3.The silk hydrogel/scaffold complex with stem cell at the concentration of 1.0×10~7/mL has demonstrated the highest new bone area and most trabecular bone formation in the rat calvarial defect area.The cell-tracing experiment shows that the encapsulated stem cells still survive and actively participate in new bone formation after implanted in vivo for 8 weeks.[Conclusion]The strategy of using the cell encapsulated silk hydrogel/scaffold for bone regeneration could effectively retain the stem cells'viability and support them to actively participate in local bone formation.The pre-mineralized silk hydrogel/scaffold with encapsulated cell at the concentration of 1.0×10~7/mL has achieved fast bone regeneration with well-distributed trabecular bone.