| BACKGROUDBone defects caused by trauma,infection,tumor and implant revision remain a significant challenge in orthopedics.Autologous bone graft and allogeneic bone graft still cannot fully meet the clinical need.Bone tissue engineering involves the use of biomaterials,cells and bioactive factors and represents a promising approach for bone regeneration.In the first and second chapters of this study,the periodontal ligament stem cells(hPDLSCs)were incorporated into the calcium phosphate cement(CPC)scaffold by constructing hydrogel microbeads for cell delivery,which could enhance the bone regeneration capacity of the CPC scaffold.Infectious bone defects,whose main cause is chronic inflammatory diseases such as periodontitis and osteomyelitis,are the most difficult to treat in clinic.The third and fourth chapter of this study combined doxycycline(DOX),chitosan and CPC to develop a bone tissue engineering scaffolds with antibacterial activity,and incorporate the platelet-rich lysate(hPL)into the scaffolds.The antibacterial CPC scaffolds exhibited excellent antibacterial activity and could promoting the osteogenic differentiation of stem cells via delivering growth factors,indicating that the CPC scaffolds can play a role in controlling infection in the treatment of infectious bone defects,as well as promoting the bone regeneration of infectious bone defects.Part-1Objective:Tissue engineering is promising for dental and craniofacial regeneration.The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells(hPDLSCs)for dental regeneration,and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement.Methods:hPDLSCswere cultured with Dulbecco’s modified eagle medium(DMEM),DMEM+10%fetal bovine serum(FBS),and DMEM+hPL(1%,2.5%,and 5%).hPDLSCs were encapsulated in alginate-fibrin microbeads(Alg+Fib),alginate-hPL microbeads(Alg+hPL),or alginate-fibrin-hPL microbeads(Alg+Fib+hPL).hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS.Quantitative real-time polymerase chain reaction(qRT-PCR),alkaline phosphatase(ALP)activity,ALP staining,and alizarin red(ARS)staining was investigated.Results:hPDLSCs were released faster from Alg+Fib+hPL than fromAlg+hPL.At 14 days,ALP activity was 44.1±7.61 mU/mg for Alg+Fib+hPL group,higher than 28.07±5.15 mU/mg of Alg+Fib(p<0.05)and 0.95±0.2 mU/mg of control(p<0.01).At 7 days,osteogenic genes(ALP,RUNX2,COL1,and OPN)inAlg+Fib+hPL and Alg+Fib were 3-10 folds those of control.At 21 days,the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group,respectively.Conclusions:The 2.5%hPL was determined to be optimal for hPDLSCs.AddinghPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads.The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct,showing a promising xeno-free approach for delivering hPDLSCs to enhance dental,craniofacial and orthopedic regenerations.Part-2Objectives:(1)Develop a novel construct of human periodontal ligament stem cells(hPDLSCs)encapsulated in hydrogel microbeads with human platelet lysate(hPL)and calcium phosphate cement(CPC);(2)Investigate the proliferation and osteogenic differentiation of hPDLSCs in CPC with hPL as axeno-free supplementand animal serumreplacement for bone tissue engineering applications.Methods:hPDLSCs were encapsulated in alginate-fibrin microbeads(Alg+Fib),alginate-hPL microbeads(Alg+hPL),or alginate-fibrin-hPL microbeads(Alg+Fib+hPL).The proliferation and osteogenic differentiation of hPDLSCs were investigated in culturing with CPC scaffold.Results:The flexural strength of CPC scaffold was 8.4±0.91 MPa,and the elastic modulus was 1.56 ± 0.1 GPa,exceeding the reported values of cancellous bone.hPDLSCs showedhigher viability in Alg+Fib+hPLgroup than in Alg+Fib group.ALP activity was 69.97±16.96 mU/mg for CPC+Alg+Fib+hPL group,higher than 30.68±2.86 mU/mg of CPC+Alg+Fib(p<0.05)and 4.12±1.65 mU/mg of control(p<0.01).At 7 days,osteogenic gene expressions(ALP,RUNX2,COL1,and OPN)in CPC+Alg+Fib+hPL and CPC+Alg+Fib were 4-11 folds that of control.At 21 days,hPDLSC-synthesized bone mineral amount in CPC+Alg+Fib+hPL and CPC+Alg+Fib was 13.2 folds and 11.1 folds that of control,respectively.Conclusions:The hPL-incorporatingalginate hydrogel is promising to protect and deliver hPDLSCs with CPC.The hPL-based medium significantly enhanced the osteogenic differentiation of hPDLSCs in CPC+Alg+Fib+hPL construct,suggesting a promising xeno-free approach for bone tissue regeneration..Part-3Objectives:(1)Develop a novel antibacterial scaffold of chitosan-reinforced calcium phosphate cementdeliveringdoxycyclinehyclate(CPCC+DOX)to combat infections;(2)Investigate proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)encapsulated in alginate beads and cocultured with CPCC+DOX scaffold.Methods:CPCC+DOX scaffolds were fabricated to contain different concentrations of doxycycline hyclate(DOX).Mechanical properties,drug release,and antibacterial activity againstStaphylococcus aureus(S.aureus)and Porphyromonasgingivalis(P.gingivalis)were evaluated.The proliferation and osteogenic differentiation of hPDLSCs encapsulated in alginate hydrogel microbeads were investigated in culture with CPCC+DOX scaffolds.Results:Flexural strength of CPCC+DOX ranged from(5.56±0.70)to(6.2±0.72)MPa,which exceeded the reported strength of cancellous bone.Scaffolds exhibited continual DOX release,reaching 80%at 21 d.Scaffold with 5mg/mL DOX(CPCC+DOX5mg)had a strong antibacterial effect,with a 4-log CFU reduction against S.aureus and P.gingivalis.CPCC+DOX5mg had no negative effect on proliferation of hPDLSCs.Alkaline phosphatase(ALP)activity,mineral synthesis,and osteogenic gene expressions for CPCC+DOX5mg group were much higher than control group.DOX did not compromise the osteogenic induction.Conclusions:The novel CPCC+DOX scaffold exhibited excellent mechanical properties and strong antibacterial activity,while supporting the proliferation and osteogenic differentiation of hPDLSCs.The CPCC+DOX+hPDLSCs construct is promising to enhance bone regeneration and combat bone infections in dental,craniofacial and orthopedic applications.Part-4Objectives:This study developed for the first time a calcium phosphate cement(CPC)for delivery of doxycycline(DOX)and human platelet lysate(hPL)to fight against S.aureus infection and enhance the osteogenesis of human periodontal ligament stem cells(hPDLSCs).Methods:Chitosan-containing CPC scaffolds were fabricated in the absence(CPCC)or presence of DOX(CPCC+DOX).In addition,hPL was encapsulated in alginate microbeads and incorporated into CPCC+DOX(CPCC+DOX+hPL).The hPDLSCs encapsulated in microbeads were co-cultured with the CPCs.Results:Flexural strength of CPCC+DOX+hPL was(5.56±0.55)MPa,lower than(8.26±1.6)MPa of CPCC+DOX(p<0.05),but exceeding the reported strength of cancellous bone.CPCC+DOX and CPCC+DOX+hPL exhibited strong antibacterial activity against S.aureus,reducing biofilm CFU by 4 orders of magnitude.The hPDLSCs were able to be released from the microbeads and showed a high proliferation rate,increasing by about 8 folds at 14 days for all groups.The hPL was released from the scaffold and promoted the osteogenic differentiation of hPDLSCs.ALP activity was 28.07±5.15 mU/mg for CPCC+DOX+hPL,higher than 17.36±2.37 mU/mg and 1.34±0.37 mU/mg of CPCC+DOX and CPCC,respectively(p<0.05).The amount of hPDLSC-synthesized bone mineral with CPCC+DOX+hPL was 3.8 folds that of CPCC(p<0.05).Conclusions:The novel CPC+DOX+hPL-hPDLSCs scaffold exhibited strong antibacterial activity,excellent cytocompatibility and hPDLSC osteogenic differentiation,showing a promising approach for treatment and prevention of bone infection and enhancement of bone regeneration. |
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