The Encapsulation Of Synthetic Oxygen Carrier PFTBA In ADSCs-VECs Based Hydrogel Promotes Bone Regeneration

Bone fracture,especially large bone defect,is still a main challenge for orthopedist.Bone fracture results in the distortion of bone architecture and the rupture of blood vessels in fracture site.The damaged vasculatures fail to provide sufficient oxygen and nutrients to the injury tissue,which causes the degradation and necrosis of the surrounding tissues.It is reported that the oxygen tension in normal bone is about 12.5%,and rapidly decreases into ~1% when bone fracture or bone defect happens.Oxygen is considered as the critical element in bone fracture healing process since it is requisite in many bone related cellular activities.For example,the alkaline phosphatase(ALP)activity and gene expression of rat osteoblasts were sharply decreased when cultured in hypoxia environment(2% O2).Furthermore,bone mineralization activity was almost abolishedwhen the p O2 was 0.2%.In addition,oxygen is essential for the activation of many enzymes,such as hydroxylases,oxygenases,and cyclooxygenase,which are related to bone regeneration.Moreover,hypoxia inhibits the synthesis of collagen,which is the major organic component of bone.It is reported that oxygen is required for the hydroxylation of lysine and proline during collagen crosslinking and bundle formation.So oxygen supply is important for bone fracture area.In the present study,we encapsulatethe synthetic oxygen carrier PFTBA into VECs-ADSCs based Gel MA hydrogel,to investigate the effect of PFTBA on the functions of ADSCs and VECs under hypoxia environment,such as osteogenic differentiation of ADSCs and tube formation of VECs.We also place this VECs-ADSCs based Gel MA-Gel MA hydrogel into rat calvarial defect to investigate the effect of this hybrid hydrogel on vascularization and bone regeneration.Part 1: The PFTBA-Gel MA hydrogel preparationand properties evaluationBackgrounds: Bone is a typical complex tissue with hierarchical structurethat consists of approximately 70% of nano hydroxyapatite(nano-HAP,Ca10(PO4)6(OH2))and 30% of collagen by weight.Bone fracture may destroy the bone architecture and blood vessels,which results in the hypoxia environment in the bone defect site.Long-term inadequate oxygen supply is an important contributing factor for delayed fracture healing or nonunion.Various treatment options are availableto promote bone defects repair such as autograft,allograftand xenograft.However,there are some disadvantages for these treatments,such as insufficient donor sites,chance of transmissible disease,immunological rejection and so on.Tissue engineering bone is a promising alternative for bone defect repair.As one of the basic elements,scaffold is crucial for successful bone defect repair.Gelatin methacryloyl(Gel MA)hydrogel scaffold has been widely applied for various biomedical applications due to their suitable biological and tunable physical characteristics.However,the low oxygen availability within the scaffold and the hypoxia environment in bone defect site are still an obstacle for bone regeneration.To overcoming the hypoxia situation,oxygen-releasing hydrogel scaffold is an optimal option.In this study,we encapsulated the oxygen carrier material PFTBA in the Gel MA hydrogel,to investigate the effect on cell viability under hypoxia.Objective: To fabricate the oxygen-releasing PFTBA-GleMA hydrogel and further evaluate the corresponding properties.Methods: GelMA was synthesized by the direction of Gelatin with MA in phosphate buffer.And then we encapsulated different concentration of PFTBA(0%,5%,10%)in the Gel MA hydrogel.Different UV exposure time(15 s,20 s,25 s,30 s)was applied to crosslink the PFTBA-Gel MA solution.The hydrogel stiffness was examined by Instron 5542 mechanical tester.ADSCs were cultured in this 3D hydrogel and placed in hypoxia incubator(1% oxygen).The proliferation of ADSCs was examined by Presto Blue assay.The cell viability was examined by Live/Dead assay,and the percentage of live cells was calculated.Result: GelMA hydrogel which exposed in different UV light timeshowed different stiffness: 15 s is 13.87 ± 1.30 Kpa,20 s is 19.76 ± 1.61 Kpa,25 s is 25.43 ± 1.89 Kpa,30 s is 31.23 ± 3.21 Kpa.Presto Blue assay showed that 10% PFTBA promoted the proliferation of ADSCs.Live/Dead assay showed that cell spread well in 10% PFTBA-Gel MA group and the percentage of live cell is 0.82 ± 0.06.Conclusion: The stiffness of PFTBA-Gel MA hydrogel can be controlled by the UV exposure time.This oxygen released hydrogel could provide enough oxygen to cell for their proliferation and function under hypoxia.Part 2: The effect of PFTBA-Gel MA hydrogel on osteogenic differentiation of ADSCs and vascularization of VECs under hypoxia in vitroIntroduction: Bone fracture destroys the blood vessels in fracture site,which results in insufficient oxygen supply for the bone healing process.Oxygen is considered as the critical element for cell metabolism.It is reported that long-term hypoxia inhibits osteogenic differentiation and vascularization.Researchers are searching for the oxygen released materials which could improve the oxygen level in bone fracture site.In this study,we choose PFTBA as the oxygen carrier material,and investigate the effect of PFTBA-Gel MA hydrogel on osteogenic differentiation and vascularization.Methods: ADSCs were cultured in this PFTBA-GelMA hydrogel and then placed in hypoxia incubator(1% O2).Group: Hypoxia,Hypoxia+5% PFTBA,Hypoxia+10% PFTBA,Normoxia.After 7 and 14 days,the ALP assay and Alizarin Red S staining assay were finished to investigate the osteogenic ability of ADSCs in different concentration of PFTBA under hypoxia.F-actin/OPN double staining showed the osteogenic differentiation of ADSCs in 3D hydrogel under hypoxia.Tube formation assay was conducted to investigated the vascularization of ECs under hypoxia.The bone and vascular related gene expression were conducted by RT-PCR.Then we co-culture VECs with ADSCs in PFTBA-Gel MA hydrogel under hypoxia to investigate if this osteogenic and vascularization effect were enhanced or not when compare with monolayer cell.Results: ALP activity assay and mineralization assay demonstrated that cells grow in 10% PFTBA group showed higher osteogenic effect compare with Hypoxia and 5% PFTBA groups.F-actin/OPN double staining showed cells could spread well and show more OPN staining in 10% PFTBA and Normoxia groups.For gene expression,bone(ALP,RUNX2,OPN,BMP-2)and vascular related genes(CD31,VEGF)in 10% PFTBA group were significant higher than Hypoxia and 5% PFTBA groups,similar with Normoxia group.And gene expression showed that the vascular and osteogenic effect was enhanced when we co-culture these cells.Conclusion: 10% PFTBA-Gel MA hydrogel shows excellent osteogenic and vascularization effect on ADSCs and VECs under hypoxia.And the osteogenic and vascular effect are enhanced when ADSCs co-culture with VECs.Part 3: The encapsulation of ADSCs-VECs in PFTBA-Gel MA hydrogel promotes bone regenerationin vivoBackgrounds: Tissue engineering bone scaffolds have been demonstrated to be realistic alternatives in bone defect repair.However,the low oxygen availability within bone defect site is still an obstacle for bone regeneration.In our previous research,we fabricated the oxygen released hydrogel PFTBA-Gel MA,and investigated its osteogenic and vascular effect on ADSCs and VECs in vitro.However,the effect of this ADSCs-VECs based PFTBA-Gel MA hydrogel on bone regenerationin vivo is still unknown.Objective: To investigated the efficacy of the ADSCs-VECs based PFTBA-GelMA hydrogel on bone regeneration in vivoMethods: Groups: Control group(Gel MA hydrogel),PFTBA-Gel MA group(10% PFTBA-Gel MA hydrogel),ADSCs-VECs based PFTBA-Gel MA group.Different groups of hydrogel were transplanted subcutaneously on the back of nude mice to examine ectopic bone formation for 2 weeks and 4 weeks.Different groups of hydrogel were used to repair the 5 mm rat calvarial defect.Specimens were collected at 2 and 4 weeks after operation to evaluate the osteogenic effect.The gene expression of BMP-2,ALP,OPN,RUNX2,VEGF and CD31 was conducted to examine the osteogenic differentiation and vascularization.Results: HE staning showed that the new bone formation capacity was gradually increased from Control group to Cells+PFTBA group,which were 2.74 ± 0.64% and 4.96 ± 0.93% in Control group,6.62 ± 0.92 and 9.86 ± 1.92% in ADSCs+VECs group,and 14.31 ± 1.47% and 23.47 ± 3.21% in Cells+PFTBA group.The results of Micro-CT scan demonstratedthat the restorative effect(new bone formation)in Cells+PFTBA group is significant higher that Control and ADSCs+VECs group.Gene expression showed that the osteogenesis and angiogenesis both were enhanced in Cells+PFTBA group compare with Control and ADSCs+VECs group.Conclusion: The ADSCs-VECs based PFTBA-Gel MA hydrogel could promote ectopic bone formation and rat calvarial defect regeneration.