Triple Cross-Linked Hyaluronic Acid Based On Tri-Hyal Technique Has More Durable Effect On Dermal Renewal

Background:Cross-linked hyaluronic acid（HA）is widely used for correction of wrinkles,filling of facial volume loss and facial remodeling.Many studies have found that except the effect of replenishing volume,cross-linked HA also show some biological effects on the skin rejuvenation.Many efforts have been made to improve HA cross-linking technology on prolonging the duration in aesthetic indications,promoting the effect on the skin rejuvenation,as well as minimizing the amount of cross-linking agent to reduce the incidence of adverse reactions.As a new patented cross-linking technology,Tri-Hyal technology nicely combined the long chain,very-long chain and free HA together to achieve the desired rheological characteristics in the dermal filler.Based on this technique,HA filler obtained better properties for the treatment of skin rejuvenation,which improved the facial volume loss by adding volume or shape restoration of the aging face.More importantly,the slow release of the non-cross-linked HAs to the dermis,which were trapped among the very-long chain and long chain cross-linked HA,could provide more suitable environment for dermal fibroblasts to produce several extracellular matrix components for skin self-renewal continuously.while the cross-linked HA would persist for a long time in the tissue.Additionally,the mixture of three HAs with three different molecular weights has the characteristic of a natural entanglement in Tri-Hyal technique.The presence of natural entanglement could minimize the amount of cross-linking agent.The mechanism for the stimulation of skin self-renewal by cross-linked HA products is not well understood,especially one with the Tri-Hyal,which is a triple cross-linking technique for sustained release of free HAs.Objective:To evaluate the efficiency and persistence of HAs with Tri-Hyal on skin rejuvenation and further investigate underlying mechanisms,we compared the performances of cross-linked HA（AF）based on Tri-Hyal with another highly acceptable HA filler（Res）in vivo and in vitro.Method:In vivo experiments:1.Animal grouping:a total of 12 skin injection points on the back of each balb/c mouse（n=6）were divided into 3 groups:Vehicle Group（4）,AF group（4）and res group（4）.AF group was treated with art filler universal,res group with RestylaneⅡ,and vehicle group with PBS.2.Animal treatment:balb/c mice were routinely fed with standard diet and injected subcutaneously with AF,res and PBS at multiple points on the back,50 for eachμl。At 4Points in each group,skin tissues were taken for biopsy at the corresponding points on the0,30,90 and 180 days respectively.3.Hematoxylin eosin staining（H&E）was used to compare the effects of HA products with different cross-linking technologies on skin thickness4.The effects of HA products with different cross-linking technologies on dermal elastic fibers were compared by elastic fiber staining.5.Immunohistochemical staining was used to compare the effects of different crosslinking techniques on the production of vascular endothelial cell adhesion molecules（CD31）,the production of transforming growth factor-β（TGF-β）,and the expression of matrix metalloproteinase 9（MMP9）.In vitro experiments:1.Human dermal fibroblast cells are divided into 3 groups:AF test group,HA control group,and Vehicle group.2.Cell treatment:human dermal fibroblasts were seeded on precoated culture plates with AF and Res in advance（HA concentration is 5 mg/ml）.3.Detection of proliferation activity of primary human skin fibroblasts by CCK-8.4.Theβ-galactosidase（SA-β-gal）staining kit was used to detect the senescence of skin fibroblasts.5.The Western Blot was used to detect the protein levels of autophagy marker LC3II/I and P62,HA receptor CD44 and UPR-related pathway proteins.6.GFP-RFP-LC3 adenovirus was used for autophagic flux detection.7.Flow cytometry was used to detect the reactive oxygen species（ROS）level of human dermal fibroblasts.8.ER-Hyper adenovirus was used to detect the level of H₂O₂ in the endoplasmic reticulum of human dermal fibroblasts.9.Inhibition of perk pathway,detection of autophagy flow level,aging,ROS level and cell viability level.Result:In vivo experiments:1.AF Based on Tri-Hyal Technique Increased Skin Thickness and Formation of Elastic Fibers in Mouse Dorsal Skin More Persistently.Data from dermal skin biopsies with H&E and MT staining after the respective treatments showed that both cross-linked HA injection could stimulate dermal growth as early as 30days and there was no difference between two groups.From 90 to 180 days,we observed a slight decrease of skin thickness and elastic fibers in skin injected with PBS.From 30 days on,HA injection promoted dermal thickness and elastic fibers formation.This elevating trend reached peak in 90 days in HA injection group,while the trend was kept until 180 days in AF group.That judgement is proved by histological analysis.Totally,AF with sustained release Tri-Hyal technique consistently stimulates the dermal growth lasting for more than half a year without efficiency attenuation.2.AF with Tri-Hyal Technique Increased the Expression Level of CD31 and TGF-βand Decreased the Expression of MMP9 More Durably.Quantitative assessment of vascular density by IHC for CD31 showed a reduction of the cutaneous microvasculature in skin injected with vehicle and increased blood vessels in the mid-to-deep dermis in both filler-injected specimens after 30 days.In particularly,AF group showed much more increased vessels staining than Res treatment.The vascular density increased even 90 days after the AF injection.TGF-βexpression was gradually decreased in the vehicle group and increased in the other two HA groups from 90 days after injection.From 90 days to 180 days,the expression of TGF-βin Res group began to decrease,while in the AF group which still showed a upward trend.Conversely,MMP9 expression levels increased gradually over time in the vehicle control,while decreased in the other two HA-treating groups.In vitro experiments:1.AF with Tri-Hyal Technique Delayed the Fibroblasts Senescence in Vitro.The cell proliferation activity was detected by CCK8 experiment.The results showed that compared with vehicle group,the proliferation activity of fibroblasts co incubated in res group and AF group was significantly higher,and AF group was more significant than res group.Data from SA-β-gal staining showed that AF treatment robustly decreased the number of positive-stained cells compared with Res and vehicle.Consistent with this,AF significantly inhibited the SA-β-gal activity compared with the Res and vehicle.2.AF with Tri-Hyal Technique Reduced the ROS Level in Fibroblasts Through Inducing Autophagy.Data from western blot showed that AF and Res both increased the expression of CD44 and the ratio of LC3II to LC3I,decreased the expression of P62,which were molecular markers of autophagy.Autophagy flux also showed that AF and Res promoted autophagy in fibroblasts compared with vehicle,while the effect of AF was more significant than Res.Data from flow cytometry with DCFH-DA showed that AF could significantly reduce the ROS level in fibroblast compared with Res treatment,although Res also has the inhibitory effect on ROS level.3.AF with Tri-Hyal Technique induced autophagy and improves cellular senescence by initiating unfolded protein response（UPR）.ER-Hyper adenovirus was manipulated to detect H₂O₂ level in the endoplasmic reticulum（ER）.Data showed that the levels of endoplasmic reticulum in the Res group and AF group were significantly lower than those in the Vehicle group,and the AF group was more significant than that in the Res group.Western Blot showed that the Res and AF treatment significantly activated UPR.After inhibiting the PERK pathway,the effects of AF on inducing autophagy,delaying senescence,reducing ROS,as well as promoting cell viability were partially or completely blocked.Conclusion:In this study in vivo,our data showed that cross-linked AF with triple sustained-release cross-linking Tri-Hyal technique consistently stimulated the dermal growth lasting for more than half year without efficiency attenuation,which effect lasted more persistently than that of traditional HA.In addition,AF with this unique technology could be associated with the proliferation,viability and activity of human primary fibroblasts in vitro.Mechnismly,AF reduced the level of H₂O₂ in ER and then initiated the UPR pathway,which would induced the cellular autophagy to decrease the total ROS in the cell.