A Comparative Study On The Efficacy And Their Impact On Surrounding Tissue Of Different Soft Tissue Fillers

Background:In recent decades,a conceptual shift in the approach to facial rejuvenation has occurred,from subtraction surgery to additive surgery.The facial injection has become the most commonly used facial rejuvenation surgery because of its minimally invasive and convenient operation.At present,there are many kinds of soft tissue fillers in the market,among which hyaluronic acid-related preparations are the most widely used.As a kind of glycosaminoglycan widely existing in the human body,hyaluronic acid has the advantages of biodegradability and high biocompatibility.However,its degradation rate in vivo is fast,not easy to retain,and can not obtain a stable efficiency.So,it needs to be modified by a cross-linking agent to play a better therapeutic effect.The effects of these cross-linking agents on the human body need to be tracked and verified by more research.And the repeated injection of the fillers may lead to the accumulation of cross-linking agents.In recent years,the application of absorbable microsphere products that can stimulate tissue regeneration has gradually increased,such as hydroxyapatite microspheres,poly-L-lactic acid microspheres,and so on.Compared with traditional soft tissue fillers,this kind of material can not only provide volume but also repair tissue defects through immunoinflammatory reactions.It can provide a more prolonged effect and reduce the complications caused by repeated injections.In addition,with the continuous development of tissue engineering and regenerative medicine,some new composites containing bioactive components,such as extracellular matrix and platelet-rich plasma,have gradually come into the view of researchers and aroused great attention.These materials can stimulate tissue regeneration through their cellular components or biological factors,so they are the ideal materials for soft tissue fillers and has a good application prospect in the future.Although these materials have done relevant animal studies before clinical application to verify their safety and effectiveness,they may have limitations,such as short observation time or single detection methods,which need further verification.In clinical application,we have observed significant differences in properties and histological reactions among different soft tissue fillers,each of which has its own advantages and disadvantages.If the surgeons appropriately use them,the patients may be unable to achieve their expected effect after receiving injection treatment and even have some adverse reactions.Therefore,in this experiment,we selected hyaluronic acid,calcium hydroxyapatite microsphere,and extracellular matrix as three representative soft tissue fillers to conduct a detailed comparative study in order to fully understand their basic characteristics,the intensity of the immuno-inflammatory response and the ability to stimulate tissue regeneration.It is intended to provide a theoretical basis for plastic surgeons to select soft tissue fillers in the clinic reasonably and provide a reference for the development of soft tissue fillers in the future.Objectives:1.With normal saline as the control group,compare the properties of ECM gel,HA gel,and CaHA microspheres.And observe the local appearance and texture changes of the three materials after injection.The volume retention rates of different soft tissue filling materials were measured and compared at immediate time,1 week,4 weeks,8 weeks,16 weeks,and 24 weeks after injection.2.In this study,the changes in the number and types of the inflammatory cells around the soft tissue fillers were observed at 1 week,4 weeks,8 weeks,16 weeks,and 24 weeks after injection,and the immuno-inflammatory reaction was scored to understand the changing trend of the immuno-inflammatory response At the same time,the levels of inflammatory factors in the vicinity of soft tissue fillers in each group were quantitatively analyzed from the gene and protein levels,and the changing trend was observed.3.The stimulation effects of three kinds of soft tissue fillers on the regeneration of surrounding tissues were quantitatively analyzed and compared from the aspects of collagen generation and angiogenesis,and the changing trend was observed.Methods:Part Ⅰ:The injection model of C57BL/6N mice was established in this part.The injection was divided into four groups:control group(normal saline),ECM gel group,HA gel group,and CaHA microsphere group.Six-week-old female C57BL/6N mice were used in the experiment after a week of adaptation in the IVC laboratory.To reduce the difference caused by different injection sites,the four groups of fillers were injected into the subcutaneous back of 32 mice in a clockwise direction according to the volume of 0.1ml.The general shape and texture of the injection site and the local reaction of the injection site were recorded at immediate time,1 week,4 weeks,8 weeks,16 weeks,and 24 weeks after injection.The vernier caliper was used to measure the fillers’ length,width,and height.And the volume formula for hemiellipse was used to calculate the volume and the retention rate of the mass.Part Ⅱ:In this part of the study,we evaluated the immuno-inflammatory reaction of the surrounding tissues after the injection of fillers in each group.The mice were killed at 1 week,4 weeks,8 weeks,16 weeks and 24 weeks after injection.And the test site was removed completely for further experiments,including the implant,the capsule,and the whole skin.HE staining was performed to observe the changes in the number and types of inflammatory cells around the injection material.Two pathologists were invited to blindly evaluate the local foreign-body response of all slides according to the Duranti’ histologic score.And the thickness of the fibrous capsule wall in each group was measured at 8 weeks.Then,PCR and ELISA were used to detect the gene and protein expression levels of TNFα,IL-1β,and IL-6 at each time point for quantitative and comparative analysis.The expression trend of inflammatory factors was monitored.Part Ⅲ:In this part,the effects of the HA gel group,CaHA microsphere group,and ECM gel group on peripheral tissue regeneration were evaluated through collagen generation and angiogenesis.The specimens were stained with Masson trichrome(for collagen)and CD31(for neovascularization)staining according to standard immunohistochemistry protocols.Also,the double immunofluorescence staining of type Ⅰcollagen and type Ⅲ collagen was performed.Collagen production and angiogenesis were observed,and the thickness of the dermis and the density of collagen fibers in the dermis were measured.Then,the expression levels of type I collagen,type Ⅲ collagen,and VEGF in the surrounding tissues were detected by PCR and ELISA for quantitative and comparative analysis.Results:Part Ⅰ:ECM gel,HA gel,and CaHA microspheres have good biocompatibility and will not cause obvious adverse reactions in the injection site.Among the three groups,ECM gel has the best fluidity.It is easy to push during the injection.And it is hard to maintain the original shape due to the ECM gel could hardly be encapsulated after injection.After ECM gel was injected,the texture of the injection site was the softest and most similar to the soft tissue,followed by the HA group,and the CaHA group was the hardest.Both HA gel and CaHA microspheres can remain in vivo for more than 24 weeks,and a thin fiber capsule can be formed around the two materials.They can maintain a certain volume and shape for a long time.The volume of the HA gel groups maintained a relatively stable decline from the beginning to the end.The CaHA microsphere groups experienced a rapid volume loss in the early stage,but their long-term volume retention is better than that of HA gel.Part Ⅱ:During the experiment,the immune inflammatory reactions of the three groups of materials were all within the range of normal physiological responses.There were no adverse reactions,such as foreign body granuloma and tissue necrosis.Among them,the immuno-inflammatory response caused by HA and ECM was slight and disappeared quickly.The immuno-inflammatory reaction induced by CaHA microspheres was more severe than that in the above two groups,but the Duranti score decreased gradually with the increase of injection time.HA gel and ECM gel could temporarily stimulate the secretion of inflammatory factors in the surrounding tissue within 4 weeks after injection,while the level of inflammatory factors in CaHA groups was higher than that in the control group within 16 weeks.Finally,the expression level of inflammatory factors in the three experimental groups decreased gradually with the injection time and eventually returned to the average level.Part Ⅲ:HA gel,ECM gel,and CaHA microspheres can significantly improve the quality of local skin and increase the thickness and the density of collagen fibers of the dermis.All three experimental groups could continuously stimulate collagen production and local angiogenesis in nearby tissues and showed a cumulative effect with the stimulation time.The new collagen was mainly type Ⅲ collagen.Generally speaking,the ECM,which causes the most minor immuno-inflammatory response,has the least efficiency on the regeneration of surrounding tissue among the three experimental groups.Although CAHA causes the most severe immuno-inflammatory reaction,it is the most effective in stimulating surrounding tissue regenerations.It is suggested that the stimulation of immuno-inflammatory response may play a decisive role in the tissue regeneration induced by soft tissue fillers.Conclusions:This study comprehensively compares three kinds of soft tissue fillers with great attention in the market,providing a theoretical basis for clinical application.All the experimental fillers have been proven to be biocompatible and histologically safe and have their own advantages and disadvantages.The ECM gel is flexible,highly similar to natural soft tissue,and has the least immune response.However,it is difficult to maintain the original volume and position after injection,and the ability to promote tissue regeneration is the weakest.HA has good biocompatibility and stable degradation and absorption,but its long-term volume retention rate is lower than CaHA.The immuno-inflammatory reaction caused by CaHA is the most severe,and there is a rapid volume loss in the short term.However,the filling efficiency of CaHA over a long time is better,and its ability to induce collagen production and angiogenesis is the strongest.In this study,it was found that the materials with severe immuno-inflammatory responses were more efficient in promoting tissue regeneration.Therefore,accurately manipulating the immunoinflammatory response around the fillers may lead to better results of soft tissue augmentation.