| Objective:Pathological scar（PS）is the abnormal wound-healing outcomes that develop excessive fibrosis after skin trauma,which is manifested as skin bulge and skin color change,often accompanied by itching and pain symptoms,and brings a series of physiological,psychological,cosmetic and other problems to patients.Its pathogenesis is that dermal fibroblasts are over-activated into myofibroblasts,leading to excessive deposition of extracellular matrix（ECM）.At present,intra-scar injection of triamcinolone acetonide（TA）is the conventional method for the treatment of PS.However,the pain during injection is severe,and the long-term and high-dose application of TA in large scars can lead to a variety of adverse reactions such as tissue atrophy,metabolic disorders,osteoporosis and so on.Although clinical methods can be used to relieve pain,such as local topical lidocaine cream before injection,drug mixed with lidocaine injection in proportion,or small needle injection,there are still shortcomings of long operation time and poor effect.Therefore,there is an urgent need for a safe,effective,painless and non-invasive method for the treatment of PS using TA.According to current scientific research,adipose-derived stem cells（ADSCs）can prevent fibrosis after tissue injury,which provide a potential strategy for treating excessive scar formation.However,the clinical application of ADSCs remains challenging.Microneedles（MNs）,as micron-scale medical devices,can penetrate the stratum corneum to form micro-channels under the condition of slight pain,resulting in high transdermal drug delivery efficiency,which have great advantages over traditional intralesional injection.Keratin is a natural protein derived from skin and skin appendage,which has excellent natural biocompatibility and degradation,and is one of the preferred materials for making microneedles matrix.Keratin is different from common natural proteins in that its disulfide bond content is high,and the disulfide bond has the characteristics of reoxidation after reduction.Through the reorganization of disulfide bond,it lays the foundation for the production of MNs with excellent mechanical properties.In this regard,a keratin MN patch carrying stem cell components and TA was designed in this study,and its therapeutic effect and mechanism on PS were explored through cell experiments and animal experiments.The proposed MNs provide a theoretical basis and a new idea for the clinical treatment of PS,which has a wide application prospect.Methods:1.ADSCs were isolated from adipose tissue of healthy liposuction patients,and the successful extraction of stem cells was confirmed by immunofluorescence and induced differentiation.Adipose-derived stem cell conditioned medium（ADSC-CM）was collected and freeze-dried by a freeze-dryer to obtain adipose-derived stem cell concentrated conditioned medium（ADSCC-CM）powder containing stem cell secretion factors.BCA method was used to detect the protein concentration of ADSCC-CM powder with different concentrations.2.Human hair keratin was extracted by chemical reduction method.Fourier transform infrared spectroscopy（FT-IR）was used to verify the success of keratin extraction.The biocompatibility was confirmed by co-culture with human gingival fibroblasts（h GFs）.Different concentrations of ADSCC-CM were used to interact with keratin to form hydrogels and were compared with hydrogels formed by cysteine（Cys）or DMEM with keratin.The gelation time of these hydrogels was observed.After lyophilization,the external morphological changes of the hydrogels were observed and the network structure was analyzed by scanning electron microscopy（SEM）.3.Due to the low solubility of TA in aqueous solution,it is often not uniformly distributed in aqueous solution.To solve this problem,sonication is used in this study.The TA solution was sonicated before MNs was fabricated,the dispersion was observed,and the settling time was recorded.The sonicated TA was loaded into the hydrogel formed by ADSCC-CM and keratin,and the hydrogel was lyophilized and scanned by SEM to observe the distribution of TA.4.The MNs were made using PDMS microneedle mold by reflection printing method.The appearance of MNs were observed by SEM.After labeling keratin with rhodamine B,the distribution of keratin in MNs was observed under a fluorescence microscope.The MNs were inserted into the porcine skin,and a confocal laser scanning microscopy（CLSM）was utilized to scan cross section of the skin sample to evaluate the depth of penetration of the MNs.After the MNs were inserted into the rat skin,the distribution of micropores was observed by methylene blue staining,and the punctured epidermis was observed by H&E staining.The mechanical properties of the MNs were tested by the load-bearing experiment of 500 g weight and compression experiment of universal machine.5.ADSCC-CM was labeled with Cy5.5,and then the labeled MNs was punctured into the back of mice.Compared with the ordinary subcutaneous injection method,the fluorescence disappearance time was observed by in vivo imaging of small animals at regular intervals to detect the release rate of ADSCC-CM.MNs patches were punctured into fresh porcine skin,and the MNs patches were removed at regular intervals.The remaining MNs was ground by a grinder and the TA was extracted with methanol,and then the content was determined by HPLC to detect the release rate of TA.6.Human keloid fibroblasts（HKFs）were isolated from keloid tissue by tissue explant method.HKFs were co-cultured with ADSCC-CM and different concentrations of TA（80μg/m L,160μg/m L,320μg/m L）,and the cell viability was tested by CCK-8.Cell immunofluorescence was used to observe the effect of ADSCC-CM and TA on the expression ofα-SMA protein in HKFs.In addition,the leaching liquor of MNs containing different components were co-cultured with HKFs to observe the effect on cell migration.7.The rabbit ear scar model was established and divided into control group,MNs group（Drug-free group,ADSCC-CM group,TA group,ADSCC-CM+TA group）and TA-Injection group（positive control group）.The treatment was given once a week for 4weeks.Appearance photograph,ultrasound test and translucency test were performed before operation,before treatment and every week after treatment to evaluate the therapeutic effect of scars in each group.At the 2nd and 4th week after treatment,specimens were cut at the edge of scar tissue for H&E,Masson,Sirius red,and immunohistochemical staining（WNT-2,TGF-β1,TIMP-1）.At the 4th week,tissue specimens were obtained for universal machine tensile test.Results:1.Under light microscope,ADSCs showed typical spindle shape of fibroblasts,which were easy to proliferate when cultured in conventional medium in vitro.Cellular immunofluorescence analysis showed that mesenchymal stem cell specific surface markers CD44 and CD105 were positive,while hematopoietic stem cell surface marker CD45 was negative.Oil red O staining and alkaline phosphatase staining confirmed the successful induction of ADSCs into adipocytes and osteoblasts.These results indicated that ADSCs were successfully isolated in this study.The total protein content of ADSCC-CM powder determined by BCA increased significantly with the increase of ADSCC-CM powder concentration.2.After dialysis and freeze-drying,the extracted human hair keratin was flocculent powder.FT-IR detected characteristic fronts of amide I(1600-1700 cm-1),amide II(1520-1540 cm-1),and amide III(1220-1300 cm-1)of keratin.This study also found that keratin preserved for 1 month and 9 months still had good gelatinization,which proved that its chemical properties were stable and easy to preserve.CCK-8 analysis showed that the cell viability was above 90%,which proved that keratin had good biocompatibility and was an ideal MN matrix material.3.The results of comparison of hydrogels prepared by different concentrations of ADSCC-CM and other methods with keratin showed that the gel formation time was shorter with the increase of the concentration of ADSCC-CM,and the gelation time of 5wt%ADSCC-CM and cysteine with keratin were all within 1 hour,while the gelation time of DMEM was the slowest（36h）.After freeze-drying of different hydrogels,it was found that the hydrogel formed by 10 wt%ADSCC-CM and keratin had the least cracks on the surface,followed by the hydrogel formed by 5 wt%ADSCC-CM and keratin;the hydrogel formed by cysteine and keratin had the most surface cracks,while the hydrogel formed by DMEM and keratin fell off due to brittleness.The micropore distribution of the hydrogel formed by 5 wt%ADSCC-CM and keratin was the most uniform.The above results indicated that ADSCC-CM can improve the mechanical characteristics of keratin hydrogel and was a new gelation method.4.It was found that the dispersion of TA in aqueous solution could be improved obviously by sonication,and no obvious settlement of TA could be observed within 1 h.After sonication,TA was loaded in the hydrogel formed by ADSCC-CM and keratin,and SEM image showed that TA could be uniformly distributed in the hydrogel.5.The results of SEM showed that all the MNs were conical and composed of needle tip and substrate.Fluorescence microscope showed that rhodamine B labeled keratin was evenly distributed in MNs.CLSM revealed that the fabricated MN patch could penetrate330μm of porcine skin,meeting the requirement of drug delivery to the dermis.The H&E result demonstrated that MNs could penetrate skin well.Load-bearing experiments confirmed that MN patches made of hydrogel formed by ADSCC-CM and keratin can maintain good MN shape even if bearing 500 g weight.The proposed MN patch also showed the most outstanding mechanical properties in the compression experiment of universal machine.6.In vivo imaging of mice showed that,compared with subcutaneous injection,MNs method could preserve ADSCC-CM locally for up to 5 days,while local injection was only preserved for 1 day,demonstrating that the proposed MN patch could prolong the action time of the drug at the scar and reduce the number of administration.The results of HPLC analysis showed that about 80%of TA was released at 16 h,which could avoid the side effects caused by subcutaneous injection of TA burst release.7.In vitro trials,both ADSCC-CM and TA had a good inhibition effect on HKFs cell activity,and the inhibition effect became more obvious with the increase of TA concentration.The results of cell scratch assay showed that ADSCC-CM combined with TA had the most obvious inhibitory effect on HKFs migration.Cellular immunofluorescence assay showed that both ADSCC-CM and TA could inhibit the expression ofα-SMA in HKFs,but the combination of ADSCC-CM and TA had the best inhibitory effect.The above results indicated that proved that ADSCC-CM and TA have synergistic effect in PS treatment.8.In vivo experiments,the results of appearance photography showed that the scars in the ADSCC-CM+TA group and TA-injection group were significantly flatter.The results of ultrasonic scar thickness test showed that SEI in TA-Injection group was the lowest at all test points compared with other groups,but there was no statistical difference between TA-Injection group and ADSCC-CM+TA group.In the light transmission experiment,it was observed that the ADSCC-CM+TA group and the TA-Injection group gradually approached the light transmission of normal skin after four treatments,while the control group and Drug-free group still showed obvious redness and severe inflammatory reaction.In the universal machine stretching experiment of scar tissue,the maximum tensile strength of the ADSCC-CM+TA group was significantly higher than that of other groups,although there was still a significant difference compared with that of normal skin（p<0.5）,among which the maximum tensile strength of the TA-Injection group was the lowest.In the H&E staining results,it was obviously found that the inflammatory cell infiltration was reduced in the ADSCC-CM+TA group and the TA-Injection group after the 4th treatment,and the epidermal thickness of the two groups was very close to that of the normal group.The Masson staining results showed that,one week after the 2nd treatment,red muscle fibers were dominant in the control group and Drug-free group,while blue collagen was dominant in the ADSCC-CM+TA group,which proved that the combination of ADSCC-CM and TA could obviously inhibit the transformation of myofibroblasts during scar formation.After the 4th treatment,the collagen in the control group and the Drug-free group increased significantly,and the collagen arrangement in the ADSCC-CM+TA group was similar to that in the normal group.However,the collagen expression of the TA-Injection group was the least during the whole process.Sirius red staining showed that,after 4th treatments,the ratio of Col-I to Col-III in scar tissue of the ADSCC-CM+TA group tended to balance,approaching that of normal tissue（Col-I:Col-III=3.8:1）,and the ratio of the TA-Injection group changed most obviously（Col-I:Col-III=7.8:1）.Immunohistochemical analysis results showed that the proposed MN patch could inhibit the expression of scar-promoting factors（WNT-2,TGF-β1,TIMP-1）.Conclusions:1.In this study,ADSCs were successfully isolated and cultured from human adipose tissue to obtain stem cell factor-enriched ADSCC-CM,and human hair keratin was successfully extracted.This study found for the first time that ADSCC-CM and keratin could be cross-linked to form hydrogels,and the MNs constructed by the hydrogel had good mechanical properties.2.After sonication was used to improve the dispersibility of TA solution,TA could be cleverly and evenly encapsulated by the hydrogel formed by ADSCC-CM and keratin,which increased the loading of TA and achieved slow release in the MNs.3.In vitro experiments,both ADSCC-CM and TA inhibited the phenotype of HKFs,and the combination of the two had a synergistic effect.This study cleverly realized the dual“role”of ADSCC-CM as a hydrogel cross-linking agent and an effective ingredient for PS treatment.4.In vivo experiments,the designed MNs had a good therapeutic effect,which can reduce SEI,reduce inflammation,reduce epidermal thickness,improve collagen content and the ratio of Col-I to Col-III collagen,and increase the maximum tensile force,so as to realize the biomimetic repair of PS.It was proved that the designed MNs could promote PS repair by inhibiting WNT signaling pathway,TGF-βsignaling pathway and TIMP-related protein expression. |
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