| Background: As a novel paradigm in regenerative medicine,mesenchymal stem cell-based acellular therapies have been widely exploited in managing hypertrophic scar.The conditioned medium contains stem cells paracrine cytokines rather than cellular components.Given the effectiveness and no risk of tumorigenesis,it may be a better alternative to stem cell therapy.However,low maintenance dose and transitory therapeutic effects during topical medication remain a thorny issue.Herein,the polysaccharide hydrogel scaffold and freeze-dried method were united,aiming to optimize the curative effect of adipose-derived stem cells conditioned medium(ADSC-CM)in hypertrophic scarring prophylaxis.Methods: In the present study,ADSC-CM was concentrated via the freeze-drying procedure,and the total protein content of ADSC-CM was detected using the BCA kit.The efficacy of different dose groups(CM,CM5,CM10)was conducted on the proliferation,apoptosis,and α-smooth muscle actin(α-SMA)expression of human keloid fibroblasts(HKFs)in vitro.Simultaneously,the gel-forming properties of polysaccharide hydrogels were preliminarily studied,and the microstructure inside the gel was observed by scanning electron microscope.Incorporation of adiposederived stem cells concentrated conditioned medium(ADSCC-CM)into polysaccharide hydrogel was investigated in rabbit ear in vivo under 4-month observation period.Haematoxylin-eosin(H&E)staining was performed to evaluate scar hyperplasia of the skin’s general structure and its appendages,and Masson’s trichrome staining was used to assess the arrangement of collagen fibres.The scar hyperplasia was measured using the scar elevation index(SEI).Protein mass spectrometry has captured the top 50 proteins with higher abundance in ADSCCCM,enabling us to analyze and explore possible cytokines in depth.Results: The freeze-drying method has been implemented to enrich cytokines in stem cell-conditioned medium.The content of total protein in the concentrated solution is proportional to the concentration ratio.We noted that ADSCC-CM could downregulate the α-SMA expression of HKFs in a dose-dependent manner.In the rabbit ear model,the scar hyperplasia in the medium-dose group(CM5)and highdose group(CM10)was inhibited with reduced SEI under 4-months of observation.It is noteworthy that the union of CM5 and polysaccharide hydrogel(CM5+H)yielded the best preventive effect on scar hyperplasia.Briefly,melanin,height,vascularity and pliability in the CM5+H group were better than those of the control group.Collagen was evenly distributed,and skin appendages could be regenerated.Scanning electron microscopy shows that the polysaccharide hydrogel contains a network-like structure,which may slow-release cytokines in a rhythmical form.The high abundance of candidate proteins in ADSCC-CM were detected by protein mass spectrometry,such as HSP90AA1,HSP90AB1,Myh9,and ENO1.Among them,HSP90α and PDI are probably involved in wound repair,and TAC and EF-1α may be related to scar inhibition.Conclusions: Altogether,ADSCC-CM can downregulate the expression of α-SMA due to its anti-fibrosis effect and promote the rearrangement of collagen fibres,which is integral to scar precaution.The in situ cross bonding of ADSCC-CM and polysaccharide hydrogel could remarkably enhance the therapeutic outcomes in inhibiting scar proliferation.Combining the two can hinder fibroblasts’ growth and survival,promote collagen fibres’ rearrangement,and induce skin appendages’ repair and reconstruction.Hence,the alliance of ADSCC-CM and hydrogel may become a potential alternative in long-term hypertrophic scar prophylaxis. |
