Polydopamine-modified Decellularized Intestinal Scaffolds Loaded With Adipose Derived Stem Cells Promote Intestinal Repair

Background and aimThe small intestine is difficult to regenerate or reconstruct due to its complex functions and structure.For some serious intestinal diseases such as inflammatory bowel disease and short bowel syndrome,although advances in enteral or parenteral nutrition treatment have greatly improved the quality of life of patients,intestinal failure and various complications are still inevitable and ultimately life-threatening.Small bowel transplantation is an ideal treatment for intestinal failure.Due to the advances in transplantation techniques and immunosuppression protocols,the survival rate of small intestine transplantation patients has been greatly improved,but the long-term efficacy is not ideal.In addition,small intestine transplantation involves the safety of donor and recipient,especially the limitation of the site and size of donor intestine,which hinders the development of small intestine transplantation.At present,intestinal grafts formed by combining specific cells to biomaterials with a similar natural intestinal structure using tissue engineering techniques provide the possibility of intestinal regeneration in vitro.Among them,decellularized whole organ scaffolds show obvious advantages due to the preservation of natural extracellular matrix(ECM)structure and vascular system of the original tissue.Both the decellularized scaffolds themselves or various biological scaffolds derived from them are utilized for reconstruction and regeneration of organs and tissues.To date,relatively simple organ grafts,such as vascular,segments of upper airways and urethras,have been used with great success in humans.However,for complex organs,including the esophagus,stomach and small intestine,there is more to explore due to their unique physiological microenvironment.With the rapid development of decellularization technology,decellularized scaffolds of the stomach and intestine with full-layer structures can be prepared,which greatly improve the possibility of repairing full-layer defects of the digestive tract.However,previous studies have confirmed varying degrees of extracellular matrix and cytokine loss during the decellularization process,resulting in the greatly reduced function of the decellularized scaffolds.Surface modification as a method to greatly improve the biocompatibility of scaffolds is often used by researchers.Polydopamine(PDA),a mussel-inspired molecule,has been widely employed as an adhesive layer on various biomedical substrates due to its outstanding merits.Firstly,the synthesis of PDA is simple,efficient and cost-saving,which can promote the large-scale application of PDA.Secondly,PDA not only has good biocompatibility but also has high stability.PDA modification can enhance the adhesion and proliferation of engrafted cells on scaffolds and improve the biocompatibility of grafted biomaterials in vivo.In addition,studies have shown that PDA can reduce cell oxidative stress damage induced by reactive oxygen species(ROS)and improve cell viability.Furthermore,PDA allows the material surface to have good secondary reactivity,leading to continued bridging of the required biomolecules,such as antibacterial molecules,to provide the material with specific antibacterial properties.Adipose derived stem cells(ADSC),as another research hotspot of tissue engineering in recent years,have shown promising prospects in the treatment of various diseases due to their excellent therapeutic effects.Studies have confirmed that ADSC can not only differentiate into a variety of cells,such as fibrocytes and vascular endothelial cells,but also secrete a variety of growth factors,such as transforming growth factor β1(TGFβ1),vascular endothelial growth factor(VEGF)and prostaglandin E2(PGE2),which can promote collagen synthesis and accelerate the migration of fibroblasts,ultimately promoting wound healing.However,the harsh wound environment may weaken the therapeutic potential of transplanted cells.Biomaterial-based cell therapy,which can be used to transplant different types of cells into damaged areas,is often considered a good option.Moreover,co-transplantation of biomaterials can be used to fill the lesion cavity and mediate directed growth and differentiation of cells.In this study,decellularized intestinal scaffolds(DIS)were prepared by decellularization,and PDA was used for surface modification.Subsequently,ADSC were recellularized in PDA-DIS,and the composite scaffolds were used to reconstruct intestinal defects in rats to explore the feasibility of decellularized scaffolds for repairing intestinal defects.Finally,this study provided an experimental and theoretical basis for tissue engineering to realize intestinal regeneration.Methods1.Preparation and identification of DISThe porcine DIS was prepared with freeze-thaw and mechanical stirring methods.HE staining,Masson staining and Sirius Red(SR)staining were used to verify the cells removal and the preservation of ECM structure in DIS after decellularization.The important ECM proteins included collagen I,collagen IV and fibronectin were observed by immunofluorescence.The ultrastructure of DIS was observed by scanning electron microscope(SEM).Residual cells after decellularization were quantitatively detected by DNA content.Collagen content in native tissues and DIS was quantitatively analyzed by hydroxyproline,and glycosaminoglycans(GAGs)content was quantitatively analyzed by enzyme linked immunosorbent assay(ELISA).The mechanical properties of the scaffold after decellularization were tested by tensile test.2.Synthesis of PDA and preparation and evaluation of PDA-DISPDA was prepared from dopamine hydrochloride by solution oxidation method.The surface microstructure of PDA-DIS was observed by SEM after DIS was immersed in PDA solutions with different concentrations for 24 h.To verify the histocompatibility of PDA-DIS,the scaffolds were implanted subcutaneous on the back of SD rats,and removed at 1 and 4 weeks postoperatively for histological staining evaluation.The antibacterial performance of PDA-DIS was detected by resazurin and plate count of E.coli.3.Cytocompatibility evaluation of PDA and recellularization of ADSC in scaffoldsHuman ADSC were isolated according to collagenase digestion.The differentiation potential of ADSC was determined by induction of osteogenesis and adipogenesis.The surface markers of ADSC were identified by flow cytometry and immunofluorescence.The cytocompatibility of PDA was detected by Calcein-AM/PI staining,CCK-8,flow cytometry and ROS.When recellularized on the DIS and PDADIS,and the growth of ADSC on the scaffolds was evaluated by immunofluorescence.The secretion levels of TGFβ1,VEGF and PGE2 in ADSC under different conditions were detected by ELISA.4.Effects of ADSC conditioned medium on HUVECs and RAW264.7 cellsConditioned medium of ADSC cultured in plastic plate,DIS and PDA-DIS were collected.Human umbilical vein endothelial cells(HUVECs)wound healing,migration and tube-formation assay were used to verify the effect of ADSC conditioned medium on angiogenesis.The immunomodulatory effect of ADSC conditioned medium was verified by inducing the phenotype change of RAW264.7 cells.5.DIS,ADSC-DIS and ADSC-PDA-DIS were transplanted to repair intestinal defects in ratsA full-thickness defect model of ileocecal intestine was established in SD rats,and DIS,ADSC-DIS and ADSC-PDA-DIS were transplanted for intestinal repair.The rats in each group were sacrificed at 1 and 4 weeks postoperatively and the operation area was collected for histological staining to compare the effect of three different scaffolds on promoting intestinal wound healing.The blood and major organs of rats were collected to evaluate the biosafety of PDA.Results1.The porcine small intestine lost its natural color gradually until it became semitransparent after decellularization at the macroscopic level.HE staining and SEM analysis confirmed the almost complete removal of cells and maintenance of ultrastructure after the decellularization process.Quantification analysis demonstrated that the DNA content of DIS was 45.77 ± 2.08 ng/mg dry weight,in contrast to 528.9± 12.97 ng/mg for the native intestine(P<0.05).According to the results of Masson trichrome and SR staining,collagen was well preserved,and no significant differences were observed between the native tissues and DIS.The collagen content in the DIS was386.8 ± 13.07 ng/mg dry weight,compared to 470.9 ± 27.55 ng/mg dry weight in the native intestine,as determined through indirect quantification of the amino acid hydroxyproline.Based on the immunofluorescence results,the distribution and expression of key ECM proteins,including collagen I,collagen IV and fibronectin,were well maintained in DIS after decellularization.The total content of GAGs in the DIS and native tissue was 2.3 ± 0.23 and 5.2 ± 0.35 μg/mg dry weight(44.2% retained),respectively.Tensile tests showed no significant reduction in the mechanical properties of the small intestine after decellularization.2.Transmission electron microscopy(TEM)showed that the average diameter of PDA was 200 nm,with a monodispersed spherical structure.To prepare PDA-DIS,the DIS was immersed in PDA solution for 24 h,and the color of the scaffolds changed to brown.SEM characterization of the microstructures revealed a uniform nanolayer on the surface of the PDA-DIS.The DIS and PDA-DIS samples were removed from the rats at 1 and 4 weeks postoperatively and it demonstrated that both scaffolds were biocompatible.HE staining showed that,compared to the untreated DIS,there were more blood vessels observed in the PDA-DIS.The results of resazurin colorimetry and plate colony count showed that PDA-DIS could inhibit E.coli.,indicating that PDA coating had antibacterial properties.3.ADSC presented typical spindle-shaped morphology.Alizarin Red and Oil Red O staining confirmed that ADSC had osteogenic and adipogenic differentiation potential.By flow cytometry and immunofluorescence,ADSC showed high expression of the classic MSC surface markers CD29,CD44,and CD90 and the absence of CD34 and CD45.Calcein AM/PI staining,CCK-8 assay,flow cytometry and ROS level showed good cytocompatibility when PDA concentration was lower than 400 μg/ml.After 3 days of culture,it demonstrated that ADSC could not only be engrafted into the scaffolds but also showed MSC surface markers(CD29,CD44,and CD90),indicating that the function of ADSC was not affected in the DIS or PDA-DIS.When PDA concentration was less than 100ug/m L,the adhesion rate of ADSC on PDA-DIS was not significantly affected.ELISA showed that ADSC could secrete more cytokines TGFβ1,VEGF and PGE2 on PDA-DIS.4.HUVECs wound healing,migration and tube formation assays showed that conditioned medium in ADSC-PDA-DIS group had the strongest ability to stimulate HUVECs migration and tubule formation,suggesting that DIS with PDA coating could stimulate ADSC to play a stronger role in promoting angiogenesis.Immunofluorescence,quantitative real time polymerase chain reaction(q RT-PCR)and western blot(WB)results showed that conditioned medium in ADSC-PDA-DIS group significantly increased the expression of anti-inflammatory factors(Arg-1,IL-10 and CD206)and down-regulated the expression of pro-inflammatory factors(i NOS,IL6,TNFα and CD86)in RAW264.7,confirming that PDA-DIS could stimulate ADSC to play a stronger immunomodulatory function.5.The transplanted scaffolds were analyzed at 1 and 4 weeks postoperatively after intestine repair.At the macroscopic level,DIS,ADSC-DIS and ADSC-PDA-DIS could repair intestinal defects without intestinal fistula.There was no significant difference in body weight between the three groups and the unoperated rats.HE staining demonstrated that the ADSC-PDA-DIS group showed more neovascularization at both1 w and 4w postoperatively.Immunofluorescence further confirmed higher expression levels of CD31 and αSMA in the ADSC-PDA-DIS group,and the expression level of HIF-1α was also significantly increased.Large populations of vimentin+ cells were found in the scaffolds,indicating infiltration of host fibroblasts into the scaffolds.Notably,the expression level of collagen was not significantly different among the three groups.This was because the scaffolds were inherently rich in collagen.The i NOS level of M1 macrophages in the DIS group was higher than that in the other two groups,and CD206 expression in M2 macrophages in the ADSC-PDA-DIS group was stronger.Biosafety evaluation of PDA showed that there were no obvious abnormalities in biochemical indexes and major organs in those rats.Conclusions1.In DIS,not only the residual cells were almost absent,but also the whole layer structure of small intestine and ECM proteins were well preserved.2.PDA could be coated on the surface of DIS,and PDA-DIS had good histocompatibility and antibacterial properties.3.PDA not only had good cytocompatibility,but also DIS and PDA-DIS could provide suitable three-dimensional growth environment for ADSC and promote the secretion function of ADSC.4.The conditioned medium produced by ADSC-PDA-DIS not only significantly enhanced the ability to stimulate HUVECs migration and tubule formation,but also significantly up-regulated the expression of anti-inflammatory factors and downregulated the expression of pro-inflammatory factors in RAW264.7.5.ADSC-PDA-DIS could repair intestinal defects in rat better,and promote intestinal healing and regeneration through rapid angiogenesis and reduction of scar formation.