Application of Decellularized Scaffold Combined with Loaded Nanoparticles for Heart Valve Tissue Engineering in vitro

The purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-β1（TGF-β1）,by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro.Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method,and their morphology was observed under a scanning electron microscope.Decelluarized valve scaffolds,prepared by using trypsinase and TritonX-100,were modified with nanoparticles by carbodiimide,and then TGF-β1 was loaded into them by adsorption.The TGF-β1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay.Whether unseeded or reseeded with myofibroblast from rats,the morphologic,biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions.The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles.The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds.Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment,which is beneficial for an application in heart valve tissue engineering.

Orthotopic implantable liver decellularized scaffold for acute liver failure

Decellularized scaffolds have an irreplaceable role in the development of tissue engineering.Challenges in this field are concentrated on shaping macro-scale constructs and maintaining blood flow.Here,we establish a whole liver perfused decellularized scaffold cultured with hepatocytes for transplantable bioengineered liver construction.Owing to retained intact gross morphology of liver,geometric structure and biomechanical environment can support whole liver lobe transplantation.Besides,unremoved extracellular matrix(ECM)and vascular net-works can delivery continuous nutrient for cell adhesion.Based on recellularized bioengineering liver scaffold,we have demonstrated its excellent hepatic functions when it orthotopic implanted in acute liver failure(ALF)rats,along with the prolonged survival rate and improved biochemical indicators.These outstanding properties indicate that the orthotopic implantable liver scaffold has broad clinical application prospects in the treatment of ALF and other regeneration diseases.

A decellularized nerve matrix scaffold inhibits neuroma formation in the stumps of transected peripheral nerve after peripheral nerve injury

Traumatic painful neuroma is an intractable clinical disease characterized by improper extracellular matrix(ECM)deposition around the injury site.Studies have shown that the microstructure of natural nerves provides a suitable microenvironment for the nerve end to avoid abnormal hyperplasia and neuroma formation.In this study,we used a decellularized nerve matrix scaffold(DNM-S)to prevent against the formation of painful neuroma after sciatic nerve transection in rats.Our results showed that the DNM-S effectively reduced abnormal deposition of ECM,guided the regeneration and orderly arrangement of axon,and decreased the density of regenerated axons.The epineurium-perilemma barrier prevented the invasion of vascular muscular scar tissue,greatly reduced the invasion ofα-smooth muscle actin-positive myofibroblasts into nerve stumps,effectively inhibited scar formation,which guided nerve stumps to gradually transform into a benign tissue and reduced pain and autotomy behaviors in animals.These findings suggest that DNM-S-optimized neuroma microenvironment by ECM remodeling may be a promising strategy to prevent painful traumatic neuromas.

Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Immobilization of Decellularized Valve Scaffolds with Arg-Gly-Asp-containing Peptide to Promote Myofibroblast Adhesion

The cell adhesive properties of decellularized valve scaffolds were promoted by immobilization of valve scaffold with arginine-glycine-aspartic acid （RGD）-containing peptides. Porcine aortic valves were decellularized with trypsin/EDTA, and detergent Triton X-100. With the help of a coupling reagent Sulfo-LC-SPDP, the valve scaffolds were immobilized with glycine-arginine-glycine-aspartic acid-serine-proline-cysteine （GRGDSPC） peptide. X-ray photoelectron spectroscopy （XPS） was used for surface structure analysis. Myofibroblasts harvested from rats were seeded onto the valve scaffolds. Cell count by using microscopy and modified MTT assay were performed to assess cell adhesion. Based on the spectra of XPS, the conjugation of GRGDSPC peptide with decellularized valve scaffolds was confirmed. Both cell count and MTT assay showed that myofibroblasts were much easier to adhere to the modified valve scaffolds, which was also confirmed histologically. Our findings suggest that it is feasible to immobilize RGD-containing peptides onto decellularized valve scaffolds. And the technique can effectively promote cell adhesion, which is beneficial for in vitro tissue engineering of heart valves.

Immobilization of RGD Peptidcs onto Decellularized Valve Scaffolds to Promote Cell Adhesion

Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD （arginine-glycine-aspartic acid） containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

Deer antler stem cell niche: An interesting perspective

In recent years,there has been considerable exploration into methods aimed at enhancing the regenerative capacity of transplanted and/or tissue-resident cells.Biomaterials,in particular,have garnered significant interest for their potential to serve as natural scaffolds for cells.In this editorial,we provide commentary on the study by Wang et al,in a recently published issue of World J Stem Cells,which investigates the use of a decellularized xenogeneic extracellular matrix(ECM)derived from antler stem cells for repairing osteochondral defects in rat knee joints.Our focus lies specifically on the crucial role of biological scaffolds as a strategy for augmenting stem cell potential and regenerative capabilities,thanks to the establishment of a favorable microenvironment(niche).Stem cell differen-tiation heavily depends on exposure to intrinsic properties of the ECM,including its chemical and protein composition,as well as the mechanical forces it can generate.Collectively,these physicochemical cues contribute to a bio-instructive signaling environment that offers tissue-specific guidance for achieving effective repair and regeneration.The interest in mechanobiology,often conceptualized as a form of“structural memory”,is steadily gaining more validation and momen-tum,especially in light of findings such as these.

Generation and metabolomic characterization of functional ductal organoids with biliary tree networks in decellularized liver scaffolds

Developing functional ductal organoids(FDOs)is essential for liver regenerative medicine.We aimed to construct FDOs with biliary tree networks in rat decellularized liver scaffolds(DLSs)with primary cholangiocytes isolated from mouse bile ducts.The developed FDOs were dynamically characterized by functional assays and metabolomics for bioprocess clarification.FDOs were reconstructed in DLSs retaining native structure and bioactive factors with mouse primary cholangiocytes expressing enriched biomarkers.Morphological assessment showed that biliary tree-like structures gradually formed from day 3 to day 14.The cholangiocytes in FDOs maintained high viability and expressed 11 specific biomarkers.Basal-apical polarity was observed at day 14 with immunostaining for E-cadherin and acetylatedα-tubulin.The rhodamine 123 transport assay and active collection of cholyl-lysyl-fluorescein exhibited the specific functions of bile secretion and transportation at day 14 compared to those in monolayer and hydrogel culture systems.The metabolomics analysis with 1075 peak pairs showed that serotonin,as a key molecule of the tryptophan metabolism pathway linked to biliary tree reconstruction,was specifically expressed in FDOs during the whole period of culture.Such FDOs with biliary tree networks and serotonin expression may be applied for disease modeling and drug screening,which paves the way for future clinical therapeutic applications.

Three-dimensional decellularized tumor extracellular matrices with different stiffness as bioengineered tumor scaffolds

In the three-dimensional(3D)tumor microenvironment,matrix stiffness is associated with the regulation of tumor cells behaviors.In vitro tumor models with appropriate matrix stiffness are urgently desired.Herein,we prepare 3D decellularized extracellular matrix(DECM)scaffolds with different stiffness to mimic the microenvironment of human breast tumor tissue,especially the matrix stiffness,components and structure of ECM.Furthermore,the effects of matrix stiffness on the drug resistance of human breast cancer cells are explored with these developed scaffolds as case studies.Our results confirm that DECM scaffolds with diverse stiffness can be generated by tumor cells with different lysyl oxidase(LOX)expression levels,while the barely intact structure and major components of the ECM are maintained without cells.This versatile 3D tumor model with suitable stiffness can be used as a bioengineered tumor scaffold to investigate the role of the microenvironment in tumor progression and to screen drugs prior to clinical use to a certain extent.