Proteomics as a tool to gain next level insights into photo-crosslinkable biopolymer modifications

The distribution of photo-crosslinkable moieties onto a protein backbone can affect a biomaterial’s crosslinking behavior, and therefore also its mechanical and biological properties. A profound insight in this respect is essential for biomaterials exploited in tissue engineering and regenerative medicine. In the present work, photo-crosslinkable moieties have been introduced on the primary amine groups of: (i) a recombinant collagen peptide (RCPhC1) with a known amino acid (AA) sequence, and (ii) bovine skin collagen (COL BS) with an unknown AA sequence. The degree of substitution (DS) was quantified with two conventional techniques: an ortho-phthalic dialdehyde (OPA) assay and ^(1)H NMR spectroscopy. However, neither of both provides information on the exact type and location of the modified AAs. Therefore, for the first time, proteomic analysis was evaluated herein as a tool to identify functionalized AAs as well as the exact position of photo-crosslinkable moieties along the AA sequence, thereby enabling an in-depth, unprecedented characterization of functionalized photo-crosslinkable biopolymers. Moreover, our strategy enabled to visualize the spatial distribution of the modifications within the overall structure of the protein. Proteomics has proven to provide unprecedented insight in the distribution of photo-crosslinkable moieties along the protein backbone, undoubtedly contributing to superior functional biomaterial design to serve regenerative medicine.

Large-sized bone defect repair by combining a decalcified bone matrix framework and bone regeneration units based on photo-crosslinkable osteogenic microgels

Physiological repair of large-sized bone defects is great challenging in clinic due to a lack of ideal grafts suitable for bone regeneration.Decalcified bone matrix(DBM)is considered as an ideal bone regeneration scaffold,but low cell seeding efficiency and a poor osteoinductive microenvironment greatly restrict its application in large-sized bone regeneration.To address these problems,we proposed a novel strategy of bone regeneration units(BRUs)based on microgels produced by photo-crosslinkable and microfluidic techniques,containing both the osteogenic ingredient DBM and vascular endothelial growth factor(VEGF)for accurate biomimic of an osteoinductive microenvironment.The physicochemical properties of microgels could be precisely controlled and the microgels effectively promoted adhesion,proliferation,and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro.BRUs were successfully constructed by seeding BMSCs onto microgels,which achieved reliable bone regeneration in vivo.Finally,by integrating the advantages of BRUs in bone regeneration and the advantages of DBM scaffolds in 3D morphology and mechanical strength,a BRU-loaded DBM framework successfully regenerated bone tissue with the desired 3D morphology and effectively repaired a large-sized bone defect of rabbit tibia.The current study developed an ideal bone biomimetic microcarrier and provided a novel strategy for bone regeneration and large-sized bone defect repair.

THE EFFECT OF PHOTO-CROSSLINKING ON THE ORIENTATION STABILITY OF POLYVINYL ALCOHOL CONTAINING 4-NITRO-4′-ALKOXYSTILBENE AND CINNAMYL PENDENT GROUPS

Crosslinking is one of the effective routes for improving the orientation stability of poled polymer films. The derivative of polyvinyl alcohol containing 4-nitro-4'-alkoxystilbene and photo-crosslinkable cinnamyl groups as side chains has been synthesized. The in-situ simultaneous photo-crosslinking poling of synthesized polymer films has teen performed, The second order nonlinear optical coefficient d(33) of poled film is 11 pm/V. The SHG measurements show that the break-over temperature of SHG signal is raised obviously after irradiation, its orientation stability is doubled as compared with that of non-crosslinking samples.

Tailor-made microstructures lead to high-performance robust PEO membrane for CO_(2)capture via green fabrication technique

Emerging excessive greenhouse gas emissions pose great threats to the ecosystem,which thus requires efficient CO_(2)capture to mitigate the disastrous issue.In this report,large molecular size bisphenol A ethoxylate diacrylate(BPA)was employed to crosslink poly(ethylene glycol)methyl ether acrylate(PEGMEA)via the green and rapid UV polymerization strategy.The microstructure of such-prepared membrane could be conveniently tailored by tuning the ratio of the two prepolymers,aiming at obtaining the optimized microstructures with suitable mesh size and PEO sol content,which was approved by a novel low-field nuclear magnetic resonance technique.The optimum membrane overcomes the tradeoff challenge:dense microstructures lower the gas permeability while loose microstructures lower high-pressure-resistance capacity,realizing a high CO_(2)permeability of 1711 Barrer and 100-h long-term running stability under 15 atm.The proposed membrane fabrication approach,hence,opens a novel gate for developing high-performance robust membranes for CO_(2)capture.

THE ROLES OF REACTION INHOMOGENEITY IN PHASE SEPARATION KINETICS AND MORPHOLOGY OF REACTIVE POLYMER BLENDS

The roles of reaction inhomogeneity in phase separation of polymer mixtures were described and summarized via two examples:photocross-link of polymer mixtures in the bulk state and photopolymerization of monomer in the liquid state. The reaction kinetics,the reaction-induced elastic strain and the phase separation kinetics were monitored respectively by UV-Vis spectroscopy,Mach-Zehnder interferometry and laser-scanning confocal microscopy.It was found that phase separation in the bulk state was strongly inf...

Highly resilient and fatigue-resistant poly(4-methyl-ε-caprolactone)porous scaffold fabricated via thiol-yne photo-crosslinking/salt-templating for soft tissue regeneration

Elastomeric scaffolds, individually customized to mimic the structural and mechanical properties of natural tissues have been used for tissue regeneration. In this regard, polyester elastic scaffolds with tunable mechanical properties and exceptional biological properties have been reported to provide mechanical support and structural integrity for tissue repair. Herein, poly(4-methyl-ε-caprolactone) (PMCL) was first double-terminated by alkynylation (PMCL-DY) as a liquid precursor at room temperature. Subsequently, three-dimensional porous scaffolds with custom shapes were fabricated from PMCL-DY via thiol-yne photocrosslinking using a practical salt template method. By manipulating the Mn of the precursor, the modulus of compression of the scaffold was easily adjusted. As evidenced by the complete recovery from 90% compression, the rapid recovery rate of >500 mm min 1, the extremely low energy loss coefficient of <0.1, and the superior fatigue resistance, the PMCL20-DY porous scaffold was confirmed to harbor excellent elastic properties. In addition, the high resilience of the scaffold was confirmed to endow it with a minimally invasive application potential. In vitro testing revealed that the 3D porous scaffold was biocompatible with rat bone marrow stromal cells (BMSCs), inducing BMSCs to differentiate into chondrogenic cells. In addition, the elastic porous scaffold demonstrated good regenerative efficiency in a 12-week rabbit cartilage defect model. Thus, the novel polyester scaffold with adaptable mechanical properties may have extensive applications in soft tissue regeneration.

Synthesis of easily-processable collagen bio-inks using ionic liquid for 3D bioprinted liver tissue models with branched vascular networks

Bioprinting has been a flouring way to fabricate complex tissue and organ mimics via precisely depositing printable cell-laden biomaterials.However,there is a limited number of biomaterials that fulfill the mechanical property of printing while meeting the responsive environment desired for the cells.Despite excellent cell compatibility and bioactivity,collagen suffers from difficulties in processing and printability which inhibited its utilization in three-dimensional(3D)bioprinting.Herein,we address this limitation by using ionic liquid as the solvent in the modification process,enabling collagens modified with quantified norbornene for chemical crosslink and extrusion-based 3D printing.With improved solubility and rheological properties,norbornene-functionalized collagen(Col-Nor)exhibited better shape fidelity in extrusion-based 3D printing compared with the one before modification.Photo-crosslinked Col-Nor hydrogel provided structural support and promoted the adhesion,proliferation,and differentiation of various types of cells,which afforded a centimeter-scale liver tissue model.This highly generalizable methodology expands printable,versatile,and tunable hydrogels developed from the natural extracellular matrix,allowing the biofabrication of 3D liver tissue model with branched vascular networks.

The effect of collagen hydrogels on chondrocyte behaviors through restricting the contraction of cell/hydrogel constructs

Collagen is a promising material for tissue engineering,but the poor mechanical properties of collagen hydrogels,which tend to cause contraction under the action of cellular activity,make its application challengeable.In this study,the amino group of type I collagen(Col I)was modified with methacrylic anhydride(MA)and the photo-crosslinkable methacrylate anhydride modified type I collagen(CM)with three different degrees of substitution(DS)was prepared.The physical properties of CM and Col I hydrogels were tested,including micromorphology,mechanical properties and degradation properties.The results showed that the storage modulus and degradation rate of hydrogels could be adjusted by changing the DS of CM.In vitro,chondrocytes were seeded into these four groups of hydrogels and subjected to fluorescein diacetate/propidium iodide(FDA/PI)staining,cell counting kit-8(CCK-8)test,histological staining and cartilage-related gene expression analysis.In vivo,these hydrogels encapsulating chondrocytes were implanted subcutaneously into nude mice,then histological staining and sulfated glycosaminoglycan(sGAG)/DNA assays were performed.The results demonstrated that contraction of hydrogels affected behaviors of chondrocytes,and CM hydrogels with suitable DS could resist contraction of hydrogels and promote the secretion of cartilage-specific matrix in vitro and in vivo.

Bidirectional differentiation of BMSCs induced by a biomimetic procallus based on a gelatin-reduced graphene oxide reinforced hydrogel for rapid bone regeneration

Developmental engineering strategy needs the biomimetic composites that can integrate the progenitor cells,biomaterial matrices and bioactive signals to mimic the natural bone healing process for faster healing and reconstruction of segmental bone defects.We prepared the gelatin-reduced graphene oxide(GOG)and constructed the composites that mimicked the procallus by combining the GOG with the photo-crosslinked gelatin hydrogel.The biological effects of the GOG-reinforced composites could induce the bi-differentiation of bone marrow stromal cells(BMSCs)for rapid bone repair.The proper ratio of GOG in the composites regulated the composites’mechanical properties to a suitable range for the adhesion and proliferation of BMSCs.Besides,the GOG-mediated bidirectional differentiation of BMSCs,including osteogenesis and angiogenesis,could be activated through Erk1/2 and AKT pathway.The methyl vanillate(MV)delivered by GOG also contributed to the bioactive signals of the biomimetic procallus through priming the osteogenesis of BMSCs.During the repair of the calvarial defect in vivo,the initial hypoxic condition due to GOG in the composites gradually transformed into a well-vasculature robust situation with the bi-differentiation of BMSCs,which mimicked the process of bone healing resulting in the rapid bone regeneration.As an inorganic constituent,GOG reinforced the organic photo-crosslinked gelatin hydrogel to form a double-phase biomimetic procallus,which provided the porous extracellular matrix microenvironment and bioactive signals for the bi-directional differentiation of BMSCs.These show a promised application of the bio-reduced graphene oxide in biomedicine with a developmental engineering strategy.

INITIATOR-FREE PHOTOCROSSLINKING OF ELECTROSPUN BIODEGRADABLE POLYESTER FIBER BASED TUBULAR SCAFFOLDS AND THEIR CELL AFFINITY FOR VASCULAR TISSUE ENGINEERING

Random copolyester of poly（ε-caprolactone-co-L-lactide） （PCLA） with a 50：50 feeding molar ratio was synthesized via the ring-opening polymerization and functionalized by the end-capping reaction with acryloyl chloride. The resulting acrylated PCLA was then fabricated into small diameter tubular scaffolds by electrospinning technique and the formed scaffolds were followed by photocrosslinking under UV irradiation in the absence of photoinitiator. The mechanical strengths including tensile, suture retention and burst pressure were greatly enhanced after the photocrosslinking. The in vitro degradation data clearly revealed that the mechanical properties of the crosslinked scaffolds still remained after one month degradation in PBS solution, while those of the non-crosslinked ones lost heavily. The cytotoxicity assay on the mouse fibroblast L929 cells was conducted via MTT measurement. Furthermore, the observation on endothelial and fibroblast cell adhesion and proliferation was also made by using scanning electron microscopy （SEM）. The initiator-free photocrosslinked tubular scaffolds show the potential to be used in vascular tissue engineering.

Preparation and Characterization of Attractive Poly(amino acid)Hydrogels Based on 2-Ureido-4[1H]-pyrimidinone

Self-healing hydrogels with the shear-thinning property are novel injectable materials and are superior to traditional injectable hydrogels.The self-healing hydrogels based on 2-ureido-4[1 H]-pyrimidinone(UPy)have recently received extensive attention due to their dynamic reversibility of UPy dimerization.However,generally,UPy-based self-healing hydrogels exhibit poor stability,cannot degrade in vivo and can hardly be excreted from the body,which considerably limit their bio-application.Here,using poly(l-glutamic acid)(PLGA)as biodegradable matrix,branchingα-hydroxy-ω-amino poly(ethylene oxide)(HAPEO)as bridging molecule to introduce UPy,and ethyl acrylate polyethylene glycol(MAPEG)to introduce double bond,the hydrogel precursors(PMHU)are prepared.A library of the self-healing hydrogels has been achieved with well self-healable and shear-thinning properties.With the increase of MAPEG grafting ratio,the storage modulus of the self-healing hydrogels decreases.The self-healing hydrogels are stable in solution only for 6 h,hard to meet the requirements of tissue regeneration.Consequently,ultraviolet(UV)photo-crosslinking is involved to obtain the dual crosslinking hydrogels with enhanced mechanical properties and stability.When MAPEG grafting ratio is 35.5%,the dual crosslinking hydrogels can maintain the shape in phosphate-buffered saline solution(PBS)for at least 8 days.Loading with adipose-derived stem cell spheroids,the self-healing hydrogels are injected and self-heal to a whole,and then they are crosslinked in situ via UV-irradiation,obtaining the dual crosslinking hydrogels/cell spheroids complex with cell viability of 86.7%±6.0%,which demonstrates excellent injectability,subcutaneous gelatinization,and biocompatibility of hydrogels as cell carriers.The novel PMHU hydrogels crosslinked by quadruple hydrogen bonding and then dual photo-crosslinking of double bond are expected to be applied for minimal invasive surgery or therapies in tissue engineering.

Facile and rapid fabrication of a novel 3D-printable,visible light-crosslinkable and bioactive polythiourethane for large-to-massive rotator cuff tendon repair

Facile and rapid 3D fabrication of strong,bioactive materials can address challenges that impede repair of large-to-massive rotator cuff tears including personalized grafts,limited mechanical support,and inadequate tissue regeneration.Herein,we developed a facile and rapid methodology that generates visible light-crosslinkable polythiourethane(PHT)pre-polymer resin(~30 min at room temperature),yielding 3D-printable scaffolds with tendon-like mechanical attributes capable of delivering tenogenic bioactive factors.Ex vivo characterization confirmed successful fabrication,robust human supraspinatus tendon(SST)-like tensile properties(strength:23 MPa,modulus:459 MPa,at least 10,000 physiological loading cycles without failure),excellent suture retention(8.62-fold lower than acellular dermal matrix(ADM)-based clinical graft),slow degradation,and controlled release of fibroblast growth factor-2(FGF-2)and transforming growth factor-β3(TGF-β3).In vitro studies showed cytocompatibility and growth factor-mediated tenogenic-like differentiation of mesenchymal stem cells.In vivo studies demonstrated biocompatibility(3-week mouse subcutaneous implantation)and ability of growth factor-containing scaffolds to notably regenerate at least 1-cm of tendon with native-like biomechanical attributes as uninjured shoulder(8-week,large-to-massive 1-cm gap rabbit rotator cuff injury).This study demonstrates use of a 3D-printable,strong,and bioactive material to provide mechanical support and pro-regenerative cues for challenging injuries such as large-to-massive rotator cuff tears.