Biomaterial–Related Cell Microenvironment in Tissue Engineering and Regenerative Medicine

An appropriate cell microenvironment is key to tissue engineering and regenerative medicine.Revealing the factors that influence the cell microenvironment is a fundamental research topic in the fields of cell biology,biomaterials,tissue engineering,and regenerative medicine.The cell microenvironment consists of not only its surrounding cells and soluble factors,but also its extracellular matrix(ECM)or nearby external biomaterials in tissue engineering and regeneration.This review focuses on six aspects of bioma-terial-related cell microenvironments:①chemical composition of materials,②material dimensions and architecture,③material-controlled cell geometry,④effects of material charges on cells,⑤matrix stiff-ness and biomechanical microenvironment,and⑥surface modification of materials.The present chal-lenges in tissue engineering are also mentioned,and eight perspectives are predicted.

Finding MicroRNA Targets in Plants: Current Status and Perspectives

MicroRNAs （miRNAs）, a class of ～20-24 nt long non-coding RNAs, have critical roles in diverse biological processes including devel- opment, proliferation, stress response, etc. With the development and availability of experimental technologies and computational approaches, the field of miRNA biology has advanced tremendously over the last decade. By sequence complementarity, miRNAs have been estimated to regulate certain mRNA transcripts. Although it was once thought to be simple and straightforward to find plant miR NA targets, this viewpoint is being challenged by genetic and biochemical studies. In this review, we summarize recent progress in plant miRNA target recognition mechanisms, principles of target prediction, and introduce current experimental and computational tools for plant miRNA target prediction. At the end, we also present our thinking on the outlook for future directions in the development of plant miRNA target finding methods.

Effects of serum proteins on corrosion rates and product bioabsorbability of biodegradable metals

Corrodible metals are the newest kind of biodegradable materials and raise a new problem of the corrosion products.However,the removal of the precipitated products has been unclear and even largely ignored in publications.Herein,we find that albumin,an abundant macromolecule in serum,enhances the solubility of corrosion products of iron in blood mimetic Hank’s solution significantly.This is universal for other main biodegradable metals such as magnesium,zinc and polyester-coated iron.Albumin also influences corrosion rates in diverse trends in Hank’s solution and normal saline.Based on quantitative study theoretically and experimentally,both the effects on corrosion rates and soluble fractions are interpreted by a unified mechanism,and the key factor leading to different corrosion behaviors in corrosion media is the interference of albumin to the Ca/P passivation layer on the metal surface.This work has illustrated that the interactions between metals and media macromolecules should be taken into consideration in the design of the next-generation metal-based biodegradable medical devices in the formulism of precision medicine.The improved Hank’s solution in the presence of albumin and with a higher content of initial calcium salt is suggested to access biodegradable metals potentially for cardiovascular medical devices,where the content of calcium salt is calculated after consideration of chelating of calcium ions by albumin,resulting in the physiological concentration of free calcium ions.

Autophagy inhibition mediated via an injectable and NO-releasing hydrogelfor amplifying the antitumor efficacy of mild magnetic hyperthermia

While mild hyperthermia holds great potential in the treatment of solid tumors, the thermal stress-triggered selfrepairingautophagy significantly compromises its efficacy. To circumvent this obstacle, an injectable hydrogel(NO-Gel) composed of thermosensitive poly(ethylene glycol)-polypeptide copolymers modified with abundantNO donors on their side chains is developed. Meanwhile, ferrimagnetic Zn0.5Fe2.5O4 magnetic nanoparticles(MNPs) with high magnetic-heat conversion efficiency are synthesized and loaded into NO-Gel to obtainMNPs@NO-Gel. The MNPs@NO-Gel system exhibits a sol-gel transition upon heating, and has the ability toperform multiple magnetic hyperthermia therapy (MHT) after only one administration due to the even distributionand strong immobilization of MNPs in NO-Gel. NO can be continuously liberated from NO-Gel and thisprocess is markedly accelerated by MHT. Additionally, MNPs@NO-Gel maintains its integrity in vivo for over onemonth and the released MNPs are metabolized by the spleen. After a single administration of MNPs@NO-Gel atthe tumor site, three mild MHT treatments with similar effects are fulfilled, and the sufficient supply of NOeffectively inhibits MHT-induced autophagic flux via blocking the formation of autophagosomes and synchronouslydestroying lysosomes, thereby substantially boosting the efficacy of mild MHT. As a consequence, CT-26colon tumors are completely eliminated without causing severe side-effects.

Recent advances in regenerative biomaterials

Nowadays,biomaterials have evolved from the inert supports or functional substitutes to the bioactive materials able to trigger or promote the regenerative potential of tissues.The interdisciplinary progress has broadened the definition of‘biomaterials’,and a typical new insight is the concept of tissue induction biomaterials.The term‘regenerative biomaterials’and thus the contents of this article are relevant to yet beyond tissue induction biomaterials.This review summarizes the recent progress of medical materials including metals,ceramics,hydrogels,other polymers and bio-derived materials.As the application aspects are concerned,this article introduces regenerative biomaterials for bone and cartilage regeneration,cardiovascular repair,3D bioprinting,wound healing and medical cosmetology.Cell-biomaterial interactions are highlighted.Since the global pandemic of coronavirus disease 2019,the review particularly mentions biomaterials for public health emergency.In the last section,perspectives are suggested:(i)creation of new materials is the source of innovation;(ii)modification of existing materials is an effective strategy for performance improvement;(iii)biomaterial degradation and tissue regeneration are required to be harmonious with each other;(iv)host responses can significantly influence the clinical outcomes;(v)the long-term outcomes should be paid more attention to;(vi)the noninvasive approaches for monitoring in vivo dynamic evolution are required to be developed;(vii)public health emergencies call for more research and development of biomaterials;and(viii)clinical translation needs to be pushed forward in a full-chain way.In the future,more new insights are expected to be shed into the brilliant field-regenerative biomaterials.

Effect of porosities of bilayered porous scaffolds on spontaneous osteochondral repair in cartilage tissue engineering

Poly(lactide-co-glycolide)-bilayered scaffolds with the same porosity or different ones on the two layers were fabricated,and the porosity effect on in vivo repairing of the osteochondral defect was examined in a comparative way for the first time.The constructs of scaffolds and bone marrow-derived mesenchymal stem cells were implanted into pre-created osteochondral defects in the femoral condyle of New Zealand white rabbits.After 12 weeks,all experimental groups exhibited good cartilage repairing according to macroscopic appearance,cross-section view,haematoxylin and eosin staining,toluidine blue staining,immunohistochemical staining and real-time polymerase chain reaction of characteristic genes.The group of 92%porosity in the cartilage layer and 77%porosity in the bone layer resulted in the best efficacy,which was understood by more biomechanical mimicking of the natural cartilage and subchondral bone.This study illustrates unambiguously that cartilage tissue engineering allows for a wide range of scaffold porosity,yet some porosity group is optimal.It is also revealed that the biomechanical matching with the natural composite tissue should be taken into consideration in the design of practical biomaterials,which is especially important for porosities of a multi-compartment scaffold concerning connected tissues.

In vivo degradation and endothelialization of an iron bioresorbable scaffold

Detection of in vivo biodegradation is critical for development of next-generation medical devices such as bioresorbable stents or scaffolds(BRSs).In particular,it is urgent to establish a nondestructive approach to examine in vivo degradation of a new-generation coronary stent for interventional treatment based on mammal experiments;otherwise it is not available to semi-quantitatively monitor biodegradation in any clinical trial.Herein,we put forward a semi-quantitative approach to measure degradation of a sirolimus-eluting iron bioresorbable scaffold(IBS)based on optical coherence tomography(OCT)images;this approach was confirmed to be consistent with the present weight-loss measurements,which is,however,a destructive approach.The IBS was fabricated by a metal-polymer composite technique with a polylactide coating on an iron stent.The efficacy as a coronary stent of this new bioresorbable scaffold was compared with that of a permanent metal stent with the name of trade mark Xience,which has been widely used in clinic.The endothelial coverage on IBS was found to be greater than on Xience after implantation in a rabbit model;and our well-designed ultrathin stent exhibited less individual variation.We further examined degradation of the IBSs in both minipig coronary artery and rabbit abdominal aorta models.The present result indicated much faster iron degradation of IBS in the rabbit model than in the porcine model.The semi-quantitative approach to detect biodegradation of IBS and the finding of the species difference might be stimulating for fundamental investigation of biodegradable implants and clinical translation of the next-generation coronary stents.

‘Invisible’ orthodontics by polymeric ‘clear’ aligners molded on 3D-printed personalized dental models

The malalignment of teeth is treated classically by metal braces with alloy wires,which has an unfavorable influence on the patients appearance during the treatment.With the development of digitization,computer simulation and three-dimensional(3D)printing technology,herein,a modern treatment was tried using clear polymeric aligners,which were fabricated by molding polyurethane films via thermoforming on the 3D-printed personalized dental models.The key parameters of photocurable 3D printing of dental models and the mechanical properties of the clear aligner film material were examined.The precision of a 3D-printed dental model mainly relied on characteristics of photocurable resin,the resolution of light source and the exposure condition,which determined the eventual shape of the molded clear aligner and thus the orthodontic treatment efficacy.The biocompatibility of the polyurethane filmmaterial was confirmed through cytotoxicity and hemolysis tests in vitro.Following a series of 3D-printed personalized dental models and finite element analysis to predict and plan the fabrication and orthodontic processes,corresponding clear aligners were fabricated and applied in animal experiments,which proved the efficacy and biocompatibility in vivo.Clinical treatments of 120 orthodontic cases were finally carried out with success,which highlights the advantage of the clear aligners as an esthetic,compatible and efficient appliance.

Research and clinical translation of trilayer stent-graft of expanded polytetrafluoroethylene for interventional treatment of aortic dissection

The aortic dissection(AD)is a life-threatening disease.The transcatheter endovascular aortic repair(EVAR)affords a minimally invasive technique to save the lives of these critical patients,and an appropriate stent-graft gets to be the key medical device during an EVAR procedure.Herein,we report a trilayer stent-graft and corresponding delivery system used for the treatment of the AD disease.The stent-graft is made of nitinol stents with an asymmetric Z-wave design and two expanded polytetrafluoroethylene(ePTFE)membranes.Each of the inner and outer surfaces of the stent-graft was covered by an ePTFE membrane,and the two membranes were then sintered together.The biological studies of the sintered ePTFE membranes indicated that the stent-graft had excellent cytocompatibility and hemocompatibility in vitro.Both the stent-graft and the delivery system exhibited satisfactory mechanical properties and operability.The safety and efficacy of this stent-graft and the corresponding delivery system were demonstrated in vivo.In nine canine experiments,the blood vessels of the animals implanted with the stent-grafts were of good patency,and there were no thrombus and obvious stenosis by angiography after implantation for 6months.Furthermore,all of the nine clinical cases experienced successful implantation using the stent-graft and its postrelease delivery system,and the 1-year follow-ups indicated the preliminary safety and efficacy of the trilayer stent-graft with an asymmetric Z-wave design for interventional treatment.

Magnetic resonance imaging for non-invasive clinical evaluation of normal and regenerated cartilage

With the development of tissue engineering and regenerative medicine,it is much desired to establish bioimaging techniques to monitor the real-time regeneration efficacy in vivo in a non-invasive way.Herein,we tried magnetic resonance imaging(MRI)to evaluate knee cartilage regeneration after implanting a biomaterial scaffold seeded with chondrocytes,namely,matrix-induced autologous chondrocyte implantation(MACI).After summary of the T2 mapping and the T1-related delayed gadolinium-enhanced MRI imaging of cartilage(dGEMRIC)in vitro and in vivo in the literature,these two MRI techniques were tried clinically.In this study,18 patients were followed up for 1 year.It was found that there was a significant difference between the regeneration site and the neighboring normal site(control),and the difference gradually diminished with regeneration time up to 1 year according to both the quantitative T1 and T2 MRI methods.We further established the correlation between the quantitative evaluation of MRI and the clinical Lysholm scores for the first time.Hence,the MRI technique was confirmed to be a feasible semiquantitative yet non-invasive way to evaluate the in vivo regeneration of knee articular cartilage.

Is polydopamine beneficial for cells on the modified surface?

Since the pioneering work of Messersmith’s group discovering that polydopamine(PDA)can serve to adhere to many types of materials,the PDA coating has,as a biomimetic approach,been widely used to enhance cell adhesion by surface modification to bind biologically active substances to a bioinert substrate.Nevertheless,it is unclear whether or not the PDA itself is beneficial for cells.Herein,we report that a PDA coating decreases viability of cells under normal culture and observation conditions.Such an inhibition effect was not caused by the free PDA or any inherent cytotoxicity of this chemical substance but a contactdependent phenomenon.Human bone marrow mesenchymal stem cells were employed as the default cell type and tissue culture plates were used as the default substrate,although some other cell types and substrates were also examined to confirm the universality of such an‘abnormal’phenomenon of a superstar molecule.The viability of cells on the PDA coating exhibited time dependence,and the decreased cell viability during the normal observation time was found to come from the decrease of cell number instead of the decrease of average viability per cell.The PDA coating led to less cell global migration yet more local motility of cells.Based on the concept of‘background adhesion’of cells on a surface without significant motifs of specific cell adhesion,we supposed that cells adhered to the PDA coating better,which influenced mobility and eventually proliferation.Hence,the cell behaviors on the PDA coating are reasonable,albeit a bit complicated.

Coordination Insertion Mechanism of Ring-Opening Polymerization of Lactide Catalyzed by Stannous Octoate

Main observation and conclusion Ring-opening polymerization(ROP)of cyclic esters in the presence of stannous octoate(Sn(Oct)2)is the main way to obtain biodegradable aliphatic polyesters,an important family of biodegradable polymers which have been widely used and still rapidly developed in the fields of biomedical polymers and environment-friendly materials.

Injectable and thermosensitive hydrogels mediating a universal macromolecular contrast agent with radiopacity for noninvasive imaging of deep tissues

It is very challenging to visualize implantable medical devices made of biodegradable polymers in deep tissues.Herein,we designed a novel macromolecular contrast agent with ultrahigh radiopacity(iodinate content>50%)via polymerizing an iodinated trimethylene carbonate monomer into the two ends of poly(ethylene glycol)(PEG).A set of thermosensitive and biodegradable polyester-PEG-polyester triblock copolymers with varied polyester compositions synthesized by us,which were soluble in water at room temperature and could spontaneously form hydrogels at body temperature,were selected as the demonstration materials.The addition of macromolecular contrast agent did not obviously compromise the injectability and thermogelation properties of polymeric hydrogels,but conferred them with excellent X-ray opacity,enabling visualization of the hydrogels at clinically relevant depths through X-ray fluoroscopy or Micro-CT.In a mouse model,the 3D morphology of the radiopaque hydrogels after injection into different target sites was visible using Micro-CT imaging,and their injection volume could be accurately obtained.Furthermore,the subcutaneous degradation process of a radiopaque hydrogel could be non-invasively monitored in a real-time and quantitative manner.In particular,the corrected degradation curve based on Micro-CT imaging well matched with the degradation profile of virgin polymer hydrogel determined by the gravimetric method.These findings indicate that the macromolecular contrast agent has good universality for the construction of various radiopaque polymer hydrogels,and can nondestructively trace and quantify their degradation in vivo.Meanwhile,the present methodology developed by us affords a platform technology for deep tissue imaging of polymeric materials.

Establishment of coverage-mass equation to quantify the corrosion inhomogeneity and examination of medium effects on iron corrosion

Metal corrosion is important in the fields of biomedicine as well as construction and transportation etc.While most corrosion occurs inhomogeneously,there is so far no satisfactory parameter to characterize corrosion inhomogeneity.Herein,we employ the Poisson raindrop question to model the corrosion process and derive an equation to relate corrosion coverage and corrosion mass.The resultant equation is named coverage-mass equation,abbreviated as C-M equation.We also suggest corrosion mass at 50%coverage,termed as half-coverage mass Mcorro50%,as an inhomogeneity parameter to quantify corrosion inhomogeneity.The equation is confirmed and the half-coverage mass Mcorro50%is justified in our experiments of iron corrosion in five aqueous media,normal saline,phosphate-buffered saline,Hank’s solution,deionized water and artificial seawater,where the former three ones are biomimetic and very important in studies of biomedical materials.The half-coverage mass Mcorro50%is proved to be more comprehensive and mathematically convergent than the traditional pitting factor.Iron corrosion is detected using visual observation,scanning electron microscopy with a build-in energy dispersive spectrometer,inductive coupled plasma emission spectrometry and electrochemical measurements.Both rates and inhomogeneity extents of iron corrosion are compared among the five aqueous media.The factors underlying the medium effects on corrosion rate and inhomogeneity are discussed and interpreted.Corrosion rates of iron in the five media differ about 7-fold,and half-coverage mass values differ about 300000-fold.The fastest corrosion and the most significant inhomogeneity occur both in biomimetic media,but not the same one.The new equation(C-M equation)and the new quantity(half-coverage mass)are stimulating for dealing with a dynamic and stochastic process with global inhomogeneity including but not limited to metal corrosion.The findings are particularly meaningful for research and development of next-generation biodegradable materials.

Biaxial stretching of polytetrafluoroethylene in industrial scale to fabricate medical ePTFE membrane with node-fibril microstructure

Expanded polytetrafluoroethylene(ePTFE)is promising in biomedical fields such as covered stents and plastic surgery owing to its excellent biocompatibility and mechanical properties.However,ePTFE material prepared by the traditional biaxial stretching process is with thicker middle and thinner sides due to the bowing effect,which poses a major problem in industrial-scale fabrication.To solve this problem,we design an olive-shaped winding roller to provide the middle part of the ePTFE tape with a greater longitudinal stretching amplitude than the two sides,so as to make up for the excessive longitudinal retraction tendency of the middle part when it is transversely stretched.The as-fabricated ePTFE membrane has,as designed,uniform thickness and node-fibril microstructure.In addition,we examine the effects of mass ratio of lubricant to PTFE powder,biaxial stretching ratio and sintering temperature on the performance of the resultant ePTFE membranes.Particularly,the relation between the internal microstructure of the ePTFE membrane and its mechanical properties is revealed.Besides stable mechanical properties,the sintered ePTFE membrane exhibits satisfactory biological properties.We make a series of biological assessments including in vitro hemolysis,coagulation,bacterial reverse mutation and in vivo thrombosis,intracutaneous reactivity test,pyrogen test and subchronic systemic toxicity test;all of the results meet the relevant international standards.The muscle implantation of the sintered ePTFE membrane into rabbits indicates acceptable inflammatory reactions of our sintered ePTFE membrane fabricated on industrial scale.Such a medical-grade raw material with the unique physical form and condensed-state microstructure is expected to afford an inert biomaterial potentially for stent-graft membrane.

Left-Right Symmetry or Asymmetry of Cells on Stripe-Like Micropatterned Material Surfaces

Material surfaces can induce cell responses such as contact guidance, yet little attention has been paid to further cell orientation. Herein, we report an interesting phenomenon of cell orientation beyond the classic contact guidance on a stripe-like micropattern with cell-adhesive arginineglycine-aspartate （RGD） peptides on a nonfouling background decorated by poly（ethylene glycol） （PEG）. Such a micropattern with cell adhesion contrast led to significant contact guidance after cell seeding. What is more, the localized and elongated cells were found to be further orientated out of the adhesive stripes, and even an anticlockwise rotation was observed for rat mesenchymal stem cells （rMSCs）. The left-right asymmetry of rMSCs stood only in statistics, for we observed all cases including clockwise orientation, anticlockwise orientation or just keeping the orientation of previous contact guidance. We further found that human foreskin fibroblasts （HFFs） preferred a clockwise rotation, while human mesenchymal stem cells （hMSCs） and human umbilical vascular endothelial cells （HUVECs） exhibited no significant preference to either direction, which indicated that the left-right symmetry or asymmetry was cell-type dependent. The present report has partially confirmed the cell chirality and revealed its complexity, calling for further careful and comprehensive investigation of the challenging topic of cell chirality on material surfaces.

Critical adhesion areas of cells on micro-nanopatterns

Cell adhesion to extracellular matrices(ECM)is critical to physiological and pathological processes as well as biomedical and biotechnological applications.It has been known that a cell can adhere on an adhesive microisland only over a critical size.But no publication has concerned critical adhesion areas of cells on microislands with nanoarray decoration.Herein,we fabricated a series of micro-nanopatterns with different microisland sizes and arginine-glycine-aspartate(RGD)nanospacings on a nonfouling poly(ethylene glycol)background.Besides reproducing that nanospacing of RGD,a ligand of its receptor integrin(a membrane protein),significantly influences specific cell adhesion on bioactive nanoarrays,we confirmed that the concept of critical adhesion area originally suggested in studies of cells on micropatterns was justified also on the micro-nanopatterns,yet the latter exhibited more characteristic behaviors of cell adhesion.We found increased critical adhesion areas of human mesenchymal stem cells(hMSCs)on nanoarrayed microislands with increased RGD nanospacings.However,the numbers of nanodots with respect to the critical adhesion areas were not a constant.A unified interpretation was then put forward after combining nonspecific background adhesion and specific cell adhesion.We further carried out the asymptotic analysis of a series of micro-nanopatterned surfaces to obtain the effective RGD nanospacing on unpatterned free surfaces with densely grafted RGD,which could be estimated nonzero but has never been revealed previously without the assistance of the micro-nanopatterning techniques and the corresponding analysis.

A composite strategy to fabricate high-performance biodegradable stents for tissue regeneration

While most of the present medical implants are nonbiodegradable,biodegradable materials are thought to replace many nonbiodegradable ones in the future,which is dependent on development of the corresponding core techniques of biomaterials[1-4].Much progress has been made in the field of biodegradable materials during the last decade[5-7].

Quantitatively relating magnetic resonance T_(1)and T_(2)to glycosaminoglycan and collagen concentrations mediated by penetrated contrast agents and biomacromolecule-bound water

Magnetic resonance imaging(MRI)is a promising non-invasive method to assess cartilage regeneration based on the quantitative relationship between MRI features and concentrations of the major components in the extracellular matrix(ECM).To this end,in vitro experiments are performed to investigate the relationship and reveal the underlying mechanism.A series of collagen(COL)and glycosaminoglycan(GAG)solutions at different concentrations are prepared,and T_(1)and T_(2)relaxation times are measured with or without a contrast agent(Gd-DTPA2−)by MRI.Fourier transform infrared spectrometry is also used to measure the contents of biomacromolecule-bound water and other water,allowing theoretical derivation of the relationship between biomacromolecules and the resulting T_(2)values.It has been revealed that the MRI signal in the biomacromolecule aqueous systems is mainly influenced by the protons in hydrogens of biomacromolecule-bound water,which we divide into inner-bound water and outer-bound water.We have also found that COL results in higher sensitivity of bound water than GAG in T_(2)mapping.Owing to the charge effect,GAG regulates the penetration of the contrast agent during dialysis and has a more significant effect on T_(1)values than COL.Considering that COL and GAG are the most abundant biomacromolecules in the cartilage,this study is particularly useful for the real-time MRI-guided assessment of cartilage regeneration.A clinical case is reported as an in vivo demonstration,which is consistent with our in vitro results.The established quantitative relation plays a critical academic role in establishing an international standard ISO/TS24560-1:2022‘Clinical evaluation of regenerative knee articular cartilage using delayed gadolinium-enhanced MRI of cartilage(dGEMRIC)and T_(2)mapping’drafted by us and approved by International Standard Organization.

Design and aligner-assisted fast fabrication of a microfluidic platform for quasi-3D cell studies on an elastic polymer

While most studies of mechanical stimulation of cells are focused on two-dimensional(2D)and three-dimensional(3D)systems,it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D.Herein,we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip.The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers.To resolve the problem,we designed and assembled a fast aligner that is cost-effective and convenient to operate.To enable quasi-3D microenvironment of cells,we fabricated polydimethylsiloxane(PDMS)microwell arrays(formed by micropillars of a few microns in diameter)with the microwell diameters close to the cell sizes.An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS.We examined three types of cells in 2D,quasi-3D,and 3D microenvironments;the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells.We also constructed transgenic human mesenchymal stem cells(hMSCs);under cyclic stretching,the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate.This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies,but also inspires further studies of the topological effects of biomaterials on cells.