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.

Nanofibrous scaffolds for the regeneration of nervous tissue

Introductons The biophysical organization of extracellular matrix (ECM) plays an important role in tissue morphogenesis,remodeling and functions. In many types of tissues,e. g. ,blood vessel,nerve,heart,muscle,tendon and ligament,ECM has aniso-

Additive manufacturing of biodegradable magnesium implants and scaffolds: Review of the recent advances and research trends

Synthetic grafting needs improvements to eliminate secondary surgeries for the removal of implants after healing of the defected tissues.Tissue scaffolds are engineered to serve as temporary templates,which support the affected tissue and gradually degrade through the healing period.Beside mechanical function to withstand the anatomic loading conditions,scaffolds should also provide a decent biological function for the diffusion of nutrients and oxygen to the cells,and excretion of the wastes from the cells to promote the new tissue growth and vascularization.Moreover,the degradation byproducts of the scaffolds should be safe to the human body.Development of such multifunctional scaffolds requires selection of the right material,design,and manufacturing method.Mg has been recognized as the prominent biodegradable metal with regards to its mechanical properties matching to that of human bone,degradability in the body fluid,and its ability to stimulate new tissue growth.Scaffolds with intricate porous structures can be designed according to the patient-specific anatomic data using computer aided designs.Additive manufacturing(AM)is the right method to materialize these models rapidly with reasonably acceptable range of dimensional accuracy.Thus,the recent research trend is to develop ideal scaffolds using biodegradable Mg through AM methods.This review compiles and discusses the available literature on the AM of biodegradable Mg parts from the viewpoints of material compositions,process conditions,formation quality,dimensional accuracy,microstructure,biodegradation,and mechanical properties.The current achievements are summarized together,and future research directions are identified to promote clinical applications of biodegradable Mg through the advancement of AM.

Advanced optical methods and materials for fabricating 3D tissue scaffolds

Three-dimensional(3D)printing,also known as additive manufacturing(AM),has undergone a phase of rapid development in the fabrication of customizable and high-precision parts.Thanks to the advancements in 3D printing technologies,it is now a reality to print cells,growth factors,and various biocompatible materials altogether into arbitrarily complex 3D scaffolds with high degree of structural and functional similarities to the native tissue environment.Additionally,with overpowering advantages in molding efficiency,resolution,and a wide selection of applicable materials,optical 3D printing methods have undoubtedly become the most suitable approach for scaffold fabrication in tissue engineering(TE).In this paper,we first provide a comprehensive and up-to-date review of current optical 3D printing methods for scaffold fabrication,including traditional extrusion-based processes,selective laser sintering,stereolithography,and two-photon polymerization etc.Specifically,we review the optical design,materials,and representative applications,followed by fabrication performance comparison.Important metrics include fabrication precision,rate,materials,and application scenarios.Finally,we summarize and compare the advantages and disadvantages of each technique to guide readers in the optics and TE communities to select the most fitting printing approach under different application scenarios.

Aligned Fibrous Scaffold Induced Aligned Growth of Corneal Stroma Cells in vitro Culture

To investigate the contribution of fibre arrangement to guiding the aligned growth of corneal stroma cells,aligned and randomly oriented fibrous scaffolds of gelatin and poly-L-lactic acid（PLLA） were fabricated by electrospinning.A comparative study of two different systems with corneal stroma cells on randomly organized and aligned fibres were conducted.The efficiency of the scaffolds for inducing the aligned growth of cells was assessed by morphological observation and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide（MTT） assay.Results show that the cells cultured on both randomly oriented and aligned scaffolds maintained normal morphology and well spreading as well as long term proliferation.Importantly,corneal stroma cells grew high orderly on the aligned scaffold,while the cells grew disordered on the randomly oriented scaffold.Moreover,the cells exhibited higher viability in aligned scaffold than that in randomly oriented scaffold.These results indcate that electrospinng to prepare aligned fibrous scaffolds has provided an effective approach to the aligned growth of corneal stroma cells in vitro.Our findings that fiber arrangement plays a crucial role in guiding the aligned growth of cells may be helpful to the development of better biomaterials for tissue engineered cornea.

A Systematic Review of Animal and Clinical Studies on the Use of Scaffolds for Urethral Repair

Replacing urethral tissue with functional scaffolds has been one of the challenging problems in the field of urethra reconstruction or repair over the last several decades. Various scaffold materials have been used in animal studies, but clinical studies on use of scaffolds for urethral repair are scarce. The aim of this study was to review recent animal and clinical studies on the use of different scaffolds for urethral repair, and to evaluate these scaffolds based on the evidence from these studies. Pub Med and OVID databases were searched to identify relevant studies, in conjunction with further manual search. Studies that met the inclusion criteria were systematically evaluated. Of 555 identified studies, 38 were included for analysis. It was found that in both animal and clinical studies, scaffolds seeded with cells were used for repair of large segmental defects of the urethra, such as in tubular urethroplasty. When the defect area was small, cell-free scaffolds were more likely to be applied. A lot of pre-clinical and limited clinical evidence showed that natural or artificial materials could be used as scaffolds for urethral repair. Urinary tissue engineering is still in the immature stage, and the safety, efficacy, cost-effectiveness of the scaffolds are needed for further study.

In vitro Mineralization Behavior of the Sol-gel Derived Bioglass/Collegen Composite Porous Scaffold

The porous scaffold of the sol-gel derived bioactive glass （BG） in the system CaO-P2O5-SiO2 was treated with the type Ⅰ collagen solution. The pore walls of the scaffold were covered by the collagenous network. The in vitro mineralization behavior of the sol- gel derived bioglassl collegen composite porous scaffold was investigated by immersion in supersaturated calcification solution （ SCS ） at 37℃ for different times, XRD , FTIR, SEM/ EDAX techniques were applied to analyze the crystalline phases, morphology and composition of the minerals formed on the pore walls of the scaffold. It was found that with increasing of immersion time, the morphology of reaction products on the pore walls changed from the spherical particles of calcium phosphate to the flake-like HCA crystals.

The Construction of Heparin Sulfate-collagen Protein Based Scaffolds and Its Effects on the Neural Stem Cells

The objective of this study was to construct the heparin sulfate-collagen pro-tein-based scaffolds and to explore its effects on the proliferation and differentiation abilities of neural stem cells(NSCs).The heparin sulfate-collagen protein-based scaffolds were produced by injection molding/freezing/cross-linking technique.The proliferation ability was detected by MTT and the dif-ferentiation ability was detected by immunofluorescence technique.The results indicate that the heparin sulfate-collagen protein-based scaffolds with porous and longitudinal parallel structures are successfully constructed.The NSCs grow well in the pores of scaffold and still maintain the prolif-eration and differentiation abilities.It is concluded that the scaffolds possess stable porous and longi-tudinal parallel structures and great biocompatibility to NSCs.The combination of scaffold and NSCs may suggest a possible treatment strategy for spinal cord injury

Bi-/multi-modal pore formation of PLGA/hydroxyapatite composite scaffolds by heterogeneous nucleation in supercritical CO_2 foaming

Scaffolds with multimodal pore structure are essential to cells differentiation and proliferation in bone tissue engineering. Bi-/multi-modal porous PLGA/hydroxyapatite composite scaffolds were prepared by supercritical C02 foaming in which hydroxyapatite acted as heterogeneous nucleation agent. Bimodal porous scaffolds were prepared under certain conditions, i.e. hydroxyapatite addition of 5%, depressurization rate of 0.3 MPa. min-1, soaking temperature of 55 ℃, and pressure of 9 MPa. And scaffolds presented specific structure of small pores （122 μM ± 66 μm） in the cellular walls of large pores （552 μm ±127 μm）. Furthermore, multimodal porous PLGA scaffolds with micro-pores （37 μM ± 11μM） were obtained at low soaking pressure of 7.5 MPa. The interconnected porosity of scaffolds ranged from （52.53 ± 2.69）% to （83.08±2.42）% by adjusting depressurization rate, while compression modulus satisfied the requirement of bone tissue engineering. Solvent-free CO2 foaming method is promising to fabricate bi-/multi-modal porous scaffolds in one step, and bioactive particles for osteogenesis could serve as nucleation agents.

Bioinspired coacervate-based bioinks for construction of multiscale tissue engineering scaffolds

Engineering hydrogels that resemble biological tissues of various lengths via conventional fabrication techniques remains challenging.Three-dimensional(3D)bioprinting has emerged as an advanced approach for constructing complex biomimetic 3D architectures,which are currently restricted by the limited number of available bioinks with high printability,biomimicry,biocompatibility,and proper mechanical properties.Inspired by ubiquitous coacervation phenomena in biology,we present a unique mineral-biopolymer coacervation strategy that enables the hierarchical assembly of nanoclay and recombinant human collagen(RHC).This system was observed to undergo a coacervation transition(liquid‒liquid phase separation)spontaneously.The formed dense phase separated from its supernatant is the coacervate of clay-RHC-rich complexes,where polymer chains are sandwiched between silicate layers.Molecular dynamics simulation was first used to verify and explore the coacervation process.Then,the coacervates were demonstrated to be potential bioinks that exhibited excellent self-supporting and shear-thinning viscoelastic properties.Through extrusion-based printing,the versatility of the bioink was demonstrated by reconstructing the key features of several biological tissues,including multilayered lattice,vascular,nose,and ear-like structures,without the need for precrosslinking operations or support baths.Furthermore,the printed scaffolds were cytocompatible,elicited minimal inflammatory responses,and promoted bone regeneration in calvarial defects.

Computer aided modeling and pore distribution of bionic porous bone structure

Artificial bone with porous structure is crucial for tissue scaffold and clinic implants.Scaffold provides structure support for cells and guides tissues regeneration for final tissue structure.A computational aided process of porous bone modeling was developed which described the design and fabrication of tissue scaffolds by considering intricate architecture,porosity and pore size.To simulate intricate bone structure,different constructive units were presented.In modeling process,bone contour was gotten from computed tomography(CT)images and was divided into two levels.Each level was represented by relatively reconstructive process.Pore size distribution was controlled by using mesh generation.The whole hexahedral mesh was reduced by unit structure,when a 3D mesh with various hexahedral elements was provided.The simulation results show that constructive structure of porous scaffold can meet the needs of clinic implants in accurate and controlled way.

AB085.E-beam sterilization of recombinant human collagen-phosphorylcholine corneal implants for transplantation

Background:The sterilization of corneal implants composed of carbodiimide crosslinked recombinant human collagen type III(RHCIII)and phosphorylcholine polymers(RHCIII-MPC)is constrained by the biochemical properties of RHCIII.Early human trials used 1%chloroform in 0.1 M phosphate buffered saline(C-PBS),but require a stringent wash procedure with antibiotics to remove the chloroform.Irradiation with gamma or electron-beam(e-beam)allows a chemical-free sterilization method,but may result in crosslinking or denaturation.Here,electron-beam irradiation is evaluated as a sterilization method for RHCIII-MPC implants.Methods:Dose-finding study:RHCIII-MPC were cast in round,350µm thick,12 mm diameter molds for corneal implants and 0.5 mm thick dumbbell-shaped molds for mechanical testing.The hydrogels received an irradiation dose of 17,19,or 21 kGy and unirradiated controls were stored in C-PBS,n=3 per group.The hydrogels were tested for sterility and endotoxin,optical and mechanical properties,biodegradation,free radicals,and cell compatibility.Clinical evaluation in rabbits:RHCIII-MPC implants were e-beamed at 17 kGy or kept in C-PBS.One implant from each group was implanted into the right cornea of each rabbit by deep anterior lamellar keratoplasty,n=4 animals per group.Animals underwent preoperative and 6-month post-operative in vivo confocal microscopy(IVCM)to check nerve count and ingrowth of keratocytes.Corneal grafts and controls were assessed via histology and immunohistochemistry.Results:Dose finding study:hydrogels were sterile at all irradiation doses with no evidence of free radicals.There were no significant differences in optical or mechanical properties between the treatment groups and controls.All hydrogels supported cell growth.The 19 and 21 kGy implants had high collagenase degradation for 21 hours until they stabilized,whereas the 17 kGy and C-PBS implants had gradual degradation until 48 hours.Clinical results:the rabbits did not experience post-surgical inflammatory reactions and full epithelial coverage of the implants occurred within the first week of surgery for all animals.Mild neovascularization occurred in all animals,but resolved by 6-month follow-up.A mild 0.5-1.0 grade subepithelial haze was observed in all rabbits,but the implanted grafts remained transparent.Re-innervation occurred in all grafts with no significant differences between sterilization methods.All regenerated corneas had mucin production and were positive for cytokeratin 3 and 12.Grafted and control corneas were negative for macrophages and blood vessels.Conclusions:E-beam sterilization is a safe and effective form of sterilization for RHCIII-MPC implants.

Challenges and Solutions for the Additive Manufacturing of H) Biodegradable Magnesium Implants

Due to their capability of fabricating geometrically complex structures,additive manufacturing(AM)techniques have provided unprecedented opportunities to produce biodegradable metallic implants—especially using Mg alloys,which exhibit appropriate mechanical properties and outstanding biocompatibility.However,many challenges hinder the fabrication of AM-processed biodegradable Mg-based implants,such as the difficulty of Mg powder preparation,powder splash,and crack formation during the AM process.In the present work,the challenges of AM-processed Mg components are analyzed and solutions to these challenges are proposed.A novel Mg-based alloy(Mg-Nd-Zn-Zr alloy,JDBM)powder with a smooth surface and good roundness was first synthesized successfully,and the AM parameters for Mg-based alloys were optimized.Based on the optimized parameters,porous JDBM scaffolds with three different architectures(biomimetic,diamond,and gyroid)were then fabricated by selective laser melting(SLM),and their mechanical properties and degradation behavior were evaluated.Finally,the gyroid scaffolds with the best performance were selected for dicalcium phosphate dihydrate(DCPD)coating treatment,which greatly suppressed the degradation rate and increased the cytocompatibility,indicating a promising prospect for clinical application as bone tissue engineering scaffolds.

Preparation and Characterization of Poly(γ-glutamic acid) Hydrogels as Potential Tissue Engineering Scaffolds

In this paper, methacrylated γ-PGA（m PGA） precursor was synthesized via reaction between γ-PGA and glycidyl methacrylate（GMA）. Hydrogels from this precursor were prepared under 365 nm ultraviolet irradiation. The swelling behavior and mechanical properties were studied in detail as functions of the degree of substitution（DS）, precursor concentration, and environmental p H. Results showed that the crosslink density, swelling kinetics and mechanical properties of the hdyrogel could be tailored by adjusting the DS and concentration of the precursor as well as the environmental p H. Three-dimensional photo-encapsulation of swine cartilage chondrocytes and Live/Dead assay proved the cytocompatibility of the hydrogel.

3D Bioplotting of Gelatin/Alginate Scaffolds for Tissue Engineering:Influence of Crosslinking Degree and Pore Architecture on Physicochemical Properties

Gelatin/Alginate hydrogels were engineered for bioplotting in tissue engineering. One major drawback of hydrogel scaffolds is the lack of adequate mechanical properties. In this study, using a bioplotter, we constructed the scaffolds with different pore architectures by deposition of gelatin/alginate hydrogels layerby-layer. The scaffolds with different crosslinking degree were obtained by post-crosslinking methods. Their physicochemical properties, as well as cell viability, were assessed. Different crosslinking methods had little influence on scaffold architecture, porosity, pore size and distribution. By contrast, the water absorption ability, degradation rate and mechanical properties of the scaffolds were dramatically affected by treatment with various concentrations of crosslinking agent （glutaraldehyde）. The crosslinking process using glutaraldehyde markedly improved the stability and mechanical strength of the hydrogel scaf- folds. Besides the post-processing methods, the pore architecture can also evidently affect the mechanical properties of the scaffolds. The crosslinked gelatin/alginate scaffolds showed a good potential to encap-sulate cells or drugs.

Electrospinning and Electrospun Nanofibers

Electro-spinning is a very modern process which can be used in various purposes. We did this experimental work at Swerea IVF in Sweden during M. Sc in Textile Technology programme at University of Bor?s. We should especially thank our supervisor—Anna Thorvaldsson and course teacher—Ioannis S. Chronakis. In this report, we have tried to explain the basic manufacturing techniques of the electrospun nanofiber by the electro-spinning, how one can characterize it by SEM (Scanning Electron Microscopy) and its various applications in the practical field, e.g wound healing, Tissue Engineering Scaffold. The experimental work helped us a lot to gather sufficient knowledge about the electro-spinning process which we wanted to share with all.

Three-dimensionally Perforated Calcium Phosphate Ceramics

Porous calcium phosphate ceramics were produced by compression molding using a special mold followed by sintering. The porous calcium phosphate ceramics have three-dimensional and penetrated open pores 380-400μm in diacneter spaced at intervals of 200μm. The layers of the linear penetration pores alternately lay perpendicular to pore direction. The porosity was 59%-65% . The Ca/ P molar ratios of the porous calcium phos phate ceramics range from 1.5 to 1.85. A binder cantaining methyl cellulose was most effective for preparing the powder compact among vinyl acetate, polyvinyl alcohol, starch, stearic acid, methyl cellulose and their mixtures . Stainless steel, polystyrene, nylon and bamboo were used as the long columnar dies for the penetrated open pores. When polystyrene, nylon and bamboo were used as the long columnar male dies, the dies were burned oat during the sintering process. Using stainless steel as the male dies with the removal of the dies before heat treatment resulted in a higher level of densification of the calcium phosphate ceramic.

Efficient calculation of fluid-induced wall shear stress within tissue engineering scaffolds by an empirical model

Mechanical stimulation,such as fluid-induced wall shear stress(WSS),is known that can influence the cellular behaviours.Therefore,in some tissue engineering experiments in vitro,mechanical stimulation is applied via bioreactors to the cells in cell culturing to study cell physiology and pathology.In 3D cell culturing,porous scaffolds are used for housing the cells.It is known that the scaffold porous geometries can influence the scaffold permeability and internal WSS in a bioreactor(such as perfusion bioreactor).To calculate the WSS generated on cells within scaffolds,usually computational fluid dynamics(CFD)simulation is needed.However,the limitations of the computational method for WSS calculation are:(i)the high time cost of the CFD simulation(in particular for the highly irregular geometries);(ii)accessibility to the CFD model for some cell culturing experimentalists due to the knowledge gap.To address these limitations,this study aims to develop an empirical model for calculating the WSS based on scaffold permeability.This model can allow the tissue engineers to efficiently calculate the WSS generated within the scaffold and/or determine the bioreactor loading without performing the computational simulations.