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.

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.

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.

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.

Extracellular matrix from human umbilical cordderived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

The extracellular matrix,which includes collagens,laminin,or fibronectin,plays an important role in peripheral nerve regeneration.Recently,a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche.However,extensive clinical use of Schwann cells remains limited because of the limited origin,loss of an autologous nerve,and extended in vitro culture times.In the present study,human umbilical cord-derived mesenchymal stem cells（h UCMSCs）,which are easily accessible and more proliferative than Schwann cells,were used to prepare an extracellular matrix.We identified the morphology and function of h UCMSCs and investigated their effect on peripheral nerve regeneration.Compared with a non-coated dish tissue culture,the h UCMSC-derived extracellular matrix enhanced Schwann cell proliferation,upregulated gene and protein expression levels of brain-derived neurotrophic factor,glial cell-derived neurotrophic factor,and vascular endothelial growth factor in Schwann cells,and enhanced neurite outgrowth from dorsal root ganglion neurons.These findings suggest that the h UCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.

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.

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.

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.

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.

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.

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.

3D printing method for bone tissue engineering scaffold

3D printing technology is an emerging technology.It constructs solid bodies by stacking materials layer by layer,and can quickly and accurately prepare bone tissue engineering scaffolds with specific shapes and structures to meet the needs of different patients.The field of life sciences has received a great deal of attention.However,different 3D printing technologies and materials have their advantages and disadvantages,and there are limitations in clinical application.In this paper,the technology,materials and clinical applications of 3D printed bone tissue engineering scaffolds are reviewed,and the future development trends and challenges in this field are prospected.

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.

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.

Electrospun Scaff old of Collagen and Polycaprolactone Containing ZnO Quantum Dots for Skin Wound Regeneration

Nanofibers(NFs)have been widely used in tissue engineering such as wound healing.In this work,the antibacterial ZnO quantum dots(ZnO QDs)have been incorporated into the biocompatible poly(ε-caprolactone)/collagen(PCL/Col)fibrous scaffolds for wound healing.The as-fabricated PCL-Col/ZnO fibrous scaffolds exhibited good swelling,antibacterial activity,and biodegradation behaviors,which were beneficial for the applications as a wound dressing.Moreover,the PCL-Col/ZnO fibrous scaffolds showed excellent cytocompatibility for promoting cell proliferation.The resultant PCL-Col/ZnO fibrous scaffolds containing vascular endothelial growth factor(VEGF)also exhibited promoted wound-healing effect through promoting expression of transforming growth factor-β(TGF-β)and the vascular factor(CD31)in tissues in the early stages of wound healing.This new electrospun fibrous scaffolds with wound-healing promotion and antibacterial property should be convenient for treating wound healing.

Compatibility of olfactory cnshcathing cells with functionalized self-assembling peptide scaffold in vitro

Background Olfactory ensheathing cell （OEC） transplantation is a promising or potential therapy for spinal cord injury （SCI）. However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell miqration.

Characterization on Modification and Biocompatibility of PCL Scaffold Prepared with Near-field Direct-writing Melt Electrospinning

In this study,orthogonal experiments were designed to explore the optimal process parameters for preparing polycaprolactone(PCL)scaffolds by the near-field direct-writing melt electrospinning(NFDWMES)technology.Based on the optimal process parameters,the PCL scaffolds with different thicknesses,gaps and structures were manufactured and the corresponding hydrophilicities were characterized.The PCL scaffolds were modified by chitosan(CS)and hyaluronic acid(HA)to improve biocompatibility and hydrophilicity.Both Fourier transform infrared spectroscopy(FTIR)analysis and antibacterial experimental results show that the chitosan and hyaluronic acid adhere to the surface of PCL scaffolds,suggesting that the modification plays a positive role in biocompatibility and antibacterial effect.The PCL scaffolds were then employed as a carrier to culture cells.The morphology and distribution of the cells observed by a fluorescence microscope demonstrate that the modified PCL scaffolds have good biocompatibility,and the porous structure of the scaffolds is conducive to adhesion and deep growth of cells.