Tussah Silk Fibroin Porous Scaffolds Prepared with a Mild Self-assembly Process for Controlled Drug Release

Besides excellent biodegradability and biocompatibility,a useful tissue engineering scaffold should provide favorable surface properties,outstanding mechanical strength and controlled drug release property. In this paper,a mild process to prepare porous tussah silk fibroin( TSF) scaffolds from aqueous solution was described. The n-butanol was used to control the self-assembly of tussah silk. The scaffolds with different TSF concentrations and the same volume showed differences in pore size and distribution. The maximum porosity of the poprepared porous scaffolds was 80% in this paper. And the pore size of the prepared porous scaffolds with different concentrations was between 10μm and 230 μm. X-ray diffraction( XRD) analysis revealed that amorphous TSF was crystallized to β-sheet secondary structure upon gelatin. The TSF scaffolds for controlled drug release was studied and the result showed that the time of drug release was significantly longer. The produced TSF scaffolds with sustained drug release have potential application in tissue engineering.

Mechanistic Insights of Cells in Porous Scaffolds via Integrated Culture Technologies

This research aimed to combine 3 cell and tissue culture technologies to obtain mechanistic insights of cells in porous scaffolds. When cultivated on 2D （2-dimensional） surfaces, HDFs （human dermal fibroblasts） behaved individually and had no strict requirement on seeding density for proliferation; while HaCat cells relied heavily on initial densities for proliferation and colony formation, which was facilitated when co-cultured with HDFs. Experiments using a 3D CCIS （3-dimensional cell culture and imaging system） indicated that HDFs colonised openpores of varying sizes （125-420 ~tm） on modular substrates via bridge structures; while HaCat cells formed aperture structures and only colonised small pores （125 txm）. When co-cultured, HDFs not only facilitated HaCat attachment on the substrates, but also coordinated with HaCat cells to colonise open pores of varying sizes via bridge and aperture structures. Based on these observations, a 2-stage strategy for the culture of HDFs and HaCat cells on porous scaffolds was proposed and applied successfully on a cellulosic scaffold. This research demonstrated that cell colonisation in scaffolds was dependent on multiple factors; while the integrated 2D＆3D culture technologies and the 3D CCIS was an effective and efficient approach to obtain mechanistic insights of their influences on tissue regeneration.

Protein-spatiotemporal partition releasing gradient porous scaffolds and anti-inflammatory and antioxidant regulation remodel tissue engineered anisotropic meniscus

Meniscus is a wedge-shaped fibrocartilaginous tissue,playing important roles in maintaining joint stability and function.Meniscus injuries are difficult to heal and frequently progress into structural breakdown,which then leads to osteoarthritis.Regeneration of heterogeneous tissue engineering meniscus(TEM)continues to be a scientific and translational challenge.The morphology,tissue architecture,mechanical strength,and functional applications of the cultivated TEMs have not been able to meet clinical needs,which may due to the negligent attention on the importance of microenvironment in vitro and in vivo.Herein,we combined the 3D(three-dimensional)-printed gradient porous scaffolds,spatiotemporal partition release of growth factors,and anti-inflammatory and anti-oxidant microenvironment regulation of Ac2-26 peptide to prepare a versatile meniscus composite scaffold with heterogeneous bionic structures,excellent biomechanical properties and anti-inflammatory and anti-oxidant effects.By observing the results of cell activity and differentiation,and biomechanics under anti-inflammatory and anti-oxidant microenvironments in vitro,we explored the effects of anti-inflammatory and anti-oxidant microenvironments on construction of regional and functional heterogeneous TEM via the growth process regulation,with a view to cultivating a high-quality of TEM from bench to bedside.

A drug-loaded composite coating to improve osteogenic and antibacterial properties of Zn-1Mg porous scaffolds as biodegradable bone implants

Zinc(Zn)alloy porous scaffolds produced by additive manufacturing own customizable structures and biodegradable functions,having a great application potential for repairing bone defect.In this work,a hydroxyapatite(HA)/polydopamine(PDA)composite coating was constructed on the surface of Zn-1Mg porous scaffolds fabricated by laser powder bed fusion,and was loaded with a bioactive factor BMP2 and an antibacterial drug vancomycin.The microstructure,degradation behavior,biocompatibility,antibacterial performance and osteogenic activities were systematically investigated.Compared with as-built Zn-1Mg scaffolds,the rapid increase of Zn2+,which resulted to the deteriorated cell viability and osteogenic differentiation,was inhibited due to the physical barrier of the composite coating.In vitro cellular and bacterial assay indicated that the loaded BMP2 and vancomycin considerably enhanced the cytocompatibility and antibacterial performance.Significantly improved osteogenic and antibacterial functions were also observed according to in vivo implantation in the lateral femoral condyle of rats.The design,influence and mechanism of the composite coating were discussed accordingly.It was concluded that the additively manufactured Zn-1Mg porous scaffolds together with the composite coating could modulate biodegradable performance and contribute to effective promotion of bone recovery and antibacterial function.

PARTICLE-COLLISION AND POROGEN-LEACHING TECHNIQUE TO FABRICATE POLYMERIC POROUS SCAFFOLDS WITH MICROSCALE ROUGHNESS OF INTERIOR SURFACES

A facile technique is herein reported to fabricate three-dimensional （3D） polymeric porous scaffolds with interior surfaces of a topographic microstructure favorable for cell adhesion. As demonstration, a well-known biodegradable polymer poly（lactide-co-glycolide） （PLGA） was employed as matrix. Under the porogen-leaching strategy, the large and soft porogens of paraffin were modified by colliding with small and hard salt particles, which generated micropits on the surfaces of paraffin spheres. The eventual PLGA scaffolds after leaching the modified porogens had thus interior surfaces of microscale roughness imprinted by those micropits. The microrough scaffolds were confirmed to benefit adhesion of bone marrow stromal cells （BMSCs） of rats and meanwhile not to hamper the proliferation and osteogenic differentiation of the cells. The insight and technique might be helpful for biomaterial designing in tissue engineering and regenerative medicine.

Effect of Sr^(2+)on 3D gel-printed Sr_(3-x)Mg_(x)(PO_(4))_(2)composite scaffolds for bone tissue engineering

Porous magnesium strontium phosphate(Sr_(3-x)Mg_(x)(PO_(4))_(2))(x=2,2.5,3)composite scaffolds were successfully prepared by three dimension gel-printing(3DGP)method in this study.The results show that Sr_(0.5)Mg_(2.5)(PO_(4))_(2)scaffolds had good compressive strength,and Sr_(1.0)Mg_(2.0)(PO_(4))_(2)scaffolds had good degradation rate in vitro.The weight loss rate of Sr_(1.0)Mg_(2.0)(PO_(4))_(2)scaffolds soaked in simulated body fluid(SBF)or 6 weeks was 6.96%,and pH value varied between 7.50 and 8.61,which was within the acceptable range of human body.Preliminary biological experiment shows that MC3T3-E1 cells had good adhesion and proliferation on the surface of Sr_(3-x)Mg_(x)(PO_(4))_(2)scaffolds.Compared with pure Mg3(PO_(4))_(2)scaffolds,strontium doped scaffolds had excellent comprehensive properties,which explain that Sr_(3-x)Mg_(x)(PO_(4))_(2)composite scaffolds can be used for bone tissue engineering.

Biodegradable magnesium alloy WE43 porous scaffolds fabricated by laser powder bed fusion for orthopedic applications:Process optimization,in vitro and in vivo investigation

Laser powder bed fusion(L-PBF)of magnesium(Mg)alloy porous scaffolds is expected to solve the dual challenges from customized structures and biodegradable functions required for repairing bone defects.However,one of the key technical difficulties lies in the poor L-PBF process performance of Mg,contributed by the high susceptibility to oxidation,vaporization,thermal expansion,and powder attachment etc.This work investigated the influence of L-PBF energy input and scanning strategy on the formation quality of porous scaffolds by using WE43 powder,and characterized the microstructure,mechanical properties,biocompatibility,biodegradation and osteogenic effect of the as-built WE43 porous scaffolds.With the customized energy input and scanning strategy,the relative density of struts reached over 99.5%,and the geometrical error between the designed and the fabricated porosity declined to below 10%.Massive secondary phases including intermetallic precipitates and oxides were observed.The compressive strength(4.37-23.49 MPa)and elastic modulus(154.40-873.02 MPa)were comparable to those of cancellous bone.Good biocompatibility was observed by in vitro cell viability and in vivo implantation.The biodegradation of as-built porous scaffolds promoted the osteogenic effect,but the structural integrity devastated after 12 h by the immersion tests in Hank’s solution and after 4 weeks by the implantation in rabbits’femur,indicating an excessively rapid degradation rate.

Exploring the interconnectivity of biomimetic hierarchical porous Mg scaffolds for bone tissue engineering:Effects of pore size distribution on mechanical properties,degradation behavior and cell migration ability

Interconnectivity is the key characteristic of bone tissue engineering scaffold modulating cell migration,blood vessels invasion and transport of nutrient and waste.However,efforts and understanding of the interconnectivity of porous Mg is limited due to the diverse architectures of pore struts and pore size distribution of Mg scaffold systems.In this work,biomimetic hierarchical porous Mg scaffolds with tailored interconnectivity as well as pore size distribution were prepared by template replication of infiltration casting.Mg scaffold with better interconnectivity showed lower mechanical strength.Enlarging interconnected pores would enhance the interconnectivity of the whole scaffold and reduce the change of ion concentration,pH value and osmolality of the degradation microenvironment due to the lower specific surface area.Nevertheless,the degradation rates of five tested Mg scaffolds were no different because of the same geometry of strut unit.Direct cell culture and evaluation of cell density at both sides of four typical Mg scaffolds indicated that cell migration through hierarchical porous Mg scaffolds could be enhanced by not only bigger interconnected pore size but also larger main pore size.In summary,design of interconnectivity in terms of pore size distribution could regulate mechanical strength,microenvironment in cell culture condition and cell migration potential,and beyond that it shows great potential for personalized therapy which could facilitate the regeneration process.

Effect of porosity on mechanical properties of porous tantalum scaffolds produced by electron beam powder bed fusion

The effect of porosity on compressive,bending,and tensile properties of the porous tantalum scaffolds fabricated by electron beam powder bed fusion(EB-PBF)was investigated.The porous tantalum scaffolds with porosity from 69%to 77.8%were obtained by varying the designed porosity and adjusting the processing parameters.It is found that the pores and unfused powder decrease with the increase of deposited energy density.The decrease of porosity leads to an improvement in mechanical properties.The relevancy between compressive/bending/tensile yield strength and relative density can be described appropriately by exponential model,while the relationship between elastic modulus and relative density is in good agreement with the Gibson-Ashby model.All the porous tantalum scaffolds exhibit good ductility in compressive,bending and tensile tests.No fragmentation of struts is observed during the compression process,but cracks are formed on the strut surface after 90°bending,mainly due to the high sensibility to defects caused by the oxide.

In vitro biodegradability and biocompatibility of porous Mg-Zn scaffolds coated with nano hydroxyapatite via pulse electrodeposition

The biodegradability and biocompatibility of porous Mg-2Zn（mass fraction, %） scaffolds coated with nano hydroxyapatite（HAP） were investigated. The nano HAP coating on Mg-2Zn scaffolds was prepared by the pulse electrodeposition method. The as-deposited scaffolds were then post-treated with alkaline solution to improve the biodegradation behavior and biocompatibility for implant applications. The microstructure and composition of scaffold and nano HAP coating, as well as their degradation and cytotoxicity behavior in simulated body fluid（SBF） were investigated. The post-treated coating is composed of needle-like HAP with the diameter less than 100 nm developed almost perpendicularly to the substrate, which exhibits a similar composition to natural bone. It is found that the products of immersion in SBF are identified to be HAP,（Ca,Mg）3（PO4）2 and Mg（OH）2. The bioactivity, biocompatibility and cell viabilities for the as-coated and post-treated scaffold extracts are higher than those for the uncoated scaffold. MG63 cells are found to adhere and proliferate on the surface of the as-coated and post-treated scaffolds, making it a promising choice for medical application. The results show that the pulse electrodeposition of nano HAP coating and alkaline treatment is a useful approach to improve the biodegradability and bioactivity of porous Mg-Zn scaffolds.

Fabrication of Highly Anisotropic and Interconnected Porous Scaffolds to Promote Preosteoblast Proliferation for Bone Tissue Engineering

Mimicking the complex structure of natural bone remains a challenge for bone tissue scaffolds.In this study,a novel processing strategy was developed to prepare the bone-like scaffolds that are featured by highly oriented and fully interconnected pores.This type of biomimetic scaffolds was evolved from solid phase stretching of immiscible polycaprolactone(PCL)/poly(ethylene oxide)(PEO)blends with cocontinuous structure and the pore morphology was inherited from selective extraction of water soluble PEO phase.The pore anisotropy was readily tuned by varying the stretching strain without loss of interconnectivity.Significant promotion in preosteoblast proliferation,alkaline phosphatase activity and osteogenic gene expression was observed in the oriented porous scaffolds compared to the isotropic porous counterpart.The oriented architecture provided a topographical cue for aligned growth of preosteoblasts,which activated the Wnt/β-catenin signaling pathway.The proposed strategy enriches the toolbox for the scaffold design and fabrication for bone tissue engineering.

Fabrication of porous scaffolds with protein nanogels

A novel type of porous scaffold was fabricated from single protein nanogels. The nanogels with single protein as core and crosslinked polymer network as shell were prepared through a two-step procedure including surface acryloylation and in situ radical polymerization. The formation of single protein nanogels was verified by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. Subsequently, the porous scaffolds were fabricated through a solvent evaporating process of aqueous nanogel solutions. The porous scaffolds were characterized by Fourier transform infrared (FTIR), scanning electronic microscopy (SEM), atomic force microscopy (AFM), and fluorescence microscopy. Interestingly, the obtained porous nanogel scaffolds presented multi-level porous morphologies with macro and nano scale pores, providing better spaces and microenvironments than normal macro porous scaffolds. Cell proliferation assay of nanogels showed low cytotoxicity. Considering that both the protein species and polymer constitutes can be pre-designed and adjusted, these multi-level porous nanogel scaffolds are promising candidates for tissue culture applications.

Fabrication and Evaluation of Porous Keratin/chitosan(KCS) Scaffolds for Effectively Accelerating Wound Healing

Objective To develop a dressing with desired antibacterial activity, good water maintaining ability and mechanical properties for wound healing and skin regeneration. Methods The chitosan with different concentrations were added in keratin solution to form porous keratin/chitosan（KCS） scaffolds. The morphological characteristics, chemical composition, wettability, porosity, swelling ratio and degradation of the scaffolds were evaluated. The antibacterial activity was tested by using S. aureus and E. coli suspension for 2 h. And L929 fibroblast cells culture was used to evaluate the cytotoxicity of the KCS scaffolds. Results The adding of chitosan could increase the hydrophobicity, decrease porosity, swelling ratio and degradation rate of the KCS porous scaffolds. Mechanical properties of KCS scaffolds could be enhanced and well adjusted by chitosan. KCS scaffolds could obviously decrease bacteria number. The proliferation of fibroblast cells in porous KCS patch increased firstly and then decreased with the increase of chitosan concentration. It was appropriate to add 400 μg/m L chitosan to form porous KCS scaffold for achieving best cell attachment and proliferation compared with other samples. Conclusion The porous KCS scaffold may be used as implanted scaffold materials for promoting wound healing and skin regeneration.

Article Preclinical evaluation of acute systemic toxicity of magnesium incorporated poly(lactic-co-glycolic acid)porous scaffolds by three-dimensional printing Jing

Biodegradable polymer scaffolds combined with bioactive components which accelerate osteogenesis and angiogenesis have promise for use in clinical bone defect repair.The preclinical acute toxicity evaluation is an essential assay of implantable biomaterials to assess the biosafety for accelerating clinical translation.We have successfully developed magnesium(Mg)particles and beta-tricalcium phosphate(β-TCP)for incorporation into poly(lactic-co-glycolic acid)(PLGA)porous composite scaffolds(PTM)using low-temperature rapid prototyping three-dimensional-printing technology.The PTM scaffolds have been fully evaluated and found to exhibit excellent osteogenic capacity for bone defect repair.The preclinical evaluation of acute systemic toxicities is essential and important for development of porous scaffolds to facilitate their clinical translation.In this study,acute systemic toxicity of the PTM scaffolds was evaluated in mice by intraperitoneal injection of the extract solutions of the scaffolds.PTM composite scaffolds with different Mg andβ-TCP content(denoted as PT5M,PT10M,and PT15M)were extracted with different tissue culture media,including normal saline,phosphate-buffered saline,and serum-free minimum essential medium,to create the extract solutions.The evaluation was carried out following the National Standard.The acute toxicity was fully evaluated through the collection of extensive data,including serum/organs ion concentration,fluorescence staining,and in vivo median lethal dose measurement.Mg in major organs(heart,liver,and lung),and Mg ion concentrations in serum of mice,after intraperitoneal injection of the extract solutions,were measured and showed that the extract solutions of PT15M caused significant elevation of serum Mg ion concentrations,which exceeded the safety threshold and led to the death of the mice.In contrast,the extract solutions of PT5M and PT10M scaffolds did not cause the death of the injected mice.The median lethal dose of Mg ions in vivo for mice was determined for the first time in this study to be 110.66 mg/kg,and the safety level of serum magnesium toxicity in mice is 5.4 mM,while the calcium serum safety level is determined as 3.4 mM.The study was approved by the Animal Care and Use Committee of Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences(approval No.SIAT-IRB-170401-YGS-LYX-A0346)on April 5,2017.All these results showed that the Mg ion concentration of intraperitoneally-injected extract solutions was a determinant of mouse survival,and a high Mg ion concentration(more than 240 mM)was the pivotal factor contributing to the death of the mice,while changes in pH value showed a negligible effect.The comprehensive acute systemic toxicity evaluation for PTM porous composite scaffolds in this study provided a reference to guide the design and optimization of this composite scaffold and the results demonstrated the preclinical safety of the as-fabricated PTM scaffold with appropriate Mg content,strongly supporting the official registration process of the PTM scaffold as a medical device for clinical translation.

Extrusion-based additive manufacturing of Mg-Zn alloy scaffolds

Porous biodegradable Mg and its alloys are considered to have a great potential to serve as ideal bone substitutes.The recent progress in additive manufacturing(AM) has prompted its application to fabricate Mg scaffolds with geometrically ordered porous structures.Extrusionbased AM,followed by debinding and sintering,has been recently demonstrated as a powerful approach to fabricating such Mg scaffolds,which can avoid some crucial problems encountered when applying powder bed fusion AM techniques.However,such pure Mg scaffolds exhibit a too high rate of in vitro biodegradation.In the present research,alloying through a pre-alloyed Mg-Zn powder was ultilized to enhance the corrosion resistance and mechanical properties of AM geometrically ordered Mg-Zn scaffolds simultaneously.The in vitro biodegradation behavior,mechanical properties,and electrochemical response of the fabricated Mg-Zn scaffolds were evaluated.Moreover,the response of preosteoblasts to these scaffolds was systematically evaluated and compared with their response to pure Mg scaffolds.The Mg-Zn scaffolds with a porosity of 50.3% and strut density of 93.1% were composed of the Mg matrix and MgZn2second phase particles.The in vitro biodegradation rate of the Mg-Zn scaffolds decreased by 81% at day 1,as compared to pure Mg scaffolds.Over 28 days of static immersion in modified simulated body fluid,the corrosion rate of the Mg-Zn scaffolds decreased from 2.3± 0.9 mm/y to 0.7±0.1 mm/y.The yield strength and Young’s modulus of the Mg-Zn scaffolds were about 3 times as high as those of pure Mg scaffolds and remained within the range of those of trabecular bone throughout the biodegradation tests.Indirect culture of MC3T3-E1 preosteoblasts in Mg-Zn extracts indicated favorable cytocompatibility.In direct cell culture,some cells could spread and form filopodia on the surface of the Mg-Zn scaffolds.Overall,this study demonstrates the great potential of the extrusion-based AM Mg-Zn scaffolds to be further developed as biodegradable bone-substituting biomaterials.

Fabrication and Characterization of 3D Graded PDMS Scaffolds Using Vacuum-Assisted Resin Transfer Moulding

Graded porous scaffold can be applied to study the interactions between cells and scaffold with different pore sizes. Polydimethylsiloxane（PDMS） scaffold with an axial pore size grade was successfully manufactured via vacuum-assisted resin transfer moulding（VARTM） and particle leaching technologies. The properties of graded PDMS scaffolds, including porosity, water absorption, interconnectivity, compression modulus, as well as compression strength were investigated. The results showed that the smaller the size of the NaCl particles is, the higher the porosity and water absorption of graded PDMS scaffolds would be. The graded PDMS scaffold fabricated had a compressive modulus and a compressive strength of 19.69±1.42 kPa and 4.76±0.22 kPa, respectively. Moreover, the graded chitosan（CS）-coated PDMS scaffolds were prepared by using dip-coating technique under low vacuum and their hydrophilicity was examined. It is found that the water contact angle（WCA） will decrease with an increase in the CS solution concentration and the coating time, which indicates that CS-coated PDMS scaffolds exhibit noticeable hydrophilicity compared with graded PDMS scaffold.

Fabrication and Interfacial Structure of Ni-YSZ-LSM Porous Scaffold for Solid Oxide Fuel Cells

A novel process route using tape casting and stacking for fabricating porous scaffold of solid oxide fuel cells （SOFC） was demonstrated. The linear shrinkages of anode （Ni-YSZ, YSZ stands for 3% Y2O3 （mole fraction） stabilized ZrO2 ） and cathode （LSM-YSZ, LSM stands for La0.8Sr0.2MnO3 ） were optimized to be uniform with that of electrolyte during sintering, by controlling the content of pore former. The micromorphology and interface microstructure of the cross-section of the porous scaffold were observed by optical microscope and scanning electron microscope, respectively. The element distribution and phase composition were analyzed by energy dispersive spectrometer and X-ray diffraction, respectively. The results showed that the porous scaffold with regular pore shape and high specific surface area was obtained after sintering at 1 350℃. The fabricated porous scaffold had defect free interracial structures due to the uniform shrinkage of anode, cathode and electrolyte layers. In addition, it was shown that diffusions of Zr, Ni and La caused a progressive boundary between YSZ, Ni-YSZ and LSM-YSZ layers. The interface between anode and electrolyte （Ni-YSZ/YSZ） was mainly composed of Ni, YSZ and a small amount of NiO, and the interface between cathode and electrolyte （LSM-YSZ/YSZ） was mainly composed of YSZ, LSM and a small amount of La2Zr2O7.

Preparation and Biocompatibility of Porous Poly(vinylalcohol)-Glycosaminoglycan-Collagen Scaffold

This paper aims to prepare a PVA-GAG-COL composite with polyvinyl alcohol (PVA), glycosaminoglycan (GAG) and collagen (COL) by the method of freeze drying and to investigate the feasibility as a tissue engineering scaffold for tissue or organ repairing. In this study, SEM was used to observe the morphology. Biocompatibility was tested by cell culture with the extracted fluid of composite materials. Different proportional scaffolds could be obtained with different concentrations and alcoholysis degree of PVA. Different proportional scaffolds also had different porous structures. SEM proved that large amount of porous structure could be formed. Biocompatibility test showed that the extracted fluid of composite materials was nontoxic, which could promote the adhesion and proliferation of the fibroblast. Fibroblast could grow on the scaffold normally.A porous scaffold for tissue engineering with high water content can be fabricated by PVA, GAG and COL, which has excellent cell biocompatibility. The porous structure shows potential in tissue engineering and cell culture.

Porous bio-high entropy alloy scaffolds fabricated by direct ink writing

Porous tantalum-titanium-niobium-zirconium(Ta-Ti-Nb-Zr)bio-high entropy alloy(bioHEA)scaffolds are fabricated using direct ink writing 3D printing technology in this study.A composite ink is prepared using four metal powders as raw materials:Ta,Ti,Nb and Zr.Ink extrusion is used to build 3D scaf-folds with interconnected porous structures at room temperature,which are then sintered in a vacuum environment.The interdiffusion of metal elements yields porous bioHEA scaffolds with a body-centered cubic(BCC)structure.The fabricated scaffolds have uniform compositions with a significant alloying ef-fect and good biocompatibility.The scaffolds have a compressive strength of 70.08-149.95 MPa and an elastic modulus of 0.18-0.64 GPa,indicating that the mechanical properties can be controlled over a wide range.The scaffolds have a compressive strength close to that of human cortical bone and thus meet the requirements for porous structure characteristics and biological and mechanical properties of orthopedic implants.

Biomimetic hydroxyapatite coating on the 3D-printed bioactive porous composite ceramic scaffolds promoted osteogenic differentiation via PI3K/AKT/mTOR signaling pathways and facilitated bone regeneration in vivo

The architecture and surface modifications have been regarded as effective methods to enhance the bi-ological response of biomaterials in bone tissue engineering.The porous architecture of the implanta-tion was essential conditions for bone regeneration.Meanwhile,the design of biomimetic hydroxyap-atite(HAp)coating on porous scaffolds was demonstrated to strengthen the bioactivity and stimulate osteogenesis.However,bioactive bio-ceramics such asβ-tricalcium phosphate(β-TCP)and calcium sili-cate(CS)with superior apatite-forming ability were reported to present better osteogenic activity than that of HAp.Hence in this study,3D-printed interconnected porous bioactive ceramicsβ-TCP/CS scaf-fold was fabricated and the biomimetic HAp apatite coating were constructed in situ via hydrothermal reaction,and the effects of HAp apatite layer on the fate of mouse bone mesenchymal stem cells(mBM-SCs)and the potential mechanisms were explored.The results indicated that HAp apatite coating en-hanced cell proliferation,alkaline phosphatase(ALP)activity,and osteogenic gene expression.Further-more,PI3K/AKT/mTOR signaling pathway is proved to have an important impact on cellular functions.The present results demonstrated that the key molecules of phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT)and mammalian target of rapamycin(mTOR)were activated after the biomimetic hydrox-yapatite coating were constructed on the 3D-printed ceramic scaffolds.Besides,the activated influence on the protein expression of Runx2 and BMP2 could be suppressed after the treatment of inhibitor HY-10358.In vivo studies showed that the constructed HAp coating promoted bone formation and strengthen the bone quality.These results suggest that biomimetic HAp coating constructed on the 3D-printed bioac-tive composite scaffolds could strengthen the bioactivity and the obtained biomimetic multi-structured scaffolds might be a potential alternative bone graft for bone regeneration.