3D Composite Scaffolds Research Articles

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.

Toward Highly Dispersed Mesoporous Bioactive Glass Nanoparticles With High Cu Concentration Using Cu/Ascorbic Acid Complex as Precursor.

Copper (Cu) ions have a variety of advantageous biological functionalities, such as proangiogenic and bactericidal activities. Given the intrinsic biodegradability and biocompatibility, silicate-based mesoporous bioactive glass nanoparticles (MBGNs) are considered as promising platforms for the delivery of Cu ions. However, effective incorporation of Cu into MBGNs still faces challenges, e.g., particle aggregation, the formation of insoluble crystalline Cu-based nanoparticles, and a low loading amount of Cu. We report a novel method to synthesize chemically homogenous and highly dispersed Cu-containing MBGNs (Cu-MBGNs) with tunable Cu concentration by using ascorbic acid/Cu complexes as the precursor of Cu in a microemulsion-assisted sol-gel approach. Cu-MBGNs exhibited a sphere-like shape with a particle size between 100 and 300 nm while their pore size varied from 2 to 10 nm. The inclusion of Cu, regardless of the incorporated concentration, did not significantly affect the morphology of particles. ICP-AES results indicated that the concentration of Cu in the particles could be conveniently tuned from 0 to ~6 mol% by controlling the amount of ascorbic acid/Cu complexes added, while the formation of crystalline Cu-based nanoparticles was avoided. The amorphous feature of Cu-MBGNs was proved by XRD, while the predominant oxidation state of Cu was evidenced to be Cu2+ by XPS. The incorporation of Cu did not inhibit the apatite-forming ability (bioactivity) of the particles in contact with simulated body fluid. Cu-MBGNs exhibited the capability of releasing Cu, Si, and Ca ions over time in the physiological fluid. The concentration of released Cu ions could be controlled by selecting specific Cu-MBGNs of different Cu contents. The dissolution products of most Cu-MBGNs at the dosage of 1, 0.1, and 0.01 mg/mL did not exhibit cytotoxicity, while only 7Cu-MBGN was cytotoxic at the dosage of 1 mg/mL. This study provided a feasible strategy to synthesize highly dispersed amorphous Cu-MBGNs with high Cu concentrations for biomedical applications. The particles exhibit great potential as building blocks for developing composite 3D scaffolds, coatings, and drug carriers, particularly when a large amount of particles incorporated may compromise the properties of (polymer) matrix materials while a relatively high concentration of released Cu ions is still required.

Aligned conductive core-shell biomimetic scaffolds based on nanofiber yarns/hydrogel for enhanced 3D neurite outgrowth alignment and elongation

Aligned topographical cue has been demonstrated as a critical role in neuronal guidance, and it is highly beneficial to develop a scaffold with aligned structure for peripheral nerve tissue regeneration. Although considerable efforts have been devoted to guiding neurite alignment and extension, it remains a remarkable challenge for developing a biomimetic scaffold for enhancing 3D aligned neuronal outgrowth. Herein, we present a core-shell scaffold based on aligned conductive nanofiber yarns (NFYs) within the hydrogel to mimic the 3D hierarchically aligned structure of the native nerve tissue. The aligned NFYs assembled by a bundle of aligned nanofibers composed of polycaprolactone (PCL), silk fibroin (SF), and carbon nanotubes (CNTs) are prepared by a developed dry–wet electrospinning method, which has the ability to induce neurite alignment and elongation when PC12 cells and dorsal root ganglia (DRG) cells are cultured on their 3D peripheral surface. Particularly, such an aligned nanofibrous structure also induces aligned neurite extension and cell migration from DRG explants along the direction of nanofibers. 3D core-shell scaffolds are fabricated by encapsulating NFYs within the hydrogel shell after photocrosslinking, and these 3D aligned scaffolds are able to control cellular alignment and elongation of nerve cells in this 3D environment. Our results suggest that such 3D hierarchically aligned core-shell scaffold consists of NFYs that mimic the aligned nerve fiber structure to induce neurite alignment and extension and a hydrogel shell that mimics the epineurium layer to protect nerve cell organization within a 3D environment, which is largely promising for the design of biomimetic scaffolds in nerve tissue engineering. Statement of SignificanceDesigning scaffolds with 3D aligned structure has been paid more attention for peripheral nerve tissue regeneration, because the aligned topographical cue is able to induce neurites alignment and extension. However, developing scaffolds mimicking the hierarchically aligned structure of native nerve tissue for directing 3D aligned neuronal outgrowth without external stimulation remains challenging. This work presented a simple and efficient strategy to prepare a 3D biomimetic core-shell scaffold based on electrospun aligned conductive nanofiber yarns within photocurable hydrogel shell to mimic the hierarchically aligned structure of native nerve tissue. These 3D aligned composite scaffolds performed the ability to direct 3D cellular alignment and elongation of nerve cells along with the nanofiber yarn direction, and the hydrogel shell mimicking the epineurium layer was able to protect nerve cells organization within the 3D environment, which indicated their great potential in peripheral nerve tissue engineering applications.

Mechanical and Biochemical Stimulation of 3D Multilayered Scaffolds for Tendon Tissue Engineering.

Tendon injuries are frequent and occur in the elderly, young, and athletic populations. The inadequate number of donors combined with many challenges associated with autografts, allografts, xenografts, and prosthetic devices have added to the value of engineering biological substitutes, which can be implanted to repair the damaged tendons. Electrospun scaffolds have the potential to mimic the native tissue structure along with desired mechanical properties and, thus, have attracted noticeable attention. In order to improve the biological responses of these fibrous structures, we designed and fabricated 3D multilayered composite scaffolds, where an electrospun nanofibrous substrate was coated with a thin layer of cell-laden hydrogel. The whole construct composition was optimized to achieve adequate mechanical and physical properties as well as cell viability and proliferation. Mesenchymal stem cells (MSCs) were differentiated by the addition of bone morphogenetic protein 12 (BMP-12). To mimic the natural function of tendons, the cell-laden scaffolds were mechanically stimulated using a custom-built bioreactor. The synergistic effect of mechanical and biochemical stimulation was observed in terms of enhanced cell viability, proliferation, alignment, and tenogenic differentiation. The results suggested that the proposed constructs can be used for engineering functional tendons.

Porous Scaffolds of Poly(lactic-co-glycolic acid) and Mesoporous Hydroxyapatite Surface Modified by Poly(γ-benzyl-l-glutamate) (PBLG) for in Vivo Bone Repair.

Three-dimensional (3D) composite porous scaffolds containing hydroxyapatite (HA) and polymer matrix showed wide applications in bone repair because of their improved biocompatibility, bioactivity, and mechanical properties in our previous studies. In this work, mesoporous hydroxyapatite (MHA) surface-modified by poly(γ-benzyl-l-glutamate) (PBLG) with different amounts (from 11 to 50 wt %) was synthesized by the in situ ring opening polymerization of γ-benzyl-l-glutamate N-carboxy anhydride (BLG-NCA) and then PBLG-g-MHA/PLGA composite films were prepared to illustrate the biological performance of the composites. Furthermore, porous scaffolds of PBLG-g-MHA/PLGA were fabricated through modified solvent casting/particulate leaching (SC/PL) method to demonstrate the ability of in vivo bone defect repair. In vitro cytological assay indicated that enhanced cell expansion on PBLG-g-MHA/PLGA with 11 wt % PBLG amounts and improved osteogenic differentiation on the composites with 33 and 50 wt % PBLG amounts were achieved. And the porous scaffolds exhibited high porosity and interconnected pores. Results of the in vivo rabbit radius defect repair indicated that rapid mineralization and new bone formation could be observed on the composites with 22 and 33 wt % PBLG. This study revealed that PBLG-g-MHA/PLGA composites might have potential applications in clinical bone repair.

Gum based 3D composite scaffolds for bone tissue engineering applications

The increase in population, greater life expectancy, and lifestyle choices have caused a drastic increase in the number of bone diseases such as bone tumours, osteoarthritis and bone fractures. This results in the dire need for treatment options such as suitable bone grafts that can be easily fabricated, and are economical. In this study, fabricated composite scaffolds are made from polysaccharide biopolymers, namely gellan and guar gum, and hydroxyapatite by freeze drying method. The developed scaffolds of optimum concentration showed a maximum percentage degradation of 13.7% only until 21 days in phosphate buffered saline solution, and minimum swelling capacity. The mechanically stable scaffolds (compressive strength equivalent to cancellous bone region, ˜3–30 MPa) amongst them were then subjected to characterization tests—scanning electron microscopy, fourier transform infrared spectroscopy, X-ray diffraction, swelling ratio percentage determination, degradation profile study and water vapour transmission study. The cytotoxic evaluation of the optimised scaffolds was performed using MTT assay with murine fibroblast (L929) cells and osteosarcoma (MG63) cells. It was found that the scaffolds were non-cytotoxic and additionally, the cells had proliferated well within the scaffolds, which was confirmed by MTT assay at 1, 4 and 7 days after cell seeding onto the scaffolds.

Fabrication and Cytocompatibility Evaluation of Psyllium Husk (Isabgol)/Gelatin Composite Scaffolds.

Psyllium husk or isabgol contains xylan backbone linked with arabinose, rhamnose, and galacturonic acid units (arabinoxylans). In this study, we demonstrate the fabrication and characterization of a macroporous three-dimensional (3D) composite scaffold by mixing psyllium husk powder (PH) and gelatin (G) in different ratios, viz.100 PH, 75/25 PH/G, and 50/50 PH/G (w/w), using an EDC-NHS coupling reaction followed by freeze-drying method. The reaction was performed in aqueous as well as in alcoholic media to determine the most appropriate solvent system for this purpose. The mechanical strength of the scaffold system was improved from 151 to 438kPa. The fabricated scaffolds exhibited enhanced structural stability, remarkable swelling capacity, and escalated cell growth and proliferation. ATR-FTIR analysis showed the presence of amide and ester bonds indicating covalent crosslinking. SEM micrographs revealed the porous nature of the scaffolds with pores ranging from 30 to 150μm, and further pore size distribution curve indicated that 75/25 PH/G (w/w%) EDC-NHS-alcohol scaffold exhibited the best fit to the Gaussian distribution. Swelling capacity of the 100 PH EDC-NHS-alcohol scaffolds was found to be nearly 40% from its original weight in 48h. MTT assay using fibroblast cells revealed ~ 80% cellular proliferation by 6th day within the fabricated scaffolds in comparison to control. Graphical Abstract ᅟ.

Development of dual delivery antituberculotic system containing rifapentine microspheres and adipose stem cells seeded in hydroxyapatite/tricalcium phosphate.

BackgroundLow drug concentration in the tuberculosis (TB) lesion and bone defects or nonunion after debridement are two major problems that occur in the course of treating osteo-articular TB. Thus, the combination of drug-delivery system and bone tissue repair appears to be the most promising option for osteoarticular TB treatment.Materials and methodsHerein, we report a novel anti-TB dual delivery system based on rifapentine polylactic acid microspheres (RPMs) to treat infections, with the addition of adipose-derived mesenchymal stem cells (ASCs) seeded in hydroxyapatite/tricalcium phosphate (HA/TCP) to promote bone formation. Cell proliferation, osteogenesis, and apoptosis were performed to investigate the effects of rifapentine on ASCs. The RPMs were synthesized by emulsion-solvent evaporation method, and then the monolayer composite (ASC + RPM) and three-dimensional (3D) composite scaffold (ASC + RPM + HA/TCP) were constructed, respectively. The alkaline phosphatase (ALP) activity and real-time PCR were used for determining the osteogenic differentiation. The concentrations of rifapentine resulting from the composites were detected.ResultsThe results showed that rifapentine has no influence on ASCs proliferation and osteogenesis when the drug concentration was below 20 µg/mL, which was significantly higher than minimal inhibitory concentration. The drug loading and encapsulation efficiency of RPMs were 40.56%±2.63% and 70.24%±2.18%, respectively. The proliferation of the cells in monolayer was higher than that in 3D composite, and the addition of RPMs slightly increased the proliferation. The ALP activity and gene expression of osteocalcin and osteopontin were higher in the 3D composite than those in the monolayer. Good biocompatibility was observed by microscopic image and H&E stain. The release tests revealed that the 3D composite exhibited sustained release profiles of rifapentine for 76 days. The dual delivery systems in 3D composite could moderate the burst release and extend the length of release time when compared to single delivery in monolayers.ConclusionIn conclusion, such dual delivery antituberculotic scaffold represents a potential new strategy for TB infections and bone defects.

Fabrication and characterisation of 3D printed MWCNT composite porous scaffolds for bone regeneration

Carbon nanotubes (CNTs) with exceptional physical and chemical properties are attracting significant interest in the field of tissue engineering. Several reports investigated CNTs biocompatibility and their impact in terms of cell attachment, proliferation and differentiation mainly using polymer/CNTs membranes. However, these 2D membranes are not able to emulate the complex in vivo environment. In this paper, additive manufacturing (3D printing) is used to create composite 3D porous scaffolds containing different loadings of multi-walled carbon nanotubes (MWCNT) (0.25, 0.75 and 3 wt%) for bone tissue regeneration. Pre-processed and processed materials were extensively characterised in terms of printability, morphological and topographic characteristics and thermal, mechanical and biological properties. Scaffolds with pore sizes ranging between 366 μm and 397 μm were successfully produced and able to sustain early-stage human adipose-derived mesenchymal stem cells attachment and proliferation. Results show that MWCNTs enhances protein adsorption, mechanical and biological properties. Composite scaffolds, particularly the 3 wt% loading of MWCNTs, seem to be good candidates for bone tissue regeneration.

Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate nanoparticles.

In the present study, biocomposites based on 3D porous additively manufactured Ti6Al4V (Ti64) scaffolds modified with biocompatible calcium phosphate nanoparticles (CaPNPs) were investigated. Ti64 scaffolds were manufactured via electron beam melting technology using an Arcam machine. Electrophoretic deposition was used to modify the scaffolds with CaPNPs, which were synthesized by precipitation in the presence of polyethyleneimine (PEI). Dynamic light scattering revealed that the CaP/PEI nanoparticles had an average size of 46 ± 18 nm and a zeta potential of +22 ± 9 mV. Scanning electron microscopy (SEM) revealed that the obtained spherical CaPNPs had an average diameter of approximately 90 nm. The titanium-based scaffolds coated with CaPNPs exhibited improved hydrophilic surface properties, with a water contact angle below 5°. Cultivation of human mesenchymal stem cells (hMSCs) on the CaPNPs-coated Ti64 scaffolds indicated that the improved hydrophilicity was beneficial for the attachment and growth of cells in vitro. The Ti6Al4V/CaPNPs scaffold supported an increase in the alkaline phosphatase (ALP) activity of cells. In addition to the favourable cell proliferation and differentiation, Ti6Al4V/CaPNPs scaffolds displayed increased mineralization compared to non-coated Ti6Al4V scaffolds. Thus, the developed composite 3D scaffolds of Ti6Al4V functionalized with CaPNPs are promising materials for different applications related to bone repair.

Development of mechanically compliant 3D composite scaffolds for bone tissue engineering applications.

Cancellous bone region of the human native bone has the potential to bear significant mechanical loads. However, due to the following parameters such as low trabecular volume, more porosity, less thickness and more interconnectivity, cancellous bone is accessible to damage in accidents or when aging. This research led to the effective fabrication and engineering of cancellous region of the bone for its application in reconstruction or filling of bone voids after resection of large tumor mass. Scaffolds containing hydroxyapatite (HAp), polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) (5% polymer and HAp concentrations) at different HAp: polymer composite ratios (50:50, 70:30 and 80:20), were fabricated by freeze-drying method using only water as solvent and ribose as crosslinker for the scaffolds. The fabricated scaffolds were characterized for its mechanical (compressive) strength, chemical properties using FT-IR and XRD analyses, swelling and degradation studies, confirmation of mineralization process by immersion in simulated body fluid (SBF) for 7, 15 and 30 days, morphological analysis using scanning electron microscopy (SEM), and biocompatibility properties by performing MTT analysis. From the combined interpretation of the above mentioned tests, it was proven that 80:20 ratio of HAp to polymers was found to be the most suitable scaffold in terms of its optimal properties for its use as bone graft material for trabecular bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3267-3274, 2018.

Antibacterial activity and biocompatibility of zein scaffolds containing silver-doped bioactive glass

Composite 3D scaffolds combining natural polymers and bioceramics are promising candidates for bone tissue engineering (BTE). Zein, as a natural plant protein, offers several advantages, including biocompatibility, adequate strength properties, and low/no immunogenicity; however, it lacks bioactivity. Thus, composite zein: bioactive glass (BG) scaffolds are proposed as promising candidate for BTE applications, with silver-doping of bioactive glass providing an antibacterial effect against possible post-implantation infection. Therefore, the aim of this study was to investigate the in vitro antibacterial properties, biocompatibility, bioactivity and compressive strength of zein scaffolds containing silver-doped bioactive glass. BG nanoparticles, undoped and Ag-doped, were fabricated using the sol-gel method. 3D composite zein:BG scaffolds, containing 20 wt% BG, were prepared and their antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was assessed using the disc diffusion assay. Human osteoblast-like MG-63 cells were used to evaluate the in vitro biocompatibility of the prepared scaffold groups. In addition, the compressive strength of the scaffolds was determined using uniaxial compression strength testing and the scaffold interconnected porosity was measured using helium pycnometer. Disc diffusion assay showed that only zein scaffolds containing Ag-doped sol-gel BG are antibacterially positive against E. coli and S. aureus. Pure zein scaffolds and zein scaffolds containing sol-gel-derived BG showed no negative influence on the growth of MG-63 cells, as evident by the cells’ ability to survive, proliferate, and function on these scaffolds. Moreover, incorporating sol-gel-derived BG into zein scaffolds at zein:BG of 80:20 ratio showed bioactive properties with adequate porosity without affecting the scaffolds’ compressive strengths, which was similar to that of trabecular bone, suggesting that the new composites have potential for BTE applications in non-loaded bearing areas.

Dual-functional 3D-printed composite scaffold for inhibiting bacterial infection and promoting bone regeneration in infected bone defect models

Infection is one of the pivotal causes of nonunion in large bone defect after trauma or tumor resection. Three-dimensional (3D) composite scaffold with multifunctional-therapeutic properties offer many advantages over allogenic or xenogenic bone grafting for the restoration of challenging infected bone defects. In the previous study, we demonstrated that quaternized chitosan (HACC)-grafted polylactide-co-glycolide (PLGA)/hydroxyapatite (HA) scaffold (PLGA/HA/HACC) via 3D-printing technique exhibited significantly improved antimicrobial and osteoconductive property in vitro, together with good biocompatibility in vivo. Hence, the present study further investigated whether such an innovative bone substitute could effectively inhibit the bacterial biofilm formation and promote bone regeneration in vivo. To evaluate the bone repairing effects of the 3D-printed scaffolds on infected cortical and cancellous bone defects scenarios, eighty female Sprague Dawley rats and thirty-six female New Zealand white rabbits were used to establish infected femoral shaft defect and condyle defect model, respectively. X-ray, micro-CT, microbiological and histopathological analyses were used to assess the anti-infection and bone repairing potential of the dual-functional porous scaffolds. We observed that HACC-grafted PLGA/HA scaffolds exhibited significantly enhanced anti-infection and bone regeneration capability in different infected bone defect models. In addition, the degradation rate of the scaffolds appeared to be closely related to the progress of infection, influencing the bone repairing potential of the scaffolds in infected bone defects models. In general, this investigation is of great significance as it demonstrates promising applications of the 3D-printed dual-functional PLGA/HA/HACC scaffold for repairing different types of bone defect under infection. Statement of SignificanceCurrently, it is clinically urgent to exploit bone substitutes with potential of bacterial inhibition and bone regeneration. However, bone scaffolds with relatively low risks of bacterial resistance and tissue toxicity used for combating infected bone defects remain to be developed. We have reported that quaternized chitosan (HACC)-grafted 3D-printed PLGA/HA composite scaffold had enhanced in vitro antimicrobial and osteoconductive property, and well cytocompatibility in our published study. This continuing study further confirmed that HACC-grafted PLGA/HA scaffolds exhibited significantly enhanced anti-infection and bone regeneration efficacy in both cortical bone defect in rat and cancellous bone defect in rabbit under infection. Meanwhile, we also found that the degradation rate of the scaffolds seemed to be closely related to the progress of infection, influencing the bone repairing potential of the scaffolds in infected bone defects models. In conclusion, this study provides significant opportunities to develop a 3D-printed bone scaffold with dual functions used for infected bone defects in future plastic and orthopaedic surgery.

Novel polycaprolactone/hydroxyapatite nanocomposite fibrous scaffolds by direct melt-electrospinning writing

Melt electrospinning writing (MEW) using an automated stage has recently been developed as a direct additive manufacturing method for the fabrication of orderly, precise and complex porous 3D fibrous structures that can promote cell infiltration and growth. The further incorporation of inorganic particles within fibrous scaffolds is desirable in order to enhance bioactivity, however this remains challenging with the MEW fabrication process. To address this challenge, flexible, osteoconductive, medical grade polycaprolactone (m-PCL) - hydroxyapatite (HAp) composite 3D fibrous structures with high porosity (96–98%) and fully interconnected pore architectures were fabricated using MEW under precisely controlled parameters. The physical properties of these 3D fibrous composite scaffolds including fibre size, mechanical characteristics, and in vitro degradation rate were investigated. The results showed that the composite m-PCL/HAp fibrous scaffolds degraded in an alkaline environment at 37 °C faster than plain m-PCL and provided a favourable platform for the infiltration and growth of human osteoblasts. Moreover, confocal imaging confirmed that the scaffolds contained HAp nano-particles (NPs) which induced a more homogeneous distribution of cells within the scaffold particularly after 7 days of culture. Osteoblast activity and viability in the m-PCL/HAp composite scaffolds indicated a favourable cell/material interaction, suggesting great potential for use in mineralised tissue reconstruction / regeneration applications.

Osteogenesis effect of dynamic mechanical loading on MC3T3-E1 cells in three-dimensional printing biomimetic composite scaffolds

To observe the effect of dynamic mechanical loading on the proliferation, differentiation, and specific gene expression of MC3T3-E1 cells that on three-dimensional (3D) biomimetic composite scaffolds prepared by low temperature 3D printing technology combined with freeze-drying. The silk fibroin, collagen type Ⅰ, and nano-hydroxyapatite (HA) were mixed at a mass ratio of 3∶9∶2 and were used to prepare the 3D biomimetic composite scaffolds via low temperature 3D printing technology combined with freeze-drying. General morphology of 3D biomimetic composite scaffold was observed. Micro-CT was used to observe the pore size and porosity of the scaffolds, and the water swelling rate, stress, strain, and elastic modulus were measured. Then, the MC3T3-E1 cells were seeded on the 3D biomimetic composite scaffolds and the cell-scaffold composites were randomly divided into 2 groups. The experimental group was subjected to dynamic mechanical loading (3 500 με, 1 Hz, 15 minutes per day); the control group was not subjected to loading treatment. After 7 days and 14 days, the cell-scaffold composites of 2 groups were harvested to observe the growth of cells on the scaffolds by HE staining and scanning electron microscope. And the gene and protein expressions of collagen type Ⅰ, BMP-2, and osteocalcin (OCN) were measured by real-time fluorescent quantitative PCR and Western blot. The 3D biomimetic composite scaffold was a white cubic grid. Micro-CT detection showed the pore network structure in the scaffold material with good pore connectivity. The diameters of large pore and micro-aperture were (506.37±18.63) μm and (62.14±17.35) μm, respectively. The porosity was 97.70%±1.37%, and the water absorption swelling rate was 1 341.97%±64.41%. Mechanical tests showed that the compression displacement of the scaffold was (0.376±0.004) mm, the compressive stress was (0.016±0.002) MPa, and the elastic modulus was (162.418±18.754) kPa when the scaffold was compressed to 10%. At 7 days and 14 days, HE staining and scanning electron microscope observation showed that the cells grew inside the scaffold, mainly distributed around the scaffold pore wall. The cells in experimental group were more than control group, and the cells morphology changed from shuttle to flat. There was no significant difference in the cell counting between 2 groups at 14 days after 200-fold microscopy ( t=-2.024， P=0.080), but significant differences were found between 2 groups at different time points under different magnifications ( P<0.05). Real-time fluorescent quantitative PCR showed that the mRNA relative expressions of collagen type Ⅰ and OCN in experimental group were significantly higher than those in control group at 7 and 14 days ( P<0.05). However, the mRNA relative expression of BMP-2 showing no significant difference between 2 groups ( P>0.05). The protein relative expressions of collagen type Ⅰ, BMP-2, and OCN in experimental group were significantly higher than those in control group at 7 and 14 days ( P<0.05). After dynamic mechanical loading, the expressions of BMP-2, collagen type Ⅰ, and OCN in MC3T3-E1 cells inoculated into 3D biomimetic composite scaffolds are significantly up-regulated, indicating that appropriate mechanical loads favor osteoblast differentiation of MC3T3-E1 cells.

Differentiation of mesenchymal stem cells into neuron-like cells using composite 3D scaffold combined with valproic acid induction.

The nervous system has little capacity for self-repair after injury because neurons cannot proliferate owing to lack of suitable microenvironment. Therefore, neural tissue engineering that combines neural stem, scaffolds, and growth factors may improve the chance of restoration of damaged neural tissues. A favorable niche for neural regeneration would be both fibrous and electrically conductive scaffolds. Human Wharton jelly-derived mesenchymal stem cells were seeded on wet-electrospun 3D scaffolds composed of poly lactic acid coated with natural polymers including alginate and gelatin, followed by a multi-wall carbon nanotube coating. The results show that a wet-electrospun poly lactic acid scaffold at a concentration of 15% w/v had higher porosity (above 80%) than other concentrations. Moreover, the coated scaffold supported the growth of human Wharton jelly-derived mesenchymal stem cells in 3D culture, and were incubated for 21 days with 1 mM valproic acid as the inducer resulted in improvement in human Wharton jelly-derived mesenchymal stem cells differentiation into neuron-like cells immunoreactivity to nestin, Map2, and neuron specific enolase (NSE), which were also consistent with reverse transcription polymerase chain reaction (RT-PCR) and quantitive Reverse transcription polymerase chain reaction (qRT-PCR) results. The conclusion is that the 3D composite nanofiber poly lactic acid scaffold improved the transdifferentiation of human Wharton jelly-derived mesenchymal stem cells into neuron-like cells.

Effect of Bone Marrow Mesenchymal Stem Cells on Hydroxyapatite/Silk Fibroin/Chitosan Composite 3D Scaffold for Rat Skull Defects Repair

Effect of Bone Marrow Mesenchymal Stem Cells on Hydroxyapatite/Silk Fibroin/Chitosan Composite 3D Scaffold for Rat Skull Defects Repair

3D printing hydrogel with graphene oxide is functional in cartilage protection by influencing the signal pathway of Rank/Rankl/OPG

3Dprinting is defined as the use of printing technology to deposit living cells, and biomaterials on a given /a substrate. Graphene oxide nanoparticles (GO-np) have been used as a delivery vehicle for small molecule drugs in order to investigate the state of GO-np within 3D tissue constructs in terms of a composite 3D printing scaffold, which in turn is relevant to the protection of cartilage. We transplanted rats with hydrogel/GO-np and hydrogel, which in turn showed that hydrogel/GO-np protected the tissue of cartilage by the signal pathway of Rank/Rankl/OPG. Those findings indicated that GO-np may be potentially used to control the release of carrier materials and influence the signal pathway of Rank/Rankl/OPG.

Towards osteogenic bioengineering of dental pulp stem induced by sodium fluoride on hydroxyapatite based biodegradable polymeric scaffold

Hydroxylapatite (HA) and sodium fluoride (NaF) are expected to have enhanced osteoblast proliferation and differentiation ability and as a result gained much attention in tissue-engineering applications. The aim of this study is to investigate the effect of NaFand/or HA addition to three-dimensional (3D) nanofibrous poly(e-caprolactone) (PCL)/poly lactic acide (PLA) scaffolds on Human dental pulp stem cells (DPSCs) proliferation, osteoblast differentiation and mineralization. For this purpose, PCL and PLA homopolymers and HA nanoparticles were synthesized. Then a 3D nanofibrous composite scaffold of PCL/PLA/HA was prepared by electrospinning method with fiber diameter in the range of around 120-300 nm. NaF was added to media during DPSCs culturing step. Cell proliferation was assessed over a 14 days period. Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the osteogenic induction. The in vitro biocompatibility tests performed with DPSCs showed excellent cell attachment (SEM), proliferation (MTT assay), and mineralization (Alizarin Red). QRT-PCR results, demonstrated significantly superior osteogenesis markers in group of DPSCs bioengineered in PCL/PLA/HA and PCL/PLA/HA/NaF. Simultaneous application of HA in nanofibrous PCL/PLA scaffolds and NaF in the media showed synergistic effects on the growth and differentiation of DPSCs, and consequently seems to be a promising scaffold for tissue regeneration in the oral cavity.

Silk fiber reinforcement modulates in vitro chondrogenesis in 3D composite scaffolds

The limited self-regenerative capacity of adult cartilage has steered the upsurge in tissue engineered replacements to combat the problem of osteoarthritis. In the present study, the potential of fiber-reinforced silk composites from mulberry (Bombyx mori) and non-mulberry (Antheraea assamensis) silk has been investigated for cartilage tissue engineering. The fabricated composites were physico-chemically characterized and analyzed for cellular viability, proliferation, extracellular matrix formation and immunocompatibility. Both mulberry and non-mulberry silk composites showed effective swelling (25%–30%) and degradation (10%–30%) behavior, owing to their interconnected porous nature. The non-mulberry fiber-reinforced composite scaffolds showed slower degradation (∼90% mass remaining) than mulberry silk over a period of 28 days. The reinforcement of silk fibers within silk solution resulted in an increased compressive modulus and stiffness (nearly eight-fold). The biochemical analysis revealed significant increase in DNA content, sulphated glycosaminoglycan (sGAG) (∼1.5 fold) and collagen (∼1.4 fold) in reinforced composites as compared to pure solution scaffolds (p ≤ 0.01). Histological and immunohistochemical (IHC) staining corroborated enhanced deposition of sGAG and localization of collagen type II in fiber-reinforced composites. This was further substantiated by real time polymerase chain reaction studies, which indicated an up-regulation (∼1.5 fold) of cartilage-specific gene markers namely collagen type II, sox-9 and aggrecan. The minimal secretion of tumor necrosis factor-α (TNF-α) by murine macrophages further demonstrated in vitro immunocompatibility of the scaffolds. Taken together, the results signified the potential of silk fiber-reinforced composite (particularly non-mulberry, A. assamensis) scaffolds as viable alternative biomaterial for cartilage tissue engineering.