Silk fibroin‐chondroitin sulfate‐alginate porous scaffolds: Structural properties and in vitro studies

The development of porous biodegradable scaffolds is of great interest in tissue engineering. In this regard, exploration of novel biocompatible materials is needed. Silk fibroin‐chondroitin sulfate‐sodium alginate (SF‐CHS‐SA) porous hybrid scaffolds were successfully prepared via lyophilization method and crosslinked by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide‐ethanol treatment. According to the scanning electron microscopy studies, mean pore diameters of the scaffolds were in the range of 60–187 μm. The porosity percentage of the scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (w/w/w %) was 92.4 ± 3%. Attenuated total reflectance Fourier transform infrared spectroscopy, X‐ray diffraction, and differential scanning calorimetry results confirmed the transition from amorphous random coil to crystalline β‐sheet in treated SF‐CHS‐SA scaffold. Compressive modulus was significantly improved in hybrid scaffold with SF‐CHS‐SA ratio of 70 : 15 : 15 (3.35 ± 0.15 MPa). Cytotoxicity assay showed that the scaffolds have no toxic effects on chondrocytes. Attachment of chondrocytes was much more improved within the SF‐CHS‐SA hybrid scaffold. Real‐time polymerase chain reaction analyses showed a significant increase in gene expression of collagen type II, aggrecan, and SOX9 and decrease in gene expression of collagen type I for SF‐CHS‐SA compared with SF scaffold. This novel hybrid scaffold can be a good candidate to be utilized as an efficient scaffold for cartilage tissue engineering. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 41048.     

Facile fabrication of near-infrared light-responsive shape memory nanocomposite scaffolds with hierarchical porous structures

In this work, the near-infrared (NIR) light-responsive shape memory scaffolds with hierarchical porous structures are designed and facilely formed by freeze drying of 3D printed viscous gel-like pickering emulsions, which are stabilized by hydrophobically modified graphene oxide (g-GO) and silica nanoparticles, and contain thermo-responsive poly(d , l -lactic acid-co-trimethylene carbonate) (PLMC) in the oil phase. The prepared scaffolds display an interconnected filament structure with hierarchical pores and high porosity. The incorporation of g-GO nanoparticles into PLMC matrix prompts that the scaffold shape memory can be triggered by NIR light with fast shape recovery. Moreover, the in vitro mineralization experiment shows that the scaffolds have biological activity, and the drug release study demonstrates that the scaffolds can be used as drug carriers with efficient drug release capacity. Furthermore, cell culture assays based on mouse bone mesenchymal stem cells exhibit that the scaffolds own good cytocompatibility. Therefore, the facile preparation and remote activation of the shape memory nanocomposite scaffolds with hierarchical porous structure and multifunctionality represents a highly attractive candidate as minimally invasive implantation scaffolds for bone tissue engineering applications.     

Bone Regeneration in rat using a gelatin/bioactive glass nanocomposite scaffold along with endothelial cells (HUVECs)

In our previous study, a three‐dimensional gelatin/bioactive glass nanocomposite scaffold with a total porosity of about 85% and pore sizes ranging from 200 to 500 μm was prepared through layer solvent casting combined with lamination technique. The aim of this study was to evaluate in vitro biocompatibility and in vivo bone regeneration potential of these scaffolds with and without endothelial cells when implanted into a critical‐sized rat calvarial defect. MTT assay, SEM observation, and DAPI staining were used to evaluate cell viability and adhesion in macroporous scaffolds and results demonstrated that the scaffolds were biocompatible enough to support cell attachment and proliferation. To investigate the in vivo osteogenesis of the scaffold, blank scaffolds and endothelial/scaffold constructs were implanted in critical‐sized defects, whereas in control group defects were left untreated. Bone regeneration and vascularization were evaluated at 1, 4, and 12 weeks postsurgery by histological, immunohistochemical, and histomorphometric analysis. It was shown that both groups facilitated bone growth into the defect area but improved bone regeneration was seen with the incorporation of endothelial cells. The data showed that the porous Gel/BaG nanocomposite scaffolds could well support new bone formation, indicating that the proposed strategy is a promising alternative for tissue‐engineered bone defects.     

3D Composite scaffolds using strontium containing bioactive glasses

In this study, it was aimed to fabricate and characterize three-dimensional composite scaffolds derived from Sr-doped bioactive glass for bone tissue engineering applications. The scaffolds were fabricated by using polymer foam replication technique and coated with gelatin to be able to improve the properties of them. The porous scaffolds were successfully synthesized using optimized process parameters. Both coated and uncoated scaffolds favored precipitation of calcium phosphate layer when they were soaked in simulated body fluid (SBF). Gelatin coating improved the mechanical properties of the scaffold and also it did not change the bioactive behavior of the scaffold. It was observed that there was a good pore interconnectivity maintained in the scaffold microstructure. Results indicated that scaffolds can deliver controlled doses of strontium toward the SBF medium. That is the determinant for bone tissue regeneration, as far as strontium is known to positively act on bone remodeling.     

Sustained release of dexamethasone from drug‐loading PLGA scaffolds with specific pore structure fabricated by supercritical CO2 foaming

Inducing differentiation of bone marrow stem cells to generate new bone tissue is highly desirable by controlling the release of some osteoinductive or osteoconductive factors from porous scaffolds. In this study, dexamethasone was selected as a representative of small molecule drugs and dexamethasone‐loading porous poly(lactide‐co‐glycolide) (PLGA) scaffolds were successfully fabricated by supercritical CO₂ foaming. Scanning electron microscopy images showed that scaffolds had rough and relatively interconnected pores facilitating cells adhesion and growth. Specially, dexamethasone which was incorporated into PLGA matrix in a molecularly dispersed state could serve as a nucleation agent to be helpful for the formation of interconnected pores. Dexamethasone‐loading porous PLGA scaffolds exhibited sustained release profile, and the delivery of dexamethasone from porous scaffolds could last for up to 2 months. The cumulative released amount of dexamethasone was relevant with drug loading capacity (1.66%–2.95%) and pore structure of scaffolds; while the release behavior was anomalous (non‐Fickian) transport by fitting with the simple exponential equation, which had a diffusional exponent n higher than 0.5. It is feasible to fabricate drug‐loading porous scaffolds by supercritical CO₂ foaming with specific pore structure and sustained release profile, which can be well applied in bone tissue engineering. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46207.     

Determination of cytocompatibility and osteogenesis properties of in situ forming collagen-based scaffolds loaded with bone synthesizing drug for bone tissue engineering

Bone tissue engineering using in situ forming 3D scaffolds can be an alternative to surgically treated scaffolds. This work aimed to develop in situ forming scaffolds using poly (lactic-co-glycolic acid) and a bone synthesizing drug (risedronate) with or without the porogenic agent (collagen). Hybrid scaffolds were formed through solvent-induced phase inversion technique and were morphologically evaluated using scanning electron microscopy (SEM). The effect of scaffolds on Saos-2 cell line viability using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test besides their effect on cell growth using fluorescence microscope was assessed. Furthermore, alkaline phosphatase (ALP) activity as well as Ca²⁺ deposition on the scaffolds was evaluated. SEM images revealed the porous structure for collagen-based scaffolds. Saos-2 cell proliferation was significantly enhanced with risedronate-loaded scaffolds compared to those lacking the drug. Porous collagen-based scaffolds were more favorable for both the cell growth and the promotion of ALP activity. Furthermore, collagen-based scaffolds promoted the Ca²⁺ deposition compared to their counterparts without collagen. Such results suggest that collagen-based scaffolds offer excellent biocompatibility for bone regeneration, where this biocompatible nature of scaffold leads to the proliferation of cells that lead to the deposition of mineral on the scaffold. Such in situ forming 3D scaffolds provide a promising noninvasive approach for bone tissue engineering.     

Dual drug spatiotemporal release from functional gradient scaffolds prepared using 3D bioprinting and electrospinning

Functional gradient scaffolds play an important role in osteochondral tissue engineering because they can meet the essential requirement for a gradual transition of both physical and chemical properties in osteochondral tissue regeneration. There is a requirement for 3D composite osteochondral regeneration scaffolds with multiscale structures that are capable of controlling release of multiple biomolecules. To this end, this article describes a 3D bioprinting platform integrated forming system designed to produce various drug‐loaded scaffolds. A novel scaffold was fabricated by the self‐developed 3D bioprinting platform combining extrusion deposition with multi‐nozzle electrospinning. For temporally controlled release of gentamycin sulfate (GS) and desferoxamine (DFO), blend electrospun GS/polyvinyl alcohol (PVA) and coaxial electrospun core (PVA‐DFO)/shell (polycaprolactone; PCL) fibers were deposited in the scaffold. After a 25‐day time‐lapse release study in vitro, results showed GS released faster than DFO during the early stages and sustained release of DFO for long periods. For spatially controlled release of DFO, the vertically gradient gelatin/sodium alginate (SA) scaffolds presented to enable the release amount of DFO in a gradient mode. The experiment and test results demonstrate the validity of the 3D bioprinting platform integrated forming system and the excellent properties of such scaffolds for performing multidrug spatiotemporal release. POLYM. ENG. SCI., 56:170–177, 2016. © 2015 Society of Plastics Engineers     

骨组织工程用PLGA多孔支架的制备及细胞毒性研究

制备能在骨组织工程研究中应用,并具有良好孔隙结构的块状聚（D,L-乳酸-CO-乙醇酸）（PLGA）多孔支架,探索出以冰粒子作为致孔剂,采用粒子滤出方法结合冷冻干燥工艺制备多孔支架的方法.首先将冰颗粒加入预冻的PLGA氯仿溶液中混合均匀,然后把混合物置于液氮中深度冷冻后冷冻干燥,制得多孔支架.对支架孔隙结构分析表明,该工艺制备的多孔支架无致孔剂残留、三维结构良好、孔径与孔隙可通过改变冰粒子的粒径和质量分数来控制;细胞毒性实验表明该多孔支架毒性在0～1级,可作为骨组织工程研究用多孔支架.     

Poly(ε‐caprolactone) composite scaffolds loaded with gentamicin‐containing β‐tricalcium phosphate/gelatin microspheres for bone tissue engineering applications

In this study, novel poly(ε‐caprolactone) (PCL) composite scaffolds were prepared for bone tissue engineering applications, where gentamicin‐loaded β‐tricalcium phosphate (β‐TCP)/gelatin microspheres were added to PCL. The effects of the amount of β‐TCP/gelatin microspheres added to the PCL scaffold on various properties, such as the gentamicin release rate, biodegradability, morphology, mechanical strength, and pore size distribution, were investigated. A higher amount of filler caused a reduction in the mechanical properties and an increase in the pore size and led to a faster release of gentamicin. Human osteosarcoma cells (Saos‐2) were seeded on the prepared composite scaffolds, and the viability of cells having alkaline phosphatase (ALP) activity was observed for all of the scaffolds after 3 weeks of incubation. Cell proliferation and differentiation enhanced the mechanical strength of the scaffolds. Promising results were obtained for the development of bone cells on the prepared biocompatible, biodegradable, and antimicrobial composite scaffolds. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40110.     

A study on retention of MWCNT in robocasted MWCNT-HAP scaffold structures using vacuum sintering technique and their characteristics

Bioceramic scaffolds are being widely employed in bone tissue engineering applications for their ability to interact with host tissues without inducing any toxicity. Additionally, bioceramics possess good biocompatibility, osteointegration, osteoinduction, and biodegradation characteristics. Hydroxyapatite (HAP) is one such bioceramic known to exhibit closeness to natural bone in terms of chemical composition. The present reports additive manufacturing of HAP and Multiwalled carbon nanotubes (MWCNTs) reinforced HAP scaffold structures for bone tissue engineering applications using the Robocasting technique. Carboxymethyl Cellulose (CMC) was employed as the polymeric binder in this study to prepare the highly viscous HAP and CNT-HAP slurry ideal for robocasting of the scaffold structures. Different percentages of MWCNT (0.5, 1 and 2 wt%) incorporated into the developed CNT-HAP scaffold structures and were vacuum sintered at 1000 °C for 15 min. Vacuum sintering was found to effectively prevent oxidation of MWCNT which is subjected to decomposition at temperatures above 400 or 500 °C in Oxygen atmosphere as per literature. Further, the retention of MWCNTs in the developed CNT-HAP structures post sintering was confirmed using FESEM imaging. The mechanical characterizations revealed that 0.5CNT-HAP structures exhibited highest compression strength (3.36 ± 0.67 MPa) in comparison to 1CNT-HAP and 2CNT-HAP structures. Also, the in vitro biological characterizations demonstrated that the developed CNT-HAP scaffold structures were cytocompatible and remained stable for about 35 days at 37 °C.     

Modified poly(caprolactone trifumarate) with embedded gelatin microparticles as a functional scaffold for bone tissue engineering

Bone tissue engineering offers high hopes in reconstructing bone defects that result from trauma, infection, tumors, and other conditions. However, there remains a need for novel scaffold materials that can effectively stimulate ossification with appropriate functional properties. Therefore, a novel injectable, biodegradable, and biocompatible scaffold made by incorporating modified poly(caprolactone trifumarate) (PCLTF) with embedded gelatin microparticles (GMPs) as porogen is developed. Specifically, in vitro and in vivo tests were carried out. For the latter, to determine the osteogenic ability of PCLTF‐GMPs scaffolds, and to characterize bone‐formation, these scaffolds were implanted into critical‐sized defects of New Zealand white rabbit craniums. Field Emission Scanning Electron Microscope (FESEM) demonstrated cells of varying shapes attached to the scaffold surface in vitro. The PCLTF‐GMPs demonstrated improved biocompatibility in vivo. Polyfluorochrome tracers detected bone growth occurring in the PCLTF‐GMPs filled defects. By incorporating PCLTF with GMPs, we have fabricated a promising self‐crosslinkable biocompatible and osteoconducive scaffold for bone tissue engineering. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43711.     

Solvent effect in phase separation for fabrication of micropatterned porous scaffold sheets

Cellular orientation control is important for tissue regeneration. Design of oriented structures for cells with suitable features can be used in tissue engineering. One of the methods of cellular orientation with the aim of regenerating which damaged tissues is utilizing oriented biocompatible substrates. This paper reports a one-step method with different solvents to fabricate porous micropatterned polyhydroxybutyrate scaffold sheets. The results indicated that the porosity and pore morphology of the scaffolds are viable with respect to proliferation rate, and a micropattern for cell alignment. Stem cells culturing proved that the scaffold sheets are suitable for cell culturing. Preliminary experiments indicate that the 2-D scaffold sheets are very promising as basis for building 3-D scaffolds.     

The effect of pore morphology and agarose coating on mechanical properties of tricalcium phosphate scaffolds

Three-dimensional biocompatible porous structures can be fabricated using different methods. However, the biological and mechanical behaviors of scaffolds are the center of focus in bone tissue engineering. In this study, tricalcium phosphate scaffolds with similar porosity contents but different pore morphologies were fabricated using two different techniques, namely, the replica method and the pore-forming agent method. The samples fabricated using the pore-forming agent showed more than two times higher compressive and bending strengths and more than three times higher compressive moduli. Furthermore, a thin layer of agarose coating improved the compressive and bending strength of both types of ceramic scaffolds. Subsequently, the samples’ capability to guide biomineralization was evaluated by immersion into a simulated body fluid that developed Ca-P nano-platelets formation and enhanced the compressive strength. Finally, the tetrazolium-based colorimetric (MTT) assay was used to evaluate L929 cell viability and proliferation on all the samples and confirmed that cell behavior was not affected by pore morphology or agarose coating. In summary, samples produced by the use of the pore-forming agent showed higher potential to be applied as bone scaffolds in tissue engineering applications.     

Impact and biocompatibility studies on Mg2+-substituted Apatite-derived 3D porous scaffolds for hard tissue engineering

The objective of this study was to develop Mg²⁺-substituted Apatite scaffolds by slip-casting method. The Apatite scaffolds were prepared as engineering constructs with interconnected pore structure with a pore size of 128-194 μm range. The physicochemical properties such as crystalline phase, functional group, microstructure, pore size distribution, and elemental compositions of the scaffolds were characterized. The bioactivity of the developed porous scaffolds was investigated in Simulated Body Fluid (SBF) for various time periods (3 and 7 days). In vitro bioactivity results confirm the hydroxyl Apatite layer formation of the scaffolds and results suggest that the developed microporous scaffold could be used as suitable candidates in bone tissue engineering.     

Supercritical carbon dioxide assisted fabrication of biomimetic sodium alginate/silk fibroin nanofibrous scaffolds

In this study, biomimetic sodium alginate (SA)/silk fibroin (SF) scaffolds were successfully fabricated by supercritical CO₂ technology. The SA/SF scaffolds exhibited an interconnected porous and extracellular matrix (ECM)-like nanofibrous structures. Moreover, the SA microparticles were embedded in the SF scaffolds. Increasing the content of SA microparticles could improve tensile strength and compressive strength of the SF scaffolds and reduce the porosity of the SF scaffolds. The addition of the SA microparticles could also regulate the degradation rate of the SA/SF scaffolds. Furthermore, the results of in vitro biocompatibility evaluation, indicated that the SA/SF scaffolds exhibited no obvious cytotoxicity and higher cell adhesion ability and were more favorable for L929 fibroblasts proliferation than pure SF scaffolds. Therefore, the SA/SF scaffolds with ECM-like nanofibrous and interconnected porous structure have potential application in skin tissue engineering.     

Improvement of mechanical properties of macroporous β-tricalcium phosphate bioceramic scaffolds with uniform and interconnected pore structures

The biocompatible and degradable macroporous bioceramic scaffolds with high mechanical properties and interconnected porous structures play an important role in hard tissue regeneration and bone tissue engineering applications. In this study, the improvement of mechanical properties of macroporous β-tricalcium phosphate [β-Ca₃(PO₄)₂, β-TCP] bioceramic scaffolds with uniform macropore size and interconnected pores were fabricated by impregnation of the synthesized β-TCP nano-powder slurry into polymeric frames. The microstructures, mechanical properties and in vitro degradation of the fabricated samples were investigated. For a comparison, β-TCP scaffolds were also fabricated from commercial micro-size powders under the same conditions. The resultant scaffolds showed porosities ∼65% with uniform macropore size ranging from 400 to 550 μm and interconnected pore size ∼100 μm. The compressive strength of the samples fabricated from nano-size powders reached 10.87 MPa, which was almost twice as high as those fabricated from commercial micro-size powders, and was comparable to the high-end value (2–10 MPa) of human cancellous bone. Furthermore, the degradation of the β-TCP bioceramics fabricated from nano-size powders was apparently lower than those fabricated from commercial micro-size powders, suggesting the possible control of the degradation of the scaffolds by regulating initial powder size. Regarding the excellent mechanical properties and porous structures, the obtained macroporous β-TCP bioceramic scaffolds can be used in hard tissue regeneration and bone tissue engineering applications.     

Preparation of chitosan-sodium alginate/bioactive glass composite cartilage scaffolds with high cell activity and bioactivity

Chitosan-sodium alginate/bioactive glass (CSB) composite cartilage scaffold with outstanding in vitro mineralization property and cytocompatibility is synthesized by freeze drying method. The effect of bioactive glass (BG) addition on the microstructure, porosity, swelling/degradation ratio, in vitro mineralization property and cytocompatibility of CSB scaffold is investigated by the characterization techniques of SEM, XRD, FTIR and BET. Results showed that CSB composite cartilage scaffold had a three-dimensional (3D) porous structure, and both porosity and average pore size met the requirements of cartilage tissue repair. Among, the typical CSB-1.0 had the largest overall pore size and lowest compressive modulus (1.083 ± 0.002 MPa). As the amount of BG increased, pore volume and porosity of CSB scaffolds gradually decreased, and the swelling and degradation ratios gradually reduced. After immersing in SBF for 3 d, cauliflower like hydroxyapatite (HA) was formed on CSB surface, indicating that the scaffold had good in vitro mineralization property. Moreover, the introduction of BG into the composite scaffold can improve the relative cell viability of MC3T3-E1 cells, and CSB-1.0 has the strongest ability to promote the proliferation of cells. Therefore, the as-obtained CSB scaffold can be used as a strong candidate for cartilage tissue engineering scaffold to meet clinical needs.     

Preparation and characterization of vancomycin releasing PHBV coated 45S5 Bioglass®-based glass–ceramic scaffolds for bone tissue engineering

Porous 45S5 Bioglass^®-based glass–ceramic scaffolds with high porosity (96%) and interconnected pore structure (average pore size 300 μm) were prepared by foam replication method. In order to improve the mechanical properties and to incorporate a drug release function, the scaffolds were coated with a drug loaded solution, consisting of PHBV and vancomycin. The mechanical properties of the scaffolds were significantly improved by the PHBV coating. The bioactivity of scaffolds upon immersion in SBF was maintained in PHBV coated scaffolds although the formation of hydroxyapatite was slightly retarded by the presence of the coating. The encapsulated drug in coated scaffolds was released in a sustained manner (99.9% in 6 days) as compared to the rapid release (99.5% in 3 days) of drug directly adsorbed on the uncoated scaffolds. The obtained drug loaded and bioactive composite scaffolds represent promising candidates for bone tissue engineering applications.     

Bi-/multi-modal pore formation of PLGA/hydroxyapatite composite scaffolds by heterogeneous nucleation in supercritical CO2 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 CO_2 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(552μm±127μm).Furthermore,multimodal porous PLGA scaffolds with micro-pores(37 μm±11 μm)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 CO_2 foaming method is promising to fabricate bi-/multi-modal porous scaffolds in one step,and bioactive particles for osteogenesis could serve as nucleation agents.     

Functionalized tricalcium phosphate and poly(3-hydroxyoctanoate) derived composite scaffolds as platforms for the controlled release of diclofenac

Novel bone substitutes such as highly porous ceramic scaffolds can serve as platforms for delivering active molecules. A common problem is to control the release of the drug, therefore, it is beneficial to use a drug-functionalized polymer coating. In this study, β-tricalcium phosphate-based porous scaffolds were obtained and coated with diclofenac-functionalized biopolymer – poly(3-hydroxyoctanoate) – P(3HO). To the best of our knowledge, studies using P(3HO) as a component in ceramic-polymer based drug delivery system for bone tissue regeneration have not yet been reported. Presented materials were comprehensively investigated by various techniques such as powder X-ray diffraction, scanning electron microscopy with energy dispersive spectroscopy, hydrostatic weighing and compression tests, pH and ionic conductivity measurements, high-performance liquid chromatography and in vitro cytotoxicity studies. The obtained diclofenac-loaded composite was not only characterised by controlled and sustained drug release, but also possessed improved mechanical properties. Moreover, the precipitation of apatite-like forms on its surface was observed after incubation in simulated body fluid, which indicates its bioactive potential. After 24 hours no cytotoxic effect on MC3T3-E1 mouse preosteoblastic cells was confirmed using indirect cytotoxicity studies. Thus, this promising multifunctional composite scaffold can be a promising candidate as an anti-inflammatory drug-delivery system in bone tissue engineering.