Development of a novel glucosamine/silk fibroin–chitosan blend porous scaffold for cartilage tissue engineering applications

Silk fibroin–chitosan blend is reported to be an attractive scaffold material for tissue engineering applications. In our earlier study, we developed a scaffold having an optimal silk fibroin–chitosan blend ratio of 80:20 and proved its potentiality for cartilage tissue engineering applications. Glucosamine is one of the major structural components of cartilage tissue. The present work investigates the effect of glucosamine components on the physicochemical and biocompatibility properties of this scaffold. To this end, varied amounts of glucosamine were added to silk fibroin–chitosan blend with the aim of improving various scaffold properties. The addition of glucosamine components did not show any significant change in physicochemical properties of silk fibroin–chitosan blend scaffolds. The composite scaffold showed an open pore structure with desired pore size and porosity. However, cell culture study using human mesenchymal stem cells derived from umbilical cord blood revealed an overall increase in cell supportive properties of glucosamine-added scaffolds. Cell viability, cell proliferation and glycosaminoglycan assays confirmed enhanced cell viability and proliferation of mesenchymal stem cells. Thus, this study demonstrated the beneficial effect of glucosamine on improving the cell supportive property of silk fibroin–chitosan blend scaffolds making it more potential for cartilage tissue regeneration. To the best of our knowledge, this is the first report on the study of glucosamine-added silk fibroin–chitosan blend porous scaffolds seeded with mesenchymal stem cells derived from umbilical cord blood.     

Preparation of Porous Scaffolds from Silk Fibroin Extracted from the Silk Gland of Bombyx mori (B. mori)

In order to use a simple and ecofriendly method to prepare porous silk scaffolds, aqueous silk fibroin solution (ASF) was extracted from silk gland of 7-day-old fifth instar larvae of Bombyx mori (B. mori). SDS-page analysis indicated that the obtained fibroin had a molecular weight higher than 200 kDa. The fabrication of porous scaffolds from ASF was achieved by using the freeze-drying method. The pore of porous scaffolds is homogenous and tends to become smaller with an increase in the concentration of ASF. Conversely, the porosity is decreased. The porous scaffolds show impressive compressive strength which can be as high as 6.9 ± 0.4 MPa. Furthermore, ASF has high cell adhesion and growth activity. It also exhibits high ALP activity. This implies that porous scaffolds prepared from ASF have biocompatibility. Therefore, the porous scaffolds prepared in this study have potential application in tissue engineering due to the impressive compressive strength and biocompatibility.     

Preparation of insoluble fibroin/collagen films without methanol treatment and the increase of its flexibility and cytocompatibility

It is still a critical challenge to increase the flexibility of regenerated fibroin materials in dry and near dry states. In this study, a novel biocompatible and water‐stable film composed of fibroin and collagen was successfully prepared from aqueous fibroin solution without methanol treatment. The result of contact angle measurement indicates that hydrophilicity is evidently increased when collagen was added to fibroin film. The elongation at break in wet state is also increased because of the blending of collagen, which implied the improvement of flexibility. More importantly, the blend films containing 20% collagen become flexible when placed at above 65% humidity in atmosphere. It means that the blend films could be fabricated to different conformations easily through adjusting humidity in atmosphere. HepG2 cells were cultured on fibroin and fibroin/collagen films to investigate the cytocompatibility of these films. Scanning electron microscopy and MTT analysis demonstrated that the adding of collagen evidently improved HepG2 proliferation in over 10 days culture. The excellent cytocompatibility, the flexibility in the near dry state as well as the green preparation process of fibroin/collagen blend films make them become the promising biomaterials for different medical applications. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Preparation of porous PLGA/HA/collagen scaffolds with supercritical CO2 and application in osteoblast cell culture

Biocompatible three-dimensional scaffolds for cell culturing may facilitate methods for the repair of damaged human tissues. A novel hybrid porous scaffold of poly(lactic-co-glycolic acid), hydroxyapatite and collagen was prepared using a supercritical CO₂ saturation technique. Expansion factors of scaffolds with different compositions were studied after supercritical CO₂ treatment to choose the optimal composition for three-dimensional culture. The supercritical CO₂ process conditions, such as saturation temperature, saturation time and saturation pressure were varied to evaluate their influence on pore structure. The results showed that the pore size and porosity of the scaffold could be controlled by manipulating these process conditions. The porous samples were characterized by environmental scanning electron microscopy, energy-dispersive X-ray spectroscope, Fourier transform infrared spectroscopy and X-ray diffractometry. Finally, MG-63 cells were successfully cultured on the porous scaffold as assessed by electron and confocal microscopy, confirming the biocompatibility of this new hybrid porous scaffold.     

Preparation of three-dimensional ordered macroporous SiCN ceramic using sacrificing template method

Three-dimensional (3D) long range well ordered macroporous SiCN ceramics were prepared by infiltrating sacrificial colloidal silica templates with the low molecular weight preceramic polymer, polysilazane. This was followed by a thermal curing step, pyrolysis at 1250 °C in a N₂ atmosphere, and finally the removal of the templates by etching with dilute HF. The produced macroporous SiCN ceramics showed high BET surface areas (pore volume) in the range 455 m²/g (0.31 cm³/g)–250 m²/g (0.16 cm³/g) with the pore sizes of 98–578 nm, which could be tailored by controlling the sizes of the sacrificial silica spheres in the range 112–650 nm. The sphere-inversed macropores were interconnected by 50 ± 30 nm windows and 3–5 nm mesopores embedded in the porous SiCN ceramic frameworks, which resulted in a trimodal pore size distribution. The surface of the achieved porous SiCN ceramic was then modified by Pt–Ru nanoparticle depositing under mild chemical conditions.     

Fabrication,characterization and cellular biocompatibility of porous biphasic calcium phosphate bioceramic scaffolds with different pore sizes

Facile wet-chemical methods are applied to synthesize hydroxyapatite and β-tricalcium phosphate nanoparticles, respectively. Porous biphasic calcium phosphate (BCP) bioceramic scaffolds are then fabricated using as-prepared HA and β-tricalcium phosphate nanoparticle powders. The macro pore diameter of BCP bioceramic scaffolds can be controlled by adjusting the amount of surfactants. The average diameter of the macro pores in BCP bioceramic scaffolds increases from 100 to 600 µm with the decrease amount of sodium dodecyl sulfate from 0.8 to 0.5 g, respectively. The BCP bioceramic scaffolds gradually degrade and the calcium-phosphate compounds fully deposit when soaking in simulated body fluid solution. Moreover, The BCP bioceramic scaffolds have outstanding biocompatibility to promote the cellular growth and proliferation of human dental pulp stem cells (hDPSCs). The hDPSCs also demonstrate favorable cellular adhering capacity on the pore surface of scaffolds, especially on the scaffolds with 100–200 µm pore diameter. The porous BCP bioceramic scaffold with inter-connected pore structure, outstanding in vitro cellular biocompatibility, favorable cell viability and adhesion ability will be a promising biomaterial for bone or dentin tissue regeneration.     

Degradable and bioactive scaffold of calcium phosphate and calcium sulphate from self-setting cement for bone regeneration

Calcium sulphate/phosphate cement (CSPC) porous scaffolds were fabricated by introduction of calcium sulphate (CS) into calcium phosphate cement utilizing particle-leaching method. The morphology, porosity and mechanical strength as well as degradation of the CSPC scaffolds were characterized. The results reveal that the CSPC with 40 wt% CS content (40 CSPC) scaffolds with a porosity of 81% showed open macropores with the pore size of 200–500 μm. In addition, the 40 CSPC scaffolds with good degree of interconnected macropores degraded 60 wt% in Tris–HCl solution after 12 weeks. The proliferation, differentiation and morphology of MG₆₃ cells on the 40 CSPC scaffolds were determined using MTT assay, ALP activity and SEM. The results suggest that the CSPC scaffolds could stimulate cell proliferation and differentiation, indicating that CSPC scaffolds were biocompatible and had no negative effects on the cells in vitro. The CSPC scaffolds were implanted in femur bone defect of rabbits, and the in vivo biocompatibility and osteogenicity of the scaffolds were investigated. The results indicate that CSPC scaffolds exhibited good biocompatibility, degradability and osteogenesis in vivo.     

Biocompatibility and strengthening of porous hydroxyapatite scaffolds using poly(l-lactic acid) coating

Hydroxyapatite (HA) is a well-known biocompatible bone substitute. Porous HA is more resorbable and osteoconductive compared with non-porous HA, and has been studied both experimentally and clinically. However, the mechanical strength of porous HA scaffolds is known to be weak. In this study, we developed a porous HA scaffold coated with a synthetic biodegradable polymer, poly(l-lactic acid) (PLLA), to strengthen the scaffold. PLLA-coated HA pellets were used to investigate the in vitro proliferation and alkaline phosphatase (ALP) activity of osteoblasts. PLLA-coated porous HA scaffolds were observed using scanning electron microscopy to investigate surface characteristics, porosity, and mechanical strength. PLLA coating concentration varied from 2 to 10 wt%. Osteoblast proliferation was higher in HA samples coated with PLLA compared with non-coated. ALP activity was highest on 8 wt% PLLA-coating after 3 days and on 4 wt% and 6 wt% PLLA after 9 and 12 days. Porous HA scaffolds with higher concentrations of PLLA were found to have a smoother, flatter surface. This enhanced proliferation and attachment of osteoblasts onto the porous HA scaffold. PLLA solution at a concentration of 10 wt% decreased scaffold porosity to half that of HA scaffolds with no PLLA coating. Scaffold mechanical strength was increased two-fold with a PLLA concentration of 2 wt%. Based on in vitro experimentation, it can be concluded that PLLA-coating on porous HA scaffolds enhances both the biocompatibility and the mechanical strength.     

Preparation of elastic chitosan/poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide-co-ε-caprolactone) macroporous scaffolds by acetylation and particulate leaching

For soft tissue engineering applications, 3-D macroporous acetylated chitosan/poly(l-lactideco-ε-caprolactone) (PLCL) scaffolds were prepared by acetylation and particulate leaching using sodium acetate in an acidic water/dioxane solution. Acetylated 5 wt% chitosan/PLCL scaffold of 90% porosity was determined and confirmed through various tests. The physiochemical properties of acetylated chitosan/PLCL hybrid scaffolds were examined by measuring water contact angles, pore morphology and interconnectivity using scanning electron microscopy (SEM), and dye release testing. In addition, mechanical properties such as tensile strength and bending stress recovery for determining the elasticity of scaffolds were measured. The fibroblast cell line NIH-3T3 was used to test relative cell affinities for the acetylated chitosan/PLCL vs. normal chitosan/PLCL films and porous scaffolds. The acetylated chitosan/PLCL films and scaffolds showed a high initial cell adhesion after 4 h of cell culture and increased cell proliferation compared to that of the control. The acetylated chitosan/PLCL scaffolds produced by particulate leaching showed a highly porous structure and improved the biocompatibility and stability of chitosan compared to that of chitosan-coated PLCL scaffolds. Thus, these scaffolds may be very useful for a variety of tissue engineering applications.     

Study on porous silk fibroin materials. II. Preparation and characteristics of spongy porous silk fibroin materials

Porous silk fibroin materials, with average pore size 10 ∼ 300 μm, pore density 1 ∼ 2000/mm², and porosity 35 ∼ 70%, were prepared by freeze drying aqueous solution of silk fibroin obtained by dissolving silk fibroin in ternary solvent CaCl₂ · CH₃CH₂OH · H₂O. Pore size distribution of such materials mostly accorded with logarithmic normal distribution. It is possible to control the aforementioned structural parameters and the physical properties of moisture permeability, compressibility, strength, elongation, etc., by adjusting freezing temperature and concentration of silk fibroin solution. Above glass transition zone (−34 ∼ −20°C) of silk fibroin, the freezing temperature has more significant effect on the structure and properties of porous silk fibroin materials. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 79: 2192–2199, 2001     

A template-free, sonochemical route to porous ZnO nano-disks

A template-free, sonochemical aqueous route was used to synthesize hexagonal-shaped ZnO nanocrystals with a combined micro- and mesoporous structure. The products are much more porous when the sonohydrolysis is carried out under argon than their sonication under air. This has been attributed to the higher average specific heat ratio γ (=C_p/C_v) of argon gas, leading to higher bubble collapse temperatures. Small-angle XRD (SAXRD) studies show that the microporosity is lost at 250 °C, while the mesoporous structure persists till a very high temperature (550 °C). The BET surface area of the products synthesized under argon and air are 35 and 13 m²/g, respectively. The pore size is distributed from 1 nm (micropore) to 3.1–3.4 nm (mesopore), while the ZnO nanoparticles are 6.3 ± 1.2 nm. The possible mechanisms of the self-assembled pore formation are attributed to the organic porous framework of basic zinc acetate. The excitonic absorption of the ZnO occurs at 349 nm. The photoluminescence (PL) spectra of the ZnO nano-disks show the red-shifted band edge exciton transitions and the presence of deep levels due to oxygen vacancies or surface-deep traps, because of the porous structure.     

Chitosan/silk fibroin composite scaffolds for wound dressing

Three‐dimensional (3D) chitosan/silk fibroin (CS/SF) porous composite scaffolds have been prepared by simply coating a thin layer of CS onto spunlaced SF scaffolds via hydrogen‐bonding assembly technique, and they were characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), X‐ray diffraction (XRD), and mechanical property measurements. The results show that porous scaffolds have a pore diameter around 50–200 μm, and improved mechanical property compared with SF, resulting from strong intermolecular hydrogen bonding interactions between CS and SF, together with the maintained β‐sheet structure of SF. The medical and biological properties of the composite scaffolds were further evaluated. The results demonstrate that they possess good biocompatibility and a broad spectrum of antimicrobial properties. The in vivo animal experiments show that the composite scaffolds promote skin regeneration of rats without any teratogenic effect and inflection, thus they are very promising in the application of wound dressings. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42503.     

Preparation and characterization of collagen/silica composite scaffolds for peripheral nerve regeneration

To improve the regeneration of peripheral nerve system, the silica nanoparticles of various concentrations were synthesized in collagen solution and formed to silica incorporated porous collagen structures. We examined various properties such as morphology, chemical composition, wettability, porosity, swelling ratio and degradation behavior of the composite scaffolds. Schwann cells culture was used to evaluate the effect of the collagen/silica composite materials on nerve regeneration. And the content of DNA in Schwann cells was measured. We ascertained that the silica nanoparticles could be incorporated into collagen scaffolds successfully. The incorporation of silica nanoparticles could increase the hydrophobicity, decrease porosity, swelling ratio and degradation rate of the collagen scaffolds. Further, the attachment and proliferation of Schwann cells on the silica incorporated porous collagen patch was much better than that of the collagen patch as control. The number and DNA contents of the cells on the composite scaffolds increased firstly and then decreased with the increment of nanoparticles concentration. It was optimal to combine silica of 25 μg/mL for achieving best cell attachment and proliferation with the highest DNA contents compared with other samples. These results indicate that silica incorporated porous collagen patch may be potentially used as implanted scaffold materials for the peripheral nerve regeneration.     

Activated carbons prepared from rice hull by one-step phosphoric acid activation

Activated carbons were prepared from rice hull by one-step phosphoric acid activation in this work. The evolution of pore structure and surface chemistry in the activation temperature range of 170–450 °C was investigated through various characterization techniques. The results showed that the development of porosity (extent of activation) was negligible at activation temperature below 300 °C, and rapid evolution occurred in 300–400 °C. Porous activated carbon with bimodel pore structure (pore < 1 nm and pore > 1 nm) and BET surface area as high as 1295 m²/g was obtained at 450 °C. The ash contents of samples prepared in this study were in the range of 5–21%. The ash contents of carbons prepared in this study initially decreased from 21.03% to 4.89% with the change of temperature from 170 to 300 °C, then increased to 8.72% at 450 °C. Boehm titration results suggested that low activation temperature (300 °C) benefits the formation of acidic surface groups. With the increase of activation temperature from 300 to 350 °C, the concentrations of strong, intermediate and weak acidic surface groups decreased from 2.23, 1.87, and 2.73 to 1.66, 1.32, and 2.16 mmol H⁺/g, respectively. Over 350 °C, the change of these groups were insignificant. FTIR results revealed the existence of carbonyl-containing, phosphorus-containing groups, and groups containing Si–O bond. The relative concentration of carbonyl-containing groups decreases with an increase in activation temperature, while that of phosphorus-containing groups follows the reverse trend. The content of Si–O decreased first, then slowly increased with the increase of activation temperature. Boehm titration and FTIR (Fourier transform infrared spectroscopy) results indicated that the surfaces of these carbons contain both temperature-sensitive and temperature-insensitive groups. The temperature-sensitive part consists mainly of carbonyl-containing groups, such as carboxylic groups, while the temperature-insensitive part is primarily phosphorus-containing groups and groups containing Si–O bond. This study demonstrated that carbon products with relative low ash content and high activation degree can be prepared from rice hull by H₃PO₄ activation at suitable temperature.     

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.     

Bioactive and Biocompatible Macroporous Scaffolds with Tunable Performances Prepared Based on 3D Printing of the Pre‐Crosslinked Sodium Alginate/Hydroxyapatite Hydrogel Ink

Bioactive and biocompatible porous scaffold materials with adjustable pore structures and drug delivery capability are one of the key elements in bone tissue engineering. In this work, bioactive and biocompatible sodium alginate (SA)/hydroxyapatite (HAP) macroporous scaffolds are facilely and effectively fabricated based on 3D printing of the pre‐crosslinked SA/HAP hydrogels followed by further crosslinking to improve the mechanical properties of scaffolds. The pore structures and porosity (>80%) of the porous scaffolds can be readily tailored by varying the formation conditions. Furthermore, the in vitro biomineralization tests show that the bioactivity of the porous scaffolds is effectively enhanced by the addition of HAP nanoparticles into the scaffold matrix. Furthermore, the anti‐inflammatory drug curcumin is loaded into the porous scaffolds and the in vitro release study shows the sustainable drug release function of the porous scaffolds. Moreover, mouse bone mesenchymal stem cells (mBMSCs) are cultured on the porous scaffolds, and the results of the in vitro biocompatibility experiment show that the mBMSCs can be adhered well on the porous scaffolds. All of the results suggest that the bioactive and biocompatible SA/HAP porous scaffolds have great application potential in bone tissue engineering.     

Influence of exchanged cations (Na, Cs, Sr and Ba) on xylene permeation through ZSM-5/SS tubular membranes

Na-ZSM-5 membranes were synthesized by secondary growth on the outer surface of stainless steel porous tubes. The membranes were ion-exchanged with Cs⁺, Ba²⁺ and Sr²⁺ to investigate their effect upon the separation of p-xylene from m-xylene and o-xylene. The permeation through the membranes was measured between 150 and 400 °C using each xylene isomer separately and a ternary mixture. All the membranes were selective to p-xylene in the temperature range studied. N₂ and xylene permeation measurements together with SEM observations were used to determine whether or not cracks and/or pinholes developed after exposure to the xylene isomers at high temperature (400 °C). Neither pore blockage nor extra-zeolitic pores developed after the ion exchange procedure and subsequent calcination. Furthermore, duplicate synthesized membranes of each cation form had similar separation factors and permeances. The duplicate values differ much less than the measurement error. The p-xylene permeation flux decreased in the order: Na-ZSM-5 > Ba-ZSM-5 > Sr-ZSM-5  Cs-ZSM-5 while the permeation flux of the m- and o-xylene decreased in the order Na-ZSM-5 > Sr-ZSM-5 > Ba-ZSM-5 > Cs-ZSM-5. The membrane that exhibited the best performance was Ba-ZSM-5, with a maximum p/o separation factor of 8.4 and a p-xylene permeance of 0.54 × 10⁻⁷ mol s⁻¹ m⁻² Pa⁻¹ at 400 °C.     

A new technique for preparing ceramics for catalyst support exhibiting high porosity and high heat resistance

A new technique for preparing magnesia ceramics of high porosity and high temperature resistance has been developed. Spray freeze drying of magnesium sulfate aqueous solution produced fine salt particles having open pores due to sublimation of ice crystals. The particles were calcined to porous magnesium oxide and formed a green body. Highly porous magnesia was produced by firing the green body. The porous magnesia exhibited a bimodal pore size distribution of macro-pores of micron order and meso-pores smaller than 100 nm. Porosity was 87–90%. After addition of an aluminum additive with an amount 3–5 mol%, the magnesia exhibited high heat resistance; surface area was greater than 20 m² g⁻¹ after 20 h exposure in a 1573 K oven. Thus, the porous magnesia is expected to be very suitable for combustion catalyst support used in a high temperature environment.     

聚乙烯醇/牛I型胶原支架材料的制备及评价

研究了一种聚乙烯醇(PVA)和胶原(COL)复合支架材料的制备方法。采用氨基硅烷对PVA海绵表面进行了氨基化修饰后,通过戊二醛溶液交联牛Ⅰ型胶原(COL),最后通过赖氨酸溶液封闭,获得一种PVA/COL复合支架材料。采用扫描电镜(SEM)、X光电子能谱仪(XPS)、傅里叶红外光谱(FT-IR)等手段对支架材料的理化性能进行表征,并通过细胞实验对支架材料的生物学性能进行评价。结果表明,经过COL修饰的PVA孔隙率为21.33%,平均孔径为168.68 ?m且均匀分布,支架材料接触角为20.03°。对支架材料的生物学评价结果表明C3A细胞在复合材料上黏附良好,优于PVA组;CCK-8增殖检测结果表明细胞在复合材料上呈增殖生长趋势,与对照组PVA相比差异显著(P?0.01)。将PVA和COL复合制备得到的支架材料具有良好的理化及生物学特性,具有广阔的应用前景。     

Sintering characteristics of in situ formed low expansion ceramics from a powder precursor in the form of hydroxy hydrogel

Lithium aluminosilicate powder precursors of compositions Li₂O:Al₂O₃:SiO₂ as 1:1:2; 1:1:2.5 and 1:1:3 were prepared in the hydroxy hydrogel form by wet interaction technique in aqueous medium followed by sintering for ultimate synthesis of low expansion ceramics. Phases formed in the sintered specimens were analyzed by XRD technique. Thermal expansion of the specimens sintered at 1100, 1200 and 1300 °C were also measured. It was found that β-spodumene, lithium aluminum oxide and silica were the predominat phases in all the specimens. Sintering was optimum up to 1200 °C beyond which no further noticeable shrinkage was observed. The sintered specimens remained highly porous even after firing at 1300 °C, whose bulk density and apparent porosity were in the range of 1.25–1.42 g/cm³ and 43–48%, respectively. Thermal expansion characteristics and density of the sintered specimens were found to be primarily related to the composition of the phases formed during sintering. A porous low expansion ceramic monolith could be prepared using the present technique.