Preparation and characterization of polycaprolactone/forsterite nanocomposite porous scaffolds designed for bone tissue regeneration

Biocomposite scaffolds made from polymers and bioceramics can provide the mechanical structure necessary for osteoinductivity in the growth of new bone. The aim of this research was to investigate the properties of a novel nanocomposite scaffold made from a combination of polycaprolactone (PCL) and forsterite nanopowder which could find use in bone tissue engineering applications. The scaffold itself was fabricated by a method of solvent casting and particle leaching. The effect of forsterite content on the mechanical properties, bioactivity, biodegradability, and cytotoxicity of the scaffolds was investigated. Significant improvement in the mechanical properties was observed in the nanocomposite scaffolds as compared to that seen in the pure PCL scaffolds. Bioactivity was also observed in the nanocomposite scaffolds, a trait which was not present in the pure PCL scaffolds. Biodegradation assay indicated that the addition of forsterite nanopowder could modulate the degradation rate of PCL. In vitro tests of cytotoxicity and osteoblast proliferation showed that the nanocomposite scaffolds were non-cytotoxic, thereby allowing cells to adhere, grow, and proliferate on the surface of these scaffolds. The results obtained in this experiment suggest that the combination of PCL with forsterite nanopowder can be used to form scaffolds suitable for use in bone tissue engineering. The exact material behavior required can be adjusted through variation of the ratio between PCL and forsterite nanopowder used to form the scaffold.     

骨组织工程多孔支架材料性质及制备技术

多孔性生物可降解支架的选择和制备是组织工程技术成功运用的关键。从骨架的材料要求、常用的骨架材料、骨架的制备技术等几个方面对组织工程和生物降解支架的研究进行了综述 ,并对该研究的前景进行了展望     

Characterization and enhancement of chondrogenesis in porous hyaluronic acid-modified scaffolds made of PLGA(75/25) blended with PEI-grafted PLGA(50/50)

Hyaluronic acid (HA) improves the quality of microfracture-mediated cartilage regeneration by recruiting bone marrow mesenchymal stem cells (BMSCs) and chondrocytes. An HA-enriched scaffold was investigated to enhance chondrogenesis by BMSCs and chondrocytes in articular cartilage tissue engineering with the microfracture technique. Pre-fabricated porous PGP2/1 [poly(d,l-lactic acid-co-glycolic acid)(75/25) blended with polyethylenimine-grafted-poly(d,l-lactic acid-co-glycolic acid)(50/50) in a 2:1 ratio] scaffolds with 72.7% porosity and a 200–400-μm pore size were generated via the gas foaming/salt leaching method. HA-modified porous PGP2/1 (HA-PGP) scaffolds were used as the HA-enriched microenvironment. The mRNA levels of chondrogenic marker genes (SOX-9, aggrecan and type II collagen) were quantified using real-time polymerase chain reaction (PCR). Sulfated glycosaminoglycan (sGAG) deposition was detected by Alcian blue staining and dimethylmethylene blue (DMMB) assays. The expression of the chondrogenic genes type II collagen and aggrecan was significantly elevated in chondrocytes and BMSCs grown on HA-PGP scaffolds after seven days of culturing. BMSCs cultured in HA-PGP scaffolds showed increased sGAG content after four weeks of culturing. These results demonstrate that HA-PGP scaffolds provide a microenvironment that induces chondrogenesis by chondrocytes and BMSCs, which may be beneficial for regenerating cartilage-like tissue in vivo with the microfracture technique.     

In vivo study of porous strontium-doped calcium polyphosphate scaffolds for bone substitute applications

The purpose of this study was to investigate in vivo biocompatibility and osteogenesis as well as degradability of the porous strontium-doped calcium polyphosphate (SCPP) scaffolds as a biomaterial for bone substitute applications. The evaluation was performed on a rabbit model over a period of 16 weeks by histology combined with image analysis, X-ray microradiography and immunohistochemistry methods. The histological and X-ray microradiographic results showed that the SCPP scaffold exhibited good biocompatibility and extensive osteoconductivity with host bone. Moreover, a significant more bone formation was observed in the SCPP group compared with that in the CPP group, especially at the initial stage after implantation. New bone volumes (NBVs) of the SCPP group determined at week 4, 8 and 16 were 14, 27 and 45%, respectively. Accordingly, NBVs of the CPP group were 10, 19 and 40%. Immunohistochemical results revealed that both the expression of collagen type I and bone morphogenetic proteins in the SCPP group were higher than that in the CPP group, which might be associated with the release of strontium ions during the implantation. In addition, during 16 weeks implantation the SCPP scaffold exhibited similar degradability with the CPP scaffold in vivo. Both scaffolds showed the greatest degradation rate for the first 4 weeks, and then the degradation rate gradually decreased. The results presented in this study demonstrated that SCPP scaffold can be considered as a biocompatible material, making it attractive for bone substitute application purposes.     

Effects of surfactants on the microstructure of porous ceramic scaffolds fabricated by foaming for bone tissue engineering

A porous scaffold comprising a β-tricalcium phosphate matrix and bioactive glass powders was fabricated by foaming method and the effects of surfactants as foaming agent on microstructure of scaffolds were investigated. Foaming capacity and foam stability of different surfactants in water firstly were carried out to evaluate their foam properties. The porous structure and pore size distribution of the scaffolds were systematically characterized by scanning electron microscopy (SEM) and an optical microscopy connected to an image analyzer. The results showed that the foam stability of surfactant has more remarkable influence on their microstructure such as pore shape, size and interconnectivity than the foaming ability of one. Porous scaffolds fabricated using nonionic surfactant Tween 80 with large foam stability exhibited higher open and total porosities, and fully interconnected porous structure with a pore size of 750-850 μm.     

Processing and characterization of porous alumina scaffolds

Bioceramic materials are used for the reconstruction or replacement of the damaged parts of the human body. In this study an improved procedure is described for producing ceramic scaffolds with controlled porosity. Bioinert alumina ceramic was used to make porous scaffolds by using indirect fused deposition modeling (FDM), a commercially available rapid prototyping (RP) technique. Porous alumina samples were coated with hydroxyapatite (HAp) to increase the biocompatibility of the scaffolds. Initial biological responses of the porous alumina scaffolds were assessed in vitro using rat pituitary tumor cells (PR1). Both porous alumina and HAp coated alumina ceramics provided favorable sites for cell attachments in a physiological solution at 37 °C, which suggests that these materials would promote good bonding while used as bone implants in vivo. Based on these preliminary studies, similar tests were performed with human osteosarcoma cells. Cell proliferation studies show that both the ceramic materials can potentially provide a non-toxic surface for bone bonding when implanted in vivo.     

Preparation and characterization of bimodal porous poly(γ-benzyl-L-glutamate) scaffolds for bone tissue engineering

An ideal scaffold in bone tissue-engineering strategy should provide biomimetic extracellular matrix-like architecture and biological properties. Poly(γ-benzyl-L-glutamate) (PBLG) has been a popular model polypeptide for various potential biomedical applications due to its good biocompatibility and biodegradability. This study developed novel bimodal porous PBLG polypeptide scaffolds via a combination of biotemplating method and in situ ring-opening polymerization of γ-benzyl-L-gIutamate N-carboxyanhydride (BLG-NCA). The PBLG scaffolds were characterized by proton nuclear magnetic resonance spectroscopy, X-ray diffraction, differential scanning calorimetry, scanning electron microscope (SEM) and mechanical test. The results showed that the semi-crystalline PBLG scaffolds exhibited an anisotropic porous structure composed of honeycomb-like channels (100–200 μm in diameter) and micropores (5–20 μm), with a very high porosity of 97.4 ± 1.6%. The compressive modulus and glass transition temperature were 402.8 ± 20.6 kPa and 20.2 °C, respectively. The in vitro biocompatibility evaluation with MC3T3-E1 cells using SEM, fluorescent staining and MTT assay revealed that the PBLG scaffolds had good biocompatibility and favored cell attachment, spread and proliferation. Therefore, the bimodal porous polypeptide scaffolds are promising for bone tissue engineering.     

Fabrication and characterization of porous calcium polyphosphate scaffolds

Porous calcium polyphosphate (CPP) scaffolds with different polymerization degree and crystalline phases were prepared, and then analyzed by scanning electron microscopy (SEM), Thermmogravimetry (TG) and X-ray diffraction (XRD). Number average polymerization degree was calculated by analyzing the calcining process of raw material Ca(H₂PO₄)₂, as a polycondensation reaction. Amorphous CPP were prepared by the quenching from the melt of Ca(H₂PO₄)₂ after calcining, and CPP with different polymerization degree was prepared by controlling the calcining time. Meanwhile, CPP with the same polymerization degree was prepared to amorphous or different crystalline phases CPP which was made from crystallization of amorphous CPP. In vitro degradation studies using 0.1 M of tris-buffered solution were performed to assess the effect of polymerization degree or crystalline phases on mechanical properties and weight loss of the samples. With the increase of polymerization degree, the weight loss during the degradation decreased, contrarily the strength of CPP increased. The degradation velocity of amorphous CPP, α-CPP, β-CPP and γ-CPP with the same polymerization degree decreased in turn at the same period. The full weight loss period of CPP can be controlled between 17 days and more than 1 year. The results of this study suggest that CPP ceramics have potential applications for bone tissue engineering.     

Preparation and characterization of 3D porous ceramic scaffolds based on portland cement for bone tissue engineering

There is a constant need for bone substitutes. This work was focused on developing a porous substrate based on Portland cement with air-voids introduced by outgassing reaction product from lime and aluminum powder. The structures were obtained through two routes of raw-materials and processing. Water absorption and compressive strength measurements and scanning electron microscopy, X-ray diffraction, and Fourier Transformed Infrared Spectroscopy assays were conducted in order to characterize the porous substrates. The substrates have shown pore size structure compatible with bone tissue colonization. Also, the mechanical strength exhibited by the scaffolds fall in the normal ranges for trabecular bone. These characteristics indicate potential use of the developed porous scaffold for bone tissue engineering which was endorsed by in vitro experiments via cell culture.     

半乳糖修饰大孔壳聚糖支架的制备及表征

生物人工肝反应器中肝细胞的数量和功能是能否有效替代已衰竭肝脏功能的关键,与此紧密相关的是细胞支架材料结构和性能的优化.采用冷冻干燥法,制备了大孔壳聚糖支架,孔隙率在90%以上,平均孔径在100～200μm之间.以肝细胞表面去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)的特异性配体-半乳糖,对材料表面进行糖基化修饰,制备了半乳糖基修饰的大孔壳聚糖支架,肝细胞在其上生长状况良好,细胞培养密度高,为高密度培养肝细胞提供了一种性能优良的支架材料.     

The effects of frequency-dependent attenuation and dispersion on sound speed measurements: applications in human trabecular bone

Sound speed may be measured by comparing the transit time of a broadband ultrasonic pulse transmitted through an object with that transmitted through a reference water path. If the speed of sound in water and the thickness of the sample are known, the speed of sound in the object may be computed. To measure the transit time differential, a marker such as a zero-crossing, may be used. A sound speed difference between the object and water shifts all markers backward or forward. Frequency-dependent attenuation and dispersion may alter the spectral characteristics of the waveform, thereby distorting the locations of markers and introducing variations in sound-speed estimates. Theory is derived to correct for this distortion for Gaussian pulses propagating through linearly attenuating, weakly dispersive media. The theory is validated using numerical analysis, measurements on a tissue mimicking phantom, and on 24 human calcaneus samples in vitro. Variations in soft tissue-like media are generally not exceptionally large for most applications but can be substantial, particularly for high bandwidth pulses propagating through media with high attenuation coefficients. At 500 kHz, variations in velocity estimates in bone can be very substantial, on the order of 40 to 50 m/s because of the high attenuation coefficient of bone. In trabecular bone, the effects of frequency-dependent attenuation are considerable, and the effects of dispersion are negligible.     

Processing and characterization of innovative scaffolds for bone tissue engineering

A new protocol, based on a modified replication method, is proposed to obtain bioactive glass scaffolds. The main feature of these samples, named "shell scaffolds", is their external surface that, like a compact and porous shell, provides both high permeability to fluids and mechanical support. In this work, two different scaffolds were prepared using the following slurry components: 59 % water, 29 % 45S5 Bioglass(?) and 12 % polyvinylic binder and 51 % water, 34 % 45S5 Bioglass(?), 10 % polyvinylic binder and 5 % polyethylene. All the proposed samples were characterized by a widespread microporosity and an interconnected macroporosity, with a total porosity of 80 % vol. After immersion in a simulated body fluid (SBF), the scaffolds showed strong ability to develop hydroxyapatite, enhanced by the high specific surface of the porous systems. Moreover preliminary biological evaluations suggested a promising role of the shell scaffolds for applications in bone tissue regeneration. As regards the mechanical behaviour, the shell scaffolds could be easily handled without damages, due to their resistant external surface. More specifically, they possessed suitable mechanical properties for bone regeneration, as proved by compression tests performed before and after immersion in SBF.     

Preparation and characterisation of poly(lactide-co-glycolide) (PLGA) and PLGA/Bioglass® composite tubular foam scaffolds for tissue engineering applications

Polylactide-co-glycolide (PLGA) and PLGA/Bioglass® foams of tubular shape have been prepared with a 1 wt.% 45S5 Bioglass® content. Porous membranes with varying thickness and porosity were fabricated via a thermally induced phase separation process, from which tubes of controlled diameter and wall thickness in the range 1.5–3 mm were produced. Scanning electron microscopy (SEM) revealed that the structure of the tubular foams consisted of radially oriented and highly interconnected pores with two distinct pore sizes, i.e. macropores ∼100-μm average diameter and interconnected micropores of 10–50-μm diameter. Foams with Bioglass® inclusions showed similarly well-defined tubular and interconnected pore morphology. Cell culture studies using mouse fibroblasts (L929) were conducted to assess the biocompatibility of the scaffolds in vitro. L929 fibroblasts cultured in medium that was pre-conditioned by incubating with PLGA tubes containing Bioglass® had a significant reduction in cell proliferation compared with fibroblasts grown in unconditioned medium (p<0.0001).The PLGA and PLGA/Bioglass® tubular foams developed here are candidate materials for soft-tissue engineering scaffolds, holding promise for the regeneration of tissues requiring a tubular shape scaffold, such as intestine, trachea and blood vessels.     

Characterization of poly(3-hydroxybutyrate)/nano-hydroxyapatite composite scaffolds fabricated without the use of organic solvents for bone tissue engineering applications

Poly(3-hydroxybutyrate)/nano-hydroxyapatite (PHB/nHA) composite scaffolds were fabricated without the use of organic solvents at different mass fractions of HA nanoparticles. HA nanoparticles were homogeneously dispersed as primary particles in the polymer matrix of the scaffolds at 10 and 15 wt.% nHA content. Agglomeration of HA nanoparticles occurred when the nHA content of the scaffolds reached 20 wt.%. All the scaffolds had high porosities with interconnected porous structure and optimized pore size ranges. Mechanical properties of all the scaffolds were in the range of mechanical properties of cancellous bone. Scaffolds were biocompatible to MG-63 cells in the indirect method of cytotoxicity evaluation. Also, the morphology of the attached MG-63 cells in direct contact with the scaffolds indicated the appropriate cell-scaffold interaction. Thus, the PHB/nHA composite scaffolds investigated in this study tend to be favorable for bone tissue engineering applications.     

Highly porous and interconnected starch-based scaffolds: Production,characterization and surface modification

A convenient and straightforward process for preparation of highly porous and interconnected fiber mesh scaffolds with 50 wt.% content of starch is described. The proposed methodology avoids some of the previous encountered problems associated with the processing of starch-based materials such as thermal degradation, starch entrapment in the material bulk and inability to control/minimise the thickness of the fibers obtained by melt spinning, or low porosity and lack of interconnectivity for the scaffolds obtained by extrusion or injection moulding with blowing agent. Topographical characterisation of the obtained fibers revealed rough surface commonly related with increased cell attachment and growth. The in vitro tests with osteoblast cell line confirmed this trend and we observed higher cell number with increasing of the culture time. These results were also associated with protein adsorption from a complex solution where predominant adsorption of vitronectin over fibronectin was detected. Finally, a model modification by plasma was also carried out in order to confirm the versatility of these scaffolds by the possibility to further upgrade them via surface functionalisation. The in vitro tests confirmed that osteoblast-like cells proliferate faster on the modified scaffolds, which allows shortening the time needed for culturing prior to implantation.     

羟基磷灰石基多孔复合支架的制备及其表征

研究利用造孔剂法制备高度贯通的多孔羟基磷灰石(HA)支架,孔隙率约为78%,并利用聚己内酯(PCL)分别复合纳米HA(nHA)或微纳米生物玻璃(nBG)粉末对其进行涂覆改性,粉末的添加量均为10%~40%(质量分数)。4种类型支架分别记为HA、PCL/HA、nHA-PCL/HA和nBG-PCL/HA。实验结果发现,nHA-PCL/HA和nBG-PCL/HA复合支架最大抗压强度分别为1.41~1.98 MPa和1.35~1.78MPa。4类支架矿化实验显示,浸泡21d后nBG-PCL/HA表面促进生成较多的磷灰石矿化物;细胞实验结果显示细胞在4类支架上均生长良好,说明支架具有良好的生物相容性。支架在实验犬背部肌肉组织内植入2个月的组织学检测显示,4种支架内均有新骨形成,尤其是nHA-PCL/HA和nBG-PCL/HA孔内有更多的新生骨组织,说明这两种支架表面复合涂层中的生物活性纳米颗粒对诱导新骨生成具有积极的促进作用。     

Fabrication and characterization of novel hydroxyapatite/porous carbon composite scaffolds

Novel hydroxyapatite (HA)/porous carbon composite scaffolds were prepared by applying sonoelectrodeposition and a subsequent hydrothermal treatment to previous carbonized phenolic resin coated polyurethane sponges. The interconnected pore network and morphology of HA/porous carbon composite scaffolds were determined by scanning electron microscope (SEM), and the whole surface of porous carbons were evenly coated with the deposited HA layer which was confirmed by EDS and XRD. The porosity (83.5 ± 0.3%) and the bulk density (0.297 ± 0.009 g·cm⁻³) of HA/porous carbon scaffolds were detected by the Archimedes method. The compressive and flexural strength of the scaffolds is 1.187 ± 0.064 MPa and 0.607 ± 0.268 MPa, respectively. Compared with the polymeric surface of 24-well cell culture plates, these novel scaffolds significantly promote the proliferation of human osteoblast-like MG-63 cells, indicating that this novel HA/porous carbon composite scaffold could be used for in vitro 3D culture of osteoblasts.     

一种新型仿生骨组织工程支架的制备与表征

利用改性生物玻璃粉体和胶原、透明质酸钠、磷酸丝氨酸等天然生物分子复合制备仿生型三维多孔骨组织工程支架材料,利用体外模拟实验结合SEM、FTIR、XRD 等测试方法对材料的显微结构、生物矿化性能进行了综合研究,研究表明该材料具有良好的孔隙结构,在模拟生理溶液(SBF)中反应24h即可在支架表面形成碳酸羟基磷灰石(HCA).     

In situ synthesis and characterization of porous polymer-ceramic composites as scaffolds for gene delivery

The use of three-dimensional scaffolds in gene delivery has emerged as a popular and necessary delivery vehicle for obtaining controlled gene delivery. In this report, techniques to synthesize composite scaffolds by combining natural polymers such as agarose and alginate with calcium phosphate (CaP) are described. The incorporation of CaP into the agarose or alginate hydrogels was performed in situ and the presence of CaP was confirmed by X-ray diffraction (XRD). The crystallite size of the CaP particles was determined to be 7.20 nm. Lyophilized, porous composites were examined under scanning electron microscopy (SEM) to estimate the size of the pores, an essential requirement for an ideal scaffold. The swelling properties of the composite samples were also investigated to study the effect of CaP incorporation on the behavior of the hydrogels. By incorporating CaP into the hydrogel, the aim is to synthesize a scaffold that is mechanically strong and chemically suitable for use as a gene delivery vehicle in tissue engineering.     

Fabrication and characterization of chitosan microspheres agglomerated scaffolds for bone tissue engineering

In the presented paper authors describe a method for bone scaffolds fabrication. The technique is based on the agglomeration of chitosan microspheres. The fabrication process is complex and consists of a few steps: chitosan spheres extrusion, scaffold formation by compression followed by the spheres agglomeration and bonding with cross-linking agent (STPP, sodium tripolyphosphate). The described method allows manufacturing of porous materials with controllable shape, pore size distribution and their interconnectivity. In this technique 3D scaffold porosity can be regulated by altering spheres diameter. Authors studied influence of cross-linker concentrations and time of cross-linking process on the scaffold morphology, mechanical properties, enzymatic degradation rate (in the presence of lysozyme) and human osteoblasts response. Surface morphology and topography were evaluated by SEM. Porosity and pore interconnectivity were observed via μCT scanning. Mechanical tests showed that chitosan scaffolds perform compression characteristic (Young Modulus) similar to natural bone. Cytotoxicity established by XTT assay confirmed that most of the developed composite materials do not show toxic properties. Osteoblast adhesion and morphology were analyzed by SEM and optical microscopy.