Compressive mechanical properties and biodegradability of porous poly(caprolactone)/chitosan scaffolds

Porous polycaprolactone(PCL)/chitosan(CH) scaffolds with large pore sizes and high porosities were fabricated via a particle-leaching technique using hexafluoro-2-propanol as a shared solvent and salt (sodium chloride) particles as porogen. By optimizing processing conditions, numerous PCL/CH scaffolds with CH proportion lower than 50 wt% and similar pore parameters were built. These scaffolds were further evaluated for their compressive mechanical properties and biodegradation behaviors. It was found that their compressive modulus and stress at 10% strain were basically maintained in their dry state in contrast to their individual components, and these scaffolds still showed well-defined compressive characteristics and dimension stability even in their hydrated state compared with pure chitosan scaffolds. After being exposed to PBS or enzymatic degradation systems in vitro for various periods up to 10 weeks, it was observed that degradation of the PCL component could be accelerated at various rates depending on the compositions of the scaffolds and the media, and the chiosan component could effectively buffer the acidic degradation products of the PCL component.     

Controlling the Pore Sizes and Related Properties of Inverse Opal Scaffolds for Tissue Engineering Applications

Inverse opal scaffolds are finding widespread use in tissue engineering and regenerative medicine. Herein, the way in which the pore sizes and related physical properties of poly(D ,L ‐lactide‐co‐glycolide) inverse opal scaffolds are affected by the fabrication conditions is systematically investigated. It is found that the window size of an inverse opal scaffold is mainly determined by the annealing temperature rather than the duration of time, and the surface pore size is largely determined by the concentration of the infiltration solution. Although scaffolds with larger pore or window sizes facilitate faster migration of cells, they show slightly lower compressive moduli than scaffolds with smaller pore or window sizes.     

Fabrication and Properties of Porous Collagen/Hyaluronic Composite Scaffolds Containing Nano-Bioactive Glass for Tissue Engineering

In this work, nano-structured scaffolds were designed for tissue engineering using collagen, hyaluronic acid (HA) and nano-bioactive glass (NBAG) as their main components. The scaffold was prepared via freeze-drying method and the properties including morphology, porosity, compressive strength, swelling ratio and cytotoxicity in-vitro, were also evaluated. The composite scaffolds showed well interconnected macropores with the pore size of ranging from 100 to 500 μm. The porosity percent and swelling ability were decreased with the introduction of NBAG into the collagen/HA hydrogel; however, the compressive strength was enhanced. The cytotoxicity in-vitro study shows that the collagen-HA/NBAG scaffolds have good biocompatibility with improving effect on fibroblastic cells growth. It could be concluded that this scaffold fulfills the main requirements to be considered as a bone substitute.     

Polyester scaffolds with bimodal pore size distribution for tissue engineering

This paper presents a method for the preparation of porous poly(L-lactide)/poly[(L-lactide)-co-glycolide] scaffolds for tissue engineering. Scaffolds were prepared by a mold pressing-salt leaching technique from structured microparticles. The total porosity was in the range 70-85%. The pore size distribution was bimodal. Large pores, susceptible for osteoblasts growth and proliferation had the dimensions 50-400 microm. Small pores, dedicated to the diffusion of nutrients or/and metabolites of bone forming cells, as well as the products of hydrolysis of polyesters from the walls of the scaffold, had sizes in the range 2 nm-5 microm. The scaffolds had good mechanical strength (compressive modulus equal to 41 MPa and a strength of 1.64 MPa for 74% porosity). Scaffolds were tested in vitro with human osteoblast-like cells (MG-63). It was found that the viability of cells seeded within the scaffolds obtained using the mold pressing-salt leaching technique from structured microparticles was better when compared to cells cultured in scaffolds obtained by traditional methods. After 34 d of culture, cells within the tested scaffolds were organized in a tissue-like structure. Photos of section of macro- and mesoporous PLLA/PLGA scaffold containing 50 wt.-% of PLGA microspheres after 34 d of culture. Dark spots mark MG-63 cells, white areas belong to the scaffold. The specimen was stained with haematoxylin/eosin. Bar = 100 microm.     

Preparation and mechanical properties of poly(chitosan‐g‐DL‐lactic acid) fibrous mesh scaffolds

DL ‐lactic acid was grafted onto chitosan to produce poly(chitosan‐g‐DL ‐lactic acid)(PCLA) without using a catalyst. These PCLAs were then spun into filaments and further fabricated into fibrous mesh scaffolds using an improved wet‐spinning technique. The diameter of filaments in different scaffolds could vary from a few micrometers to several tens of micrometers. The scaffolds exhibited various pore sizes ranging from about 20 µm to more than 200 µm and different porosities up to 80%. The several main processing conditions were optimized for obtaining the desired scaffolds with well‐controlled structures. The tensile and compressive mechanical properties of the mesh scaffolds in both dry and hydrated states were mainly examined. Significantly improved tensile strength and modulus, enhanced compressive modulus, and stress as well as the dimensional stability for these mesh scaffolds in their hydrated state were observed. Copyright © 2007 John Wiley & Sons, Ltd.     

Evaluation of 3D Printed Gelatin‐Based Scaffolds with Varying Pore Size for MSC‐Based Adipose Tissue Engineering

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion‐based 3D printing is used to fabricate photo‐crosslinkable gelatin‐based scaffolds with pore sizes in the range of 200–600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC‐based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.     

Fabrication of poly(lactide‐co‐glycolide) scaffold embedded spatially with hydroxyapatite particles on pore walls for bone tissue engineering

Poly(lactide‐co‐glycolide) (PLGA) scaffolds embedded spatially with hydroxyapatite (HA) particles on the pore walls (PLGA/HA‐S) were fabricated by using HA‐coated paraffin spheres as porogens, which were prepared by Pickering emulsion. For comparisons, PLGA scaffolds loaded with same amount of HA particles (2%) in the matrix (PLGA/HA‐M) and pure PLGA scaffolds were prepared by using pure paraffin spheres as porogens. Although the three types of scaffolds had same pore size (450–600 µm) and similar porosity (90%–93%), the PLGA/HA‐S showed the highest compression modulus. The embedment of the HA particles on the pore walls endow the PLGA/HA‐S scaffold with a stronger ability of protein adsorption and mineralization as well as a larger mechanical strength against compression. In vitro culture of rat bone marrow stem cells revealed that cell morphology and proliferation ability were similar on all the scaffolds. However, the alkaline phosphatase activity was significantly improved for the cells cultured on the PLGA/HA‐S scaffolds. Therefore, the method for fabricating scaffolds with spatially embedded nanoparticles provides a new way to obtain the bioactive scaffolds for tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd.     

3D Plotted PCL Scaffolds for Stem Cell Based Bone Tissue Engineering

The ability to control the architecture and strength of a bone tissue engineering scaffold is critical to achieve a harmony between the scaffold and the host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to satisfy this need and to create a scaffold directly from the scanned and digitized image of the defect site. Design and construction of complex structures with different shapes and sizes, at micro and macro scale, with fully interconnected pore structure and appropriate mechanical properties are possible by using RP techniques. In this study, RP was used for the production of poly(ε-caprolactone) (PCL) scaffolds. Scaffolds with four different architectures were produced by using different configurations of the fibers (basic, basic-offset, crossed and crossed-offset) within the architecture of the scaffold. The structure of the prepared scaffolds were examined by scanning electron microscopy (SEM), porosity and its distribution were analyzed by micro-computed tomography (µ-CT), stiffness and modulus values were determined by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very ordered structures with mean porosities about 60%, and having storage modulus values about 1 × 10⁷ Pa. These structures were then seeded with rat bone marrow origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold structure on the cell behavior; the proliferation and differentiation of the cells on the scaffolds were studied. It was observed that cell proliferation was higher on offset scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and stainings for actin filaments of the cells reveal successful attachment and spreading at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher for the samples with lower cell proliferation, as expected. Highest MSC differentiation was observed for crossed scaffolds indicating the influence of scaffold structure on cellular activities.     

Preparation and characterization of new materials based on silk fibroin,chitosan and nanohydroxyapatite

Abstract

In this paper, a series of porous nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds were successfully prepared using the freeze-drying method. The biomaterials were characterized by attenuated total reflection Fourier transform infrared spectroscopy, and mechanical testing and thermogravimetric analysis. Moreover, studies of porosity, pore size, swelling properties and in vitro degradation test were performed. Research has proved that micro-structure, porosity, water adsorption and compressive strength were greatly affected by the components’ concentration, in particular the content of silk fibroin. SEM observations showed that the scaffolds of nHA/SF/CTS are highly porous, with pore size in wide range from 25 to 300?µm which is suitable for cell growth. nHA/SF/CTS scaffolds have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both, the compressive modulus and compressive strength of the scaffold, decrease with the increase in silk fibroin content.     

Properties of Calcium Phosphate Scaffolds Produced by Freeze-Casting

The pore structure of three-dimensional scaffolds applied in tissue engineering may influence the mechanical properties and cellular activity. As the optimal pore size is dependent on the specifics of the biomaterial or tissue engineering application, the ability to alter the pore size over a wide range is necessary for several scaffolds in order to meets the requirements of the applications. The aim of this study is to develop methodologies to produce calcium phosphate scaffolds with acceptable pore size and defined pore-channel interconnectivity. The pore size of calcium phosphate scaffolds is established during the freeze-drying fabrication process. In this process, material suspension is simply frozen and then dried by freeze-drier, which able to produce material with unique porous architectures, where the porosity is almost a direct replica of the frozen solvent crystals. There are two different method of freeze-casting carried out in order to study the effect of freezing temperature by which in the first method; sample being soaked with liquid nitrogen (-196 °C) for about 10 minutes before been place inside a freezer (-40 °C). In the second method, the sample was directly placed inside a freezer for casting at temperature of -40 ̊C. The results show that the pore size of the scaffolds decreased as the freezing temperature was reduced. Taken together, these results demonstrate that the methodologies applied in this study can be used to produce a range of calcium phosphate scaffolds exhibiting better compressive strength, approximately 665-875 KPa for 54-64.3% of porosity with mean pore size from 102-113 μm. The methods developed in this study provide a basis for the investigation on the effects of different freezing temperature in freeze-casting process on the porosity, morphology, and compressive properties of the calcium phosphate scaffolds.     

Preparation of porous nanocomposite scaffolds with honeycomb monolith structure by one phase solution freezedrying method

Biodegradable porous nanocomposite scaffolds of poly（lactide-co-glycolide）（PLGA） and L-lactic acid（LAc） oligomer surface-grafted hydroxyapatite nanoparticles（op-HA） with a honeycomb monolith structure were fabricated with the single-phase solution freeze-drying method.The effects of different freezing temperatures on the properties of the scaffolds,such as microstructures,compressive strength,cell penetration and cell proliferation were studied.The highly porous and well interconnected scaffolds with a tunable pore structure were obtained.The effect of different freezing temperature（4℃,-20℃,-80℃and -196℃） was investigated in relation to the scaffold morphology,the porosity varied from 91.2%to 83.0%and the average pore diameter varied from（167.2±62.6）μm to（11.9±4.2）μm while theσ₁₀ increased significantly.The cell proliferation were decreased and associated with the above-mentioned properties.Uniform distribution of op-HA particles and homogeneous roughness of pore wall surfaces were found in the 4℃frozen scaffold.The 4℃frozen scaffold exhibited better cell penetration and increased cell proliferation because of its larger pore size,higher porosity and interconnection.The microstructures described here provide a new approach for the design and fabrication of op-HA/PLGA based scaffold materials with potentially broad applicability for replacement of bone defects.     

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.     

Chitosan‐g‐polycaprolactone copolymer fibrous mesh scaffolds and their related properties

Chitosan‐g‐polycaprolactone copolymers (CPCs) with desired composition proportions were synthesized by carefully controlling the weight ratio of polycaprolactone side chains changing approximately between 45 and 48 wt% so that the obtained CPCs could be further processed via different processing techniques. Aqueous acetic acid solutions and dimethyl sulfoxide were respectively employed as solvents to fabricate CPCs into fibrous mesh scaffolds that had nearly similar parameters characterized by the average porosity and pore‐size of scaffolds as well as the average diameter of filaments under optimal processing conditions. The swelling index, surface group analysis, antibacterial activity and tensile mechanical properties of these mesh scaffolds were investigated in several ways, and the scaffolds showed quite different properties due to the different processing methods employed, although the same type of CPC was used. Copyright © 2008 John Wiley & Sons, Ltd.     

Microstructure and mechanical properties of bacterial cellulose/chitosan porous scaffold

A family of polysaccharide based scaffold materials, bacterial cellulose/chitosan (BC/CTS) porous scaffolds with various weight ratios (from 20/80 to 60/40 w/w%) were prepared by freezing (−30 and −80 °C) and lyophilization of a mixture of microfibrillated BC suspension and chitosan solution. The microfibrillated BC (MFC) was subjected to 2,2,6,6-tetramethylpyperidine-1-oxyl radical (TEMPO)-mediated oxidation to introduce surface carboxyl groups before mixing. The integration of MFC within chitosan matrix was performed by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-mediated cross-linking. The covalent amide bond formation was determined by ATR-FTIR. Because of this covalent coupling, the scaffolds retain their original shapes during autoclave sterilization. The composite scaffolds are three-dimensional open pore microstructure with pore size ranging from 120 to 280 μm. The freezing temperature and mean pore size take less effect on scaffold mechanical properties. The compressive modulus and strength increased with increase in MFC content. The results show that the scaffolds of higher MFC content contribute to overall better mechanical properties.     

Evaluation of Chitosan/Biphasic Calcium Phosphate Scaffolds for Maxillofacial Bone Tissue Engineering

Porous, 3D chitosan/biphasic calcium phosphate (BCP) scaffolds were used to prepare tissue engineering constructs for maxillofacial bone tissue reconstruction. Mesenchymal stem cells (MSC's) were seeded and cultured on clinically relevant sized scaffolds. In vitro engineered constructs facilitated the healing of mandibular defects in pigs if accompanied with delivery of basic fibroblast growth factor (bFGF).     

Effects of Laminin For Osteogenesis in Porous Hydroxyapatite

Summery: As a tooth is composed of hard tissue covering pulp, it may be suitable for tooth regeneration to use porous cylindrical hydroxyapatite (HA) scaffolds with a hollow center. Generally, in vivo examination, bone marrow cell suspension for osteogenesis in cell/HA composite scaffold without subculture is prepared at a density of 1 × 10⁷ cells/ml or higher. In dentistry, stem cells would be obtained from tooth pulp. For dentine formation, a smaller number of stem cells must be used. In this study, a suspension of rat bone marrow cells at 1 × 10⁶ cells/ml of density was prepared to estimate the adhesive effect of laminin. After immersion of HA scaffold in laminin solution, bone marrow cells were seeded in the pores of the HA scaffolds by immersion in the cell suspension for preparing the cell/HA composite scaffolds. The specimens were respectively implanted in the dorsal subcutis of 7-week-old male Fischer 344 rats for 4 weeks for histological examination. Comparing with the results of in vivo examination, alkaline phosphatase activity of bone marrow cells on laminin-coated plate with and without dexamethasone cultured for 2 weeks was measured in vitro. It was considered that laminin contributed to bone formation in pores of a scaffold.     

Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.     

Nano‐hydroxyapatite/polyamide66 composite tissue‐engineering scaffolds with anisotropy in morphology and mechanical behaviors

Based on a biomimetic conception, nano‐hydroxyapatite (n‐HA)/polyamide66 (PA66) composite scaffolds were prepared with anisotropic properties both in morphology and mechanical behavior. A novel improved thermally induced phase separation (TIPS) technique was developed to generate orientation‐structured scaffolds for tissue engineering. The physiochemical, morphological, and mechanical properties of the resultant scaffolds were evaluated. According to the results, the improved TIPS method exhibited good processability and reproducibility and enabled the composite scaffolds to have a high content of inorganic fillers. The morphological study proved that the n‐HA/PA66 scaffolds exhibited unidirectional microtubular architecture with high porosity (ca. 80–85%) and an optimal pore size ranging from 200 to 500 μm. Besides, the effect of n‐HA content on the morphology of the scaffolds was studied, and the results indicated that the obtained scaffolds presented an improvement in anisotropic morphology with increase of n‐HA content. The anisotropy was also evaluated in the mechanical properties of the scaffolds, that is, the longitudinal compressive strength and modulus were ～1.5 times of the transverse ones. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 658–669, 2009     

In-Vitro Biocompatibility Evaluation of Collagen-Hyaluronic Acid/Bioactive Glass Nanocomposite Scaffold

A nano-structured scaffold was designed for bone repair using collagen, hyaluronic acid (HYA) and nano-bioactive glass (NBaG) as its main components. The collagen-HYA/NBaG scaffold was prepared by using a freeze-drying technique and characterized by scanning electron microscopy (SEM). Osteoblastls were seeded on these scaffolds and their proliferation rate, alkaline phosphatase (ALP) activity and ability to form mineralized bone nodules were compared with those osteoblasts grown on cell culture plastic surfaces. The cross-section morphology shows that the collagen-HYA/NBaG scaffold possessed a three-dimensional (3D) interconnected homogenous porous structure. The results obtained from biological assessment show that this scaffold did not negatively affect osteoblasts proliferation rate and improves osteoblasts function as shown by increasing the ALP activity and calcium deposition and formation of mineralized bone nodules. Therefore, the composite scaffolds could provide a favorable environment for initial cell adhesion, maintained cell viability and cell proliferation, and had good in-vitro biocompatibility.