Fabrication of porous poly(L-lactide) (PLLA) scaffolds for tissue engineering using liquid–liquid phase separation and freeze extraction

PLLA scaffolds were successfully fabricated using liquid–liquid phase separation with freeze extraction techniques. The effects of different processing conditions, such as method of cooling (direct quenching and pre-quenching), freezing temperature (−80°C and −196°C) and polymer concentration (3, 5 and 7 wt%) were investigated in relations to the scaffold morphology. SEM micrographs of scaffolds showed interconnected porous network with pore size ranging from 20 to 60 μm. The scaffolds had porosity values ranging from 80 to 90%. Changes to the interconnected network, porosity and pore size were observed when the method of cooling and polymer concentration was changed. Direct quenching to −80°C gave a more porous interconnected microstructure with uniform pore size compared to samples prepared using pre-quenching method. Larger pores were observed for samples quenched at −80°C compared to −196°C. Scaffolds prepared using direct quenching to −196°C had higher elastic modulus and compressive stress compared to those quenched to −80°C. The compressive elastic modulus ranged from 4 to 7 MPa and compressive stress at 10% strain was from 0.13 to 0.18 MPa.     

Freeze extrusion fabrication of 13–93 bioactive glass scaffolds for bone repair

A solid freeform fabrication technique, freeze extrusion fabrication (FEF), was investigated for the creation of three-dimensional bioactive glass (13–93) scaffolds with pre-designed porosity and pore architecture. An aqueous mixture of bioactive glass particles and polymeric additives with a paste-like consistency was extruded through a narrow nozzle, and deposited layer-by-layer in a cold environment according to a computer-aided design (CAD) file. Following sublimation of the ice in a freeze dryer, the construct was heated according to a controlled schedule to burn out the polymeric additives (below ~500°C), and to densify the glass phase at higher temperature (1 h at 700°C). The sintered scaffolds had a grid-like microstructure of interconnected pores, with a porosity of ~50%, pore width of ~300 μm, and dense glass filaments (struts) with a diameter or width of ~300 μm. The scaffolds showed an elastic response during mechanical testing in compression, with an average compressive strength of 140 MPa and an elastic modulus of 5–6 GPa, comparable to the values for human cortical bone. These bioactive glass scaffolds created by the FEF method could have potential application in the repair of load-bearing bones.     

Silk fibroin/chitosan scaffold: preparation,characterization, and culture with HepG2 cell

Tissue engineering requires the development of three-dimensional water-stable scaffolds. In this study, silk fibroin/chitosan (SFCS) scaffold was successfully prepared by freeze-drying method. The scaffold is water-stable, only swelling to a limited extent depending on its composition. Fourier Transform Infrared (FTIR) spectra and X-Ray diffraction curves confirmed the different structure of SFCS scaffolds from both chitosan and silk fibroin. The homogeneous porous structure, together with nano-scale compatibility of the two naturally derived polymers, gives rise to the controllable mechanical properties of SFCS scaffolds. By varying the composition, both the compressive modulus and compressive strength of SFCS scaffolds can be controlled. The porosity of SFCS scaffolds is above 95% when the total concentration of silk fibroin and chitosan is below 6 wt%. The pore sizes of the SFCS scaffolds range from 100 μm to 150 μm, which can be regulated by changing the total concentration. MTT assay showed that SFCS scaffolds can promote the proliferation of HepG2 cells (human hepatoma cell line) significantly. All these results make SFCS scaffold a suitable candidate for tissue engineering.     

Control of the pore architecture in three-dimensional hydroxyapatite-reinforced hydrogel scaffolds

Hydrogels (gellan or agarose) reinforced with nanocrystalline carbonated hydroxyapatite (nCHA) were prepared by the GELPOR3D technique. This simple method is characterized by compositional flexibility; it does not require expensive equipment, thermal treatment, or aggressive or toxic solvents, and yields a three-dimensional (3D) network of interconnected pores 300–900 μm in size. In addition, an interconnected porosity is generated, yielding a hierarchical porous architecture from the macro to the molecular scale. This porosity depends on both the drying/preservation technology (freeze drying or oven drying at 37 ^○C) and on the content and microstructure of the reinforcing ceramic. For freeze-dried samples, the porosities were approximately 30, 66 and below 3% for pore sizes of 600–900 μm, 100–200 μm and 50–100 nm, respectively. The pore structure depends much on the ceramic content, so that higher contents lead to the disappearance of the characteristic honeycomb structure observed in low-ceramic scaffolds and to a lower fraction of the 100–200-μm-sized pores. The nature of the hydrogel did not affect the pore size distribution but was crucial for the behavior of the scaffolds in a hydrated medium: gellan-containing scaffolds showed a higher swelling degree owing to the presence of more hydrophilic groups.     

Control of the pore architecture in three-dimensional hydroxyapatite-reinforced hydrogel scaffolds

Abstract

Hydrogels (gellan or agarose) reinforced with nanocrystalline carbonated hydroxyapatite (nCHA) were prepared by the GELPOR3D technique. This simple method is characterized by compositional flexibility; it does not require expensive equipment, thermal treatment, or aggressive or toxic solvents, and yields a three-dimensional (3D) network of interconnected pores 300–900 μm in size. In addition, an interconnected porosity is generated, yielding a hierarchical porous architecture from the macro to the molecular scale. This porosity depends on both the drying/preservation technology (freeze drying or oven drying at 37 ^○C) and on the content and microstructure of the reinforcing ceramic. For freeze-dried samples, the porosities were approximately 30, 66 and below 3% for pore sizes of 600–900 μm, 100–200 μm and 50–100 nm, respectively. The pore structure depends much on the ceramic content, so that higher contents lead to the disappearance of the characteristic honeycomb structure observed in low-ceramic scaffolds and to a lower fraction of the 100–200-μm-sized pores. The nature of the hydrogel did not affect the pore size distribution but was crucial for the behavior of the scaffolds in a hydrated medium: gellan-containing scaffolds showed a higher swelling degree owing to the presence of more hydrophilic groups.     

Proliferation and function of MC3T3-E1 cells on freeze-cast hydroxyapatite scaffolds with oriented pore architectures

Previous work by the authors showed that hydroxyapatite (HA) scaffolds with different types of oriented microstructures and a unique ‘elastic–plastic’ mechanical response could be prepared by unidirectional freezing of suspensions. The objective of the present work was to evaluate the in vitro cellular response to these freeze-cast HA scaffolds. Unidirectional scaffolds with approximately the same porosity (65–70%) but different pore architectures, described as ‘lamellar’ (pore width = 25 ± 5 μm) and ‘cellular’ (pore diameter = 100 ± 10 μm), were evaluated. Whereas both groups of scaffolds showed excellent ability to support the proliferation of MC3T3-E1 pre-osteoblastic cells on their surfaces, scaffolds with the cellular-type microstructure showed far better ability to support cell proliferation into the pores and cell function. These results indicate that freeze-cast HA scaffolds with the cellular-type microstructure have better potential for bone repair applications.     

Preparation,mechanical properties and in vitro degradability of wollastonite/tricalcium phosphate macroporous scaffolds from nanocomposite powders

A new class of scaffolds with a gain size of 200 nm was prepared from wollastonite/tricalcium phosphate (WT) nanocomposite powders (termed “nano-sintered scaffolds”) through a two-step chemical precipitation and porogen burnout techniques. For a comparison, WT scaffolds with a grain size of 2 μm were also fabricated from submicron composite powders (termed “submicron-sintered scaffolds”) under the same condition. The resultant scaffolds showed porosities between 50 ± 1.0% and 65 ± 1.0% with a pore size ranging from 100 μm to 300 μm. The WT nano-sintered scaffolds exhibited compressive strength and elastic modulus values that were about twice that of their submicron-sintered counterparts. The in vitro degradation tests demonstrated that the degradability could be regulated by the grain size of bioceramics. The decreased specific surface area of pores in the nano-sintered scaffolds led to their reduced degradation rate. The mechanical properties of the nano-sintered scaffolds exhibited less strength loss during the degradation process. The WT macroporous nano-sintered scaffolds are a promising and potential candidate for bone reconstruction applications.     

Polyelectrolyte-complex nanostructured fibrous scaffolds for tissue engineering

In the current work, polyelectrolyte complex (PEC) fibrous scaffolds for tissue engineering have been synthesized and a mechanism of their formation has been investigated. The scaffolds are synthesized using polygalacturonic acid and chitosan using the freeze drying methodology. Highly interconnected pores of sizes in the range of 5–20 µm are observed in the scaffolds. The thickness of the fibers was found to be in the range of 1–2 µm. Individual fibers have a nanogranular structure as observed using AFM imaging. In these scaffolds, PEC nanoparticles assemble together at the interface of ice crystals during freeze drying process. Further investigation shows that the freezing temperature and concentration have a remarkable effect on structure of scaffolds. Biocompatibility studies show that scaffold containing chitosan, polygalacturonic acid and hydroxyapatite promotes cell adhesion and proliferation. On the other hand, cells on scaffolds fabricated without hydroxyapatite nanoparticles showed poor adhesion.     

Preparation of porous hydroxyapatite with interconnected pore architecture

Since pore connectivity has significant effects on the biological behaviors of biomedical porous hydroxyapatite (PHA), the preparation of PHA with interconnected pore architecture is of great practical significance. In the present study, PHA with highly interconnected architecture was prepared via a simple burnout route with rod-like urea as the porogen. Microscopy and porosimetry data showed that the as-prepared PHA had open and interconnected pore structure with the average fenestration size of about 120 μm. Open pores occupied up to 98% of the total porosity. The compressive strength and modulus of the as-prepared PHA were respectively 1.3–7.6 MPa and 4.0–10.4 GPa.     

Hydroxyapatite scaffolds processed using a TBA-based freeze-gel casting/polymer sponge technique

A novel freeze-gel casting/polymer sponge technique has been introduced to fabricate porous hydroxyapatite scaffolds with controlled “designer” pore structures and improved compressive strength for bone tissue engineering applications. Tertiary-butyl alcohol (TBA) was used as a solvent in this work. The merits of each production process, freeze casting, gel casting, and polymer sponge route were characterized by the sintered microstructure and mechanical strength. A reticulated structure with large pore size of 180–360 μm, which formed on burn-out of polyurethane foam, consisted of the strut with highly interconnected, unidirectional, long pore channels (~4.5 μm in dia.) by evaporation of frozen TBA produced in freeze casting together with the dense inner walls with a few, isolated fine pores (<2 μm) by gel casting. The sintered porosity and pore size generally behaved in an opposite manner to the solid loading, i.e., a high solid loading gave low porosity and small pore size, and a thickening of the strut cross section, thus leading to higher compressive strengths.     

The influence of silica on pore diameter and distribution in PLA scaffolds produced using supercritical CO<Subscript>2</Subscript>

Macroporous polylactide (PLA) scaffolds were fabricated using a supercritical CO₂ foaming process. The addition of silica particles to the polymer matrix resulted in a significant modification in the pore size distribution exhibited by the scaffold. In the absence of silica, the scaffolds contained pores between 88 μm and 980 μm in diameter as determined using X-ray computed microtomography. The addition of silica at only 2 wt% resulted in the elimination of pores of >620 μm, with no significant influence on the total porosity of the material. This effect was attributed to the silica nucleating the formation of gas bubbles in the polymeric material. Although the addition of further silica to the scaffold resulted in a further reduction in modal pore diameter, when more than 20 wt% was added to the matrix little additional effect was noted. In addition to enabling some control over pore diameter, mineral deposition was shown to occur considerably more rapidly on the silica-modified scaffolds than on those containing no silica.     

Preparation of porous biodegradable poly(lactide-co-glycolide)/ hyaluronic acid blend scaffolds: Characterization, in vitro cells culture and degradation behaviors

A series of poly(lactide-co-glycolide) (PLGA)/ hyaluronic acid (HA) blend with different HA composition were used to fabricate scaffolds successfully. The pores of the three dimensional scaffold were prepared by particle leaching and freeze drying. The pore size was about 50–200 μ m and the porosity was about 85%. The characterizations of the scaffold, such as mechanical properties, hydrophilicity and surface morphologies were determined. Mouse 3T3 fibroblast was directly seeded on the scaffolds. The cell adhesion efficiency, cell morphology observed by scanning electron microscopy (SEM) and the degradation behavior of the blend scaffold were evaluated. In summary, the results show that the adhesion efficiency of cells on the PLGA/HA blend scaffold is higher than that on the PLGA scaffold. Moreover, the incorporation of HA in PLGA not only helps to increase the cell affinity but also tends to lead the water and nutrient into the scaffold easily.     

Fabrication and characterization of porous poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide) scaffolds using solid–liquid phase separation

Freeze-extraction, which involves phase separation principle, gave highly porous scaffolds without the time and energy consuming freeze-drying process. The presented method eliminates the problem of formation of surface skin observed in freeze-drying methods. The effects of different freezing temperature (−80 and −24°C), medium (dry ice/ethanol bath and freezer) and polymer concentrations (1, 3, and 5 wt.%) on the scaffold properties were investigated in connection with the porous morphology and physicomechanical characteristics of the final scaffolds. The FESEM micrographs showed porous PLLA scaffolds with ladder-like architecture. The size of the longitudinal pores was in the range of 20–40 μm and the scaffolds had high porosity values ranging from 90% to 98%. Variation in porosity, mechanical resistance, and degree of regularity in the spatial organization of pores were observed when polymer concentration was changed. More open scaffold architecture with enhanced pore interconnectivity was achieved when a dry ice/ethanol bath of −80°C was used. Polymer concentration played an important role in fabricating highly porous scaffolds, with ladder-like architecture only appearing at polymer concentrations of above 3 wt.%. With the freeze-extraction method used here, highly porous and interconnected poly(l-lactide) scaffolds were successfully fabricated, holding great potential for tissue engineering applications.     

Cancellous bone from porous T{i}6Al4V by multiple coating technique

A highly porous T{i}6Al4V with interconnected porous structure has been developed in our previous study. By using a so-called “Multiple coating” technique, the porous T{i}6Al4V can be tailored to resemble cancellous bone in terms of porous structure and mechanical properties. A thin layer of T{i}6Al4V slurry was coated on the struts of base porous T{i}6Al4V to improve the pore structure. After two additional coating, pore sizes ranged from 100 μm to 700 μm, and the porosity was decreased from ∼90% to ∼ 75%, while the compressive strength was increased from 10.3 ± 3.3 MPa to 59.4 ± 20.3 MPa and the Young's modulus increased from 0.8 ± 0.3 GPa to 1.8 ± 0.3 GPa. The pore size and porosity are similar to that of cancellous bone, meanwhile the compressive strength is higher than that of cancellous bone, and the Young's modulus is between that of cancellous bone and cortical bone. Porosity, pore size and mechanical properties can be controlled by the parameters in such multiple coating processes. Therefore the porous T{i}6Al4V with the characteristics of cancellous bone is expected to be a promising biomaterial for biomedical applications. Author to whom all correspondence should be addressed.     

Preparation and characterisation of calcium-phosphate porous microspheres with a uniform size for biomedical applications

In the present work, a novel route for the preparation of porous ceramic microspheres is described. Two ceramic powders, calcium-titanium-phosphate (CTP) and hydroxyapatite (HAp), were mixed with a sodium alginate solution that enabled the preparation of spherical particles, using the droplet extrusion method combined with ionotropic gelation in the presence of Ca²⁺. The spherical particles were subsequently sintered, to burn-off the polymer and obtain calcium-phosphate microspheres with a uniform size and an interconnected porous network. CTP microspheres with diameters ranging from 513 ± 24 μm to 792 ± 35 μm and with pores of approximately 40 μm were obtained. HAp microspheres presented diameters of 429 ± 46 μm and 632 ± 40 μm and pores of ca. 2 μm. Depending on the formulations tested, the structure of both calcium phosphates may become altered during the sintering process, suggesting that the ratio between the ceramic phase and the polymer solution is a critical parameter. Porous microspheres prepared using the described methodology are promising candidates as bone defect fillers and scaffolds for bone tissue regeneration.     

Composite scaffolds of nano calcium deficient hydroxyapatite/multi-(amino acid) copolymer for bone tissue regeneration

In this study, nano calcium deficient hydroxyapatite (n-DA)/multi-(amino acid) copolymer composite scaffolds were prepared by injection molding foaming method using calcium sulphate dihydrate as a foaming agent. The composite scaffolds showed well interconnected macropores with the pore size of ranging from 100 to 600 μm, porosity of 81 % and compressive strength of 12 MPa, and the compressive strength obviously affected by the porosity. The composite scaffolds could be slowly degraded in phosphate buffered solution (PBS), which lost its initial weight of 61 w % after immersion into PBS for 12 weeks, and the porosity significantly affected the degradability of the scaffolds. Moreover, it was found that the composite scaffolds could promote the MG-63 cells growth and proliferation, and enhance its alkaline phosphatase activity. The implantation of the scaffolds into the femoral bone of rabbits confirmed that the composite scaffolds were biocompatibitive, degradable, and osteoconductive in vivo.     

Fabrication of polymeric scaffolds with a controlled distribution of pores

The design of tissue engineering scaffolds must take into account many factors including successful vascularisation and the growth of cells. Research has looked at refining scaffold architecture to promote more directed growth of tissues through well-defined anisotropy in the pore structure. In many cases it is also desirable to incorporate therapeutic ingredients, such as growth factors, into the scaffold so that their release occurs as the scaffold degrades. Therefore, scaffold fabrication techniques must be found to precisely control, not only the overall porosity of scaffolds, but also the pore size, shape and spatial distribution. This work describes the use of a regularly shaped porogen, sugar spheres, to manufacture polymeric scaffolds. Results show that pre-assembling the spheres created scaffolds with a constant porosity of 60%, but with varying pores sizes from 200–800 μm, leading to a variation in the surface area and likely degradation rate of the scaffolds. Employing different polymer impregnation techniques tailored the number of pores present with a diameter of less than 100 μm to suit different functions, and altering the packing structure of the sugar spheres created scaffolds with novel layered porosity. Replacing sugar spheres with sugar strands formed scaffolds with pores aligned in one direction.     

3-D PLLA scaffolds formation by a supercritical freeze extraction assisted process

Various techniques have been reported in the literature for the fabrication of biodegradable scaffolds; but, it is very difficult to obtain in the same structure macro, micro and nanostructural characteristics. In this work we developed a supercritical freeze extraction process (SFEP) for the formation of poly(l-lactic acid) (PLLA) scaffolds, that combines the advantages of thermally induced phase separation with those of supercritical drying. We processed solutions in chloroform of two PLLA molecular weights and at different polymer concentrations ranging between 5 and 20 % w/w. Supercritical drying was performed at 35 °Cand pressures ranging between 100 and 250 bar. 3-D scaffolds characterized by high porosity (between 88 and 97.5 %), with coexisting micro and nanometric morphology were obtained. Structures generated were characterized by pores ranging between 10 and 30 μm and with a wrinkled nanostructure of about 200 nm, superimposed on the internal pore surface, that could be useful for biomedical applications. A solvent residue lower than 5 ppm was also measured.     

Optimization of macroporous 3-D silk fibroin scaffolds by salt-leaching procedure in organic solvent-free conditions

A novel all-aqueous process is described to form three-dimensional porous silk fibroin (SF) scaffolds, which not only avoided the use of organic solvents or harsh chemicals, but also can form scaffolds with various sizes and in large quantities. The scaffolds show a rough surface on the pores and the pores are highly interconnected. The porosity of the scaffolds, which varied between a large range (67.6~99.3%), can be controlled by the SF concentrations and the salt/fibroin ratio. The results of measurements indicated that this novel process can improve and enforce the transformation in SF structure from a random coil to a β-sheet. Swelling studies showed that the scaffold has excellent properties of hydrophilicity. The cell culture experiments demonstrated that the scaffolds facilitated the human osteosarcoma cells attachment and proliferation in vitro.     

Indirect rapid prototyping of biphasic calcium phosphate scaffolds as bone substitutes: influence of phase composition,macroporosity and pore geometry on mechanical properties

While various materials have been developed for bone substitute and bone tissue engineering applications over the last decades, processing techniques meeting the high demands of scaffold shaping are still under development. Individually adapted and mechanically optimised scaffolds can be derived from calcium phosphate (CaP-) ceramics via rapid prototyping (RP). In this study, porous ceramic scaffolds with a periodic pattern of interconnecting pores were prepared from hydroxyapatite, β-tricalcium phosphate and biphasic calcium phosphates using a negative-mould RP technique. Moulds predetermining various pore patterns (round and square cross section, perpendicular and 60° inclined orientation) were manufactured via a wax printer and subsequently impregnated with CaP-ceramic slurries. Different pore patterns resulted in macroporosity values ranging from about 26.0–71.9 vol% with pore diameters of approximately 340 μm. Compressive strength of the specimens (1.3–27.6 MPa) was found to be mainly influenced by the phase composition as well as the macroporosity, both exceeding the influence of the pore geometry. A maximum was found for scaffolds with 60 wt% hydroxyapatite and 26.0 vol% open porosity. It has been shown that wax ink-jet printing allows to process CaP-ceramic into scaffolds with highly defined geometry, exhibiting strength values that can be adjusted by phase composition and pore geometry. This strength level is within and above the range of human cancellous bone. Therefore, this technique is well suited to manufacture scaffolds for bone tissue engineering.