Foam Replication Technique Research Articles

HA/TCP Scaffolds Coated by PLA and Gelatin: Preliminary <i>In Vitro</i> Evaluation

The importance of this work is development of two methods to try to improve the bone tissue regeneration. The surface of scaffold was modified in order to favor cell interaction, through adhesion and proliferation. PLA and gelatin were used. PLA has shown in literature good results in bone tissue engineering. The gelatin is used as coating in cell culture plates to improve cell attachment for a variety of cell types, including osteoblast. Hydroxyapatite (HA) and tricalcium phosphate (TCP) were used due to its known properties in tissue engineering. It was used the polymer foam replication technique to produce the scaffolds. For characterization were used: scanning electron microscopy (SEM), optical microscopy (OP), stereoscopy, transmission electron microscopy (TEM), X-ray microtomography (Micro-CT), X-ray diffraction (XRD) and X-ray fluorescence (XRF). The scaffolds showed morphology with adequate porosity for tissue engineering and thein vitrotest showed evidence of not being cytotoxic.

Evaluation of borate bioactive glass scaffolds as a controlled delivery system for copper ions in stimulating osteogenesis and angiogenesis in bone healing.

Biocompatible synthetic scaffolds with enhanced osteogenic and angiogenic capacity are of great interest for the repair of large (critical size) bone defects. In this study, we investigated an approach based on the controlled delivery of copper (Cu) ions from borate bioactive glass scaffolds for stimulating angiogenesis and osteogenesis in a rodent calvarial defect model. Borate glass scaffolds (pore size = 200-400 μm) doped with varying amounts of Cu (0-3.0 wt% CuO) were created using a polymer foam replication technique. When immersed in simulated body fluid (SBF) in vitro, the scaffolds released Cu ions into the medium at a rate that was dependent on the amount of Cu in the glass and simultaneously converted to hydroxyapatite (HA). At the concentrations used, the Cu in the glass was not cytotoxic to human bone marrow derived stem cells (hBMSCs) cultured on the scaffolds and the alkaline phosphatase activity of the hBMSCs increased with increasing Cu in the glass. When implanted in rat calvarial defects for 8 weeks, the scaffolds doped with 3 wt% CuO showed a significantly better capacity to stimulate angiogenesis and regenerate bone when compared to the undoped glass scaffolds. Together, these results indicate that the controlled delivery of Cu ions from borate bioactive glass implants is a promising approach in healing bone defects.

Fabrication and Characterization of 45S5 Bioglass<sup>®</sup> Composite Scaffolds

There are many challenges in designing and fabrication of synthetic tissue scaffold for osteochondral tissue engineering. A promising scaffolding material such as 45S5 Bioglass® has been extensively considered to construct scaffolds for this purpose. The 45S5 Bioglass® scaffold was fabricated using foam replication technique. The polyurethane foam was immersed in slurry of 45S5 Bioglass® and poly vinyl alcohol (PVA). The obtained green body was exposed for 1 hour at room temperature before oven dried at 60°C for 24 hours. The 45S5 Bioglass® composite scaffold was prepared by sintering the green body at 1050 °C for 2 hours, followed by dipping in PDLLA solution and dried at room temperature for 12 hours. Characterisations of the above scaffold were carried out using Extended Pressure Scanning Electron Microscope (SEM), Universal Tensile Machine (UTM) and X-ray diffraction (XRD). The SEM micrographs revealed the porous structure of the scaffold. The X-ray diffraction (XRD) spectrum showed the presence of Na2Ca2Si3O9 and (Na2Ca4(PO4)2SiO4 phases in the sintered scaffold. The result showed that the 45S5® Bioglass scaffold has compressive strength of 0.40 MPa with porosity of 88%. Bioactivity assessment in Simulated Body Fluid (SBF) for 7, 14 and 28 hours gave the formation of apatite layer on the scaffold.

Biodegradable borosilicate bioactive glass scaffolds with a trabecular microstructure for bone repair

Three-dimensional porous scaffolds of a borosilicate bioactive glass (designated 13-93B1), with the composition 6Na2O–8K2O–8MgO–22CaO–18B2O3–36SiO2–2P2O5 (mol%), were prepared using a foam replication technique and evaluated in vitro and in vivo. Immersion of the scaffolds for 30days in a simulated body fluid in vitro resulted in partial conversion of the glass to a porous hydroxyapatite composed of fine needle-like particles. The capacity of the scaffolds to support bone formation in vivo was evaluated in non-critical sized defects created in the femoral head of rabbits. Eight weeks post-implantation, the scaffolds were partially converted to hydroxyapatite, and they were well integrated with newly-formed bone. When loaded with platelet-rich plasma (PRP), the scaffolds supported bone regeneration in segmental defects in the diaphysis of rabbit radii. The results indicate that these 13-93B1 scaffolds, loaded with PRP or without PRP, are beneficial for bone repair due to their biocompatibility, conversion to hydroxyapatite, and in vivo bone regenerative properties.

Sago starch as binder and pore-forming agent for the fabrication of porcelain foam

The aim of this study is to verify the potential of sago starch (Metroxylon sagu) as a binder and pore-forming agent for the fabrication of porcelain foam. The porcelain foam was fabricated via the polymeric foam replication technique. Initially, the porcelain raw material was mixed in water with several binders (respectively) to form a slurry. Replication starts by fully submerging a compressed sponge in the slurry. Excess slurry was then removed by hand squeezed and pressed between two parallel plates. Subsequently, the impregnated sponge was sintered at 500°C and up to 1250°C for 2h for sponge and binder removal and porcelain to mature, respectively. The results from this work showed that sago starch slurry produced porcelain with density, total porosity and compressive strength of 0.37–0.51gcm−3, 79–81%, and 0.33–0.93MPa, respectively. The increased of porosity and compressive strength are perhaps due to the addition of sago starch which changed the rheological property of the slurry from Newtonian to pseudoplastic behavior. This results in higher value of viscosity, consequently allowing the construction of smoother and uniform cell skeleton although with existence of micro-pores on the structure, which contributed to higher compressive strength. The results from this study showed that sago starch has huge potential as a binder and a pore-forming agent, whilst providing a simpler and cheaper route for the fabrication of cellular ceramics.

45S5 Bioglass®-derived scaffolds coated with organic–inorganic hybrids containing graphene

Highly porous 45S5 Bioglass®-based scaffolds fabricated by a foam replication technique were coated with electrically conductive organic–inorganic hybrid layers containing graphene by a solution method. α,ω-Triethoxysilane terminated poly (ethylene glycol) and tetraethoxysilane were used as the precursors of the organic–inorganic hybrid coatings, that contained 1.5wt.% of homogeneously dispersed graphene nanoplatelets. The resulting coated scaffolds retained their original high porosity and interconnected pore structure after coating. The presence of graphene did not impair the bioactivity of the scaffolds in simulated body fluid.Initial tests carried out using MG-63 cells demonstrated that both uncoated scaffolds and scaffolds coated with organic/inorganic hybrids containing graphene offered the cultured cells an adequate surface for cell attachment, spreading and expression of extracellular matrix. The results showed that scaffolds coated with graphene are biocompatible and they can support cellular activity. The electrical conductivity introduced by the coating might have the potential to increase tissue growth when cell culture is carried out under an applied electric field.

TENSILE PROPERTIES OF OPEN CELL CERAMIC FOAMS

Full understanding of response of highly porous (cellular) ceramic foams to different mechanical loading conditions is important because of their potential use in industry, medical and other applications. A new technique for testing tensile strength of ceramic foam materials has been developed and first knowledge is described in the paper. Testing procedure consists of fixation of ceramic foam into aluminium pots by suitable adhesive. Alumina based ceramic foam material produced by Lanik (CZ) was used for the test methodology development. Samples were produced by polyurethane foam replication technique. Foams having 10 and 60 pores per linear inch (PPI) were tested. Basic fractographic analysis was carried out by scanning electron microscopy (SEM) to get corresponding figure about specimen behaviour during tensile loading. Influence of increasing PPI (decreasing cell size) on tensile strength of specimens was experimentally proven. With increasing PPI the tensile strength decreases. Slight dependence of tensile strength on sample size of examined material was observed in the range of the tested specimen dimensions. PDLLA-coated samples were also produced and tested, exhibiting nearly 10 times higher tensile strength.

Synthesis and characterization of nanocrystalline hydroxyapatite gel and its application as scaffold aggregation

The sol-gel process is a technique used to synthesize materials from colloidal suspensions and, therefore, is suitable for preparing materials in the nanoscale. In this work hydroxyapatite was used due to its known properties in tissue engineering. Hydroxyapatite Ca10(PO4)6(OH)2 is a bioactive ceramic which is found in the mineral phase of bone tissue and is known for its great potential in tissue engineering applications. For this reason, this material can be applied as particle aggregates on ceramic slurry, coating or film on materials with a poorer biological response than hydroxyapatite. In this work, hydroxyapatite gel was obtained by the sol-gel process and applied as nanoparticle aggregation in the mixture of hydroxyapatite and tricalcium phosphate to form a ceramic slurry. This process is the polymer foam replication technique used to produce scaffolds, which are used in tissue engineering. For HA gel characterization it was used enviromental scanning electron microscopy (ESEM), transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), scanning electron microscopy (SEM), X-ray diffraction (XRD) and X-ray fluorescence (XRF). The crystallite size was calculated from XRD data using the Scherrer equation. The nanoparticles size before firing was approximately 5nm. The crystallite size calculated after calcination was approximately 63 nm. The EELS results showed that calcium phosphate was obtained before firing. After HA gel calcination at 500 ºC the XRD results showed hydroxyapatite with a small content of beta-TCP. The scaffolds obtained by polymer foam replication technique showed a morphology with adequate porosity for tissue engineering.

Biomimetic coating on bioactive glass‐derived scaffolds mimicking bone tissue

Bioceramic "shell" scaffolds, with a morphology resembling the cancellous bone microstructure, have been recently obtained by means of a new protocol, developed with the aim to overcome the limits of the conventional foam replication technique. Because of their original microstructure, the new samples combine high porosity, permeability, and manageability. In this study, for the first time, the novel bioactive glass shell scaffolds are provided with a gelatin-based biomimetic coating to realize hybrid implants which mimic the complex morphology and structure of bone tissue. Moreover, the presence of the coating completely preserves the in vitro bioactivity of the bioactive glass samples, whose surfaces are converted into hydroxyapatite after a few days of immersion in a simulated body fluid solution (SBF).

3D Composite scaffolds using strontium containing bioactive glasses

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

Hydroxyapatite scaffolds infiltrated with thermally crosslinked polycaprolactone fumarate and polycaprolactone itaconate

In this work, two unsaturated derivatives of polycaprolactone (PCL), polycaprolactone fumarate (PCLF), and polycaprolactone itaconate (PCLI), have been synthesized and used as an infiltrating polymer to improve the mechanical properties of brittle hydroxyapatite (HA) scaffolds. PCLF and PCLI were first synthesized through polyesterification of the low molecular weight PCL diols with fumaryl chloride and itaconyl chloride respectively, and then characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, gel permeation chromatography, and differential scanning calorimetry analysis. HA scaffolds were sintered using a foam replication technique, with porosity of about 60%. Polymer-HA composites were obtained by infiltrating the HA scaffolds with PCLF and PCLI solution (12.5 and 30 w/v in dichloromethane) followed by thermal crosslinking. The polymer infiltrated HA scaffolds were characterized by scanning electron microscopy, porosimetry, and gravimetrical analysis. The polyesterification reaction of PCL diols with fumarate chloride was more efficient than itaconyl chloride and dependent upon the molecular weight of the initial PCL precursor; the resultant PCLF demonstrated a degree of substitution of 1.2, 4.2, and 2.7 times higher than PCLIs. Polymer infiltration improved the compressive strength of the HA scaffolds, and based upon the type of macromer (PCLF or PCLI) and also their concentration in infiltrating solution (12.5 or 30 w/v %) compressive strength increased about 14-328%. In all studied samples, the reinforcement effect of PCLF infiltration was higher than PCLI. The macromers and their corresponding infiltrated HA scaffolds did not show any significant cytotoxicity toward human primary osteogenic sarcoma cell (G92 cell lines), in vitro.

Ag-doped 45S5 Bioglass®-based bone scaffolds by molten salt ion exchange: processing and characterisation

There is increasing interest in developing scaffolds with therapeutic and antibacterial potential for bone tissue engineering. Silver is a proven antibacterial agent which bacteria such as MRSA have little or no defense against. Using an ion exchange method, silver ions have been introduced into 45S5 Bioglass(®) based scaffolds that were fabricated using the foam replication technique. This technique allows the introduction of Ag(+) ions onto the surface of the scaffold without compromising the scaffold bioactivity and other physical properties such as porosity. Controlling the amount of Ag(+) ions introduced onto the surface of the scaffold was achieved by tailoring the ion exchange parameters to fabricate samples with repeatable and predictable Ag(+) ion release behavior. In vitro studies in simulated body fluid were carried out to ensure that the scaffolds maintained their bioactivity after the introduction of Ag(+) ions. It was also shown that the addition of low concentrations (2000:1 w/w) of silver ions supported the attachment and viability of human periodontal ligament stromal cells on the 3D scaffolds. This work has thus confirmed ion exchange as an effective technique to introduce Ag(+) ions into 45S5 Bioglass(®) scaffolds without compromising the basic properties of 45S5 Bioglass(®) which are required for applications in bone tissue engineering.

Influence of gelatin coatings on compressive strength of porous hydroxyapatite ceramics

Porous ceramics prepared by the foam replication technique have a high porosity and low mechanical stability. It has been reported that coating such porous ceramics with gelatin allows for an improved compressive strength. Little details regarding the influence of important gelatin parameters such as concentration, temperature and drying conditions as well as bloom number which is a measure of the gel rigidity, on this toughening effect are available. This paper investigates the influence of these parameters on compressive strength of gelatin coated porous hydroxyapatite ceramics. It was found that concentration in the gelatin sols has a marked impact whereas sol temperature, bloom number and differing conditions during subsequent drying have only little influence on the compressive strength of the coated ceramics.

Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. I. Preparation and in vitro degradation

Bioactive glass scaffolds with a microstructure similar to that of dry human trabecular bone but with three different compositions were evaluated for potential applications in bone repair. The preparation of the scaffolds and the effect of the glass composition on the degradation and conversion of the scaffolds to a hydroxyapatite (HA)-type material in a simulated body fluid (SBF) are reported here (Part I). The in vitro response of osteogenic cells to the scaffolds and the in vivo evaluation of the scaffolds in a rat subcutaneous implantation model are described in Part II. Scaffolds (porosity = 78-82%; pore size = 100-500 microm) were prepared using a polymer foam replication technique. The glasses consisted of a silicate (13-93) composition, a borosilicate composition (designated 13-93B1), and a borate composition (13-93B3), in which one-third or all of the SiO2 content of 13-93 was replaced by B2O3, respectively. The conversion rate of the scaffolds to HA in the SBF increased markedly with the B2O3 content of the glass. Concurrently, the pH of the SBF also increased with the B2O3 content of the scaffolds. The compressive strengths of the as-prepared scaffolds (5-11 MPa) were in the upper range of values reported for trabecular bone, but they decreased markedly with immersion time in the SBF and with increasing B2O3 content of the glass. The results show that scaffolds with a wide range of bioactivity and degradation rate can be achieved by replacing varying amounts of SiO(2) in silicate bioactive glass with B2O3.

Preparation and Characterization of Gallium Releasing 3‐D Alginate Coated 45S5 Bioglass® Based Scaffolds for Bone Tissue Engineering

AbstractThe aim of this work was to develop functional three‐dimensional scaffolds based on Bioglass® derived glass–ceramics for bone tissue engineering (TE) with prophylaxis effect against infections. The highly porous scaffolds were fabricated by the foam replication technique and then coated with sodium alginate crosslinked with Ga3+. It was confirmed that the polymer coating did not affect the interconnectivity of the scaffold pore structure. The resulting composite scaffold exhibited antibacterial effect and improved mechanical properties as well as high bioactivity. The novel Ga3+ loaded 45S5 Bioglass®‐based scaffolds coated with alginate are promising candidates for bone TE applications.

Conversion of borate-based glass scaffold to hydroxyapatite in a dilute phosphate solution**This work was presented at the Materials Science and Technology (MS&T'09) Conference Symposium: Next Generation Biomaterials, Pittsburgh, PA, 25–29 October 2009.

Porous scaffolds of a borate-based glass (composition in mol%: 6Na2O, 8K2O, 8MgO, 22CaO, 36B2O3, 18SiO2, 2P2O5), with interconnected porosity of ∼70% and pores of size 200–500 µm, were prepared by a polymer foam replication technique. The degradation of the scaffolds and conversion to a hydroxyapatite-type material in a 0.02 M K2HPO4 solution (starting pH = 7.0) at 37 °C were studied by measuring the weight loss of the scaffolds, as well as the pH and the boron concentration of the solution. X-ray diffraction, scanning electronic microscopy and energy dispersive x-ray analysis showed that a hydroxyapatite-type material was formed on the glass surface within 7 days of immersion in the phosphate solution. Cellular response to the scaffolds was assessed using murine MLO-A5 cells, an osteogenic cell line. Scanning electron microscopy showed that the scaffolds supported cell attachment and proliferation during the 6 day incubation. The results indicate that this borate-based glass could provide a promising degradable scaffold material for bone tissue engineering applications.

Multi-functional P(3HB) microsphere/45S5 Bioglass®-based composite scaffolds for bone tissue engineering

Novel multi-functional P(3HB) microsphere/45S5 Bioglass®-based composite scaffolds exhibiting potential for drug delivery were developed for bone tissue engineering. 45S5 Bioglass®-based glass–ceramic scaffolds of high interconnected porosity produced using the foam-replication technique were coated with biodegradable microspheres (size < 2 μm) made from poly(3-hydroxybutyrate), P(3HB), produced using Bacillus cereus SPV. A solid-in-oil-in-water emulsion solvent extraction/evaporation technique was used to produce these P(3HB) microspheres. A simple slurry-dipping method, using a 1 wt.% suspension of P(3HB) microspheres in water, dispersed by an ultrasonic bath, was used to coat the scaffold, producing a uniform microsphere coating throughout the three-dimensional scaffold structure. Compressive strength tests confirmed that the microsphere coating slightly enhanced the scaffold mechanical strength. It was also confirmed that the microsphere coating did not inhibit the bioactivity of the scaffold when immersed in simulated body fluid (SBF) for up to 4 weeks. The hydroxyapatite (HA) growth rate on P(3HB) microsphere-coated 45S5 Bioglass® composite scaffolds was very similar to that on the uncoated control sample, qualitatively indicating similar bioactivity. However, the surface topography of the HA surface layer was affected as shown by results obtained from white light interferometry. The roughness of the surface was much higher for the P(3HB) microsphere-coated scaffolds than for the uncoated samples, after 7 days in SBF. This feature would facilitate cell attachment and proliferation. Finally, gentamycin was successfully encapsulated into the P(3HB) microspheres to demonstrate the drug delivery capability of the scaffolds. Gentamycin release kinetics was determined using liquid chromatography–mass spectrometry. The release of the drug from the coated composite scaffolds was slow and controlled when compared to the observed fast and relatively uncontrolled drug release from the bone scaffold (without microsphere coating). Thus, this unique multifunctional bioactive composite scaffold has the potential to enhance cell attachment and to provide controlled delivery of relevant drugs for bone tissue engineering.

In vitrobiocompatibility of 45S5 Bioglass®-derived glass-ceramic scaffolds coated with poly(3-hydroxybutyrate)

The aim of this work was to study the in vitro biocompatibility of glass-ceramic scaffolds based on 45S5 Bioglass, using a human osteosarcoma cell line (HOS-TE85). The highly porous scaffolds were produced by the foam replication technique. Two different types of scaffolds with different porosities were analysed. They were coated with a biodegradable polymer, poly(3-hydroxybutyrate) (P(3HB)). The scaffold bioactivity was evaluated by soaking in a simulated body fluid (SBF) for different durations. Compression strength tests were performed before and after immersion in SBF. These experiments showed that the scaffolds are highly bioactive, as after a few days of immersion in SBF a hydroxyapatite-like layer was formed on the scaffold's surface. It was also observed that P(3HB)-coated samples exhibited higher values of compression strength than uncoated samples. Biocompatibility assessment was carried out by qualitative evaluation of cell morphology after different culture periods, using scanning electron microscopy, while cell proliferation was determined by using the AlamarBlue assay. Alkaline phosphatase (ALP) and osteocalcin (OC) assays were used as quantitative in vitro indicators of osteoblast function. Two different types of medium were used for ALP and OC tests: normal supplemented medium and osteogenic medium. HOS cells were seeded and cultured onto the scaffolds for up to 2 weeks. The AlamarBlue assay showed that cells were able to proliferate and grow on the scaffold surface. After 7 days in culture, the P(3HB)-coated samples had a higher number of cells on their surfaces than the uncoated samples. Regarding ALP- and OC-specific activity, no significant differences were found between samples with different pore sizes. All scaffolds containing osteogenic medium seemed to have a slightly higher level of ALP and OC concentration. These experiments confirmed that Bioglass/P(3HB) scaffolds have potential as osteoconductive tissue engineering substrates for maintenance and normal functioning of bone tissue.

Simple methods to fabricate Bioglass®-derived glass–ceramic scaffolds exhibiting porosity gradient

The present paper discusses different processing technologies for fabrication of novel 45S5 Bioglass®-derived glass–ceramic scaffolds with tailored porosity gradient for potential application in bone tissue engineering. Different types of scaffolds with continuous or stepwise gradient of porosity were produced by the foam replication technique, using preformed polyurethane (PU) foams as sacrificial templates. After preforming the PU foams in metallic moulds, they were dipped in a 45S5 Bioglass®-based slurry and subsequently heat treated in a chamber furnace up to 1100 °C. During heating, the organic phase is burned out and the glass sinters and partially crystallises. By using this new approach, Bioglass®-derived glass-ceramic scaffolds with different shapes and porosity profiles were designed. Scanning electron microscopy (SEM) showed that all samples have highly interconnected porous structure, with specific porosity gradients. By modifying the shape and dimensions of the metallic mould, bioactive glass–ceramic scaffolds with complex shapes and different degrees of porosity gradient could be obtained.

Mechanical and in vitro performance of 13–93 bioactive glass scaffolds prepared by a polymer foam replication technique

A polymer foam replication technique was used to prepare porous scaffolds of 13–93 bioactive glass with a microstructure similar to that of human trabecular bone. The scaffolds, with a porosity of 85 ± 2% and pore size of 100–500 μm, had a compressive strength of 11 ± 1 MPa, and an elastic modulus of 3.0 ± 0.5 GPa, approximately equal to the highest values reported for human trabecular bone. The strength was also considerably higher than the values reported for polymeric, bioactive glass–ceramic and hydroxyapatite constructs prepared by the same technique and with the equivalent level of porosity. The in vitro bioactivity of the scaffolds was observed by the conversion of the glass surface to a nanostructured hydroxyapatite layer within 7 days in simulated body fluid at 37 °C. Protein and MTT assays of in vitro cell cultures showed an excellent ability of the scaffolds to support the proliferation of MC3T3-E1 preosteoblastic cells, both on the surface and in the interior of the porous constructs. Scanning electron microscopy showed cells with a closely adhering, well-spread morphology and a continuous increase in cell density on the scaffolds during 6 days of culture. The results indicate that the 13–93 bioactive glass scaffolds could be applied to bone repair and regeneration.