Porous Ceramic Scaffolds Research Articles

In situ formation of nano-hydroxyapatite whisker reinfoced porous β-TCP scaffolds

Graphical abstractDisplay Omitted Highlights? Nano-HA whiskers were born in the porous β-TCP ceramic during in situ synthesis. ? The thermal stability of nano-HA whiskers improved significantly. ? The porous nHAW/β-TCP scaffolds exhibited higher mechanical properties. ? The nano-HA whisker could improve cell adhesion and proliferation on the scaffolds. The preparation of porous ceramic scaffolds with nano-hydroxyapatite (HA) whiskers was reported in this work. By adding kalium ion and adjusting the pH value of solution, the thermal stability of nano-HA whiskers were improved significantly. The whiskers in the ceramic scaffolds can be kept completely after sintered at 1000?C. The nano-HA whisker was about 2µm in length and 100nm in diameter. The raw powders and scaffolds were observed by SEM and TEM, and the phase composition of samples was determined by XRD. The nHAW/β-TCP/BG scaffolds exhibited interconnected porous structures and enhanced mechanical properties as compared to those β-TCP/BG scaffolds. In order to assess the efforts of nano-HA whiskers on the behavior of osteoblasts cultured in vitro, the scaffolds were seeded with MC3T3-E1 and cultured for the same period. The cell viability and proliferation was assessed by the MTT assay and observed using laser scanning confocal microscopy. The Results suggest that the nano-HA whisker could be used to improve the cell adhesion and proliferation.

Enhanced bone tissue formation by alginate gel‐assisted cell seeding in porous ceramic scaffolds and sustained release of growth factor

Increasing cell seeding efficiency in a tissue engineering construct can enhance cellular activity and tissue formation in vivo. Here, we demonstrate the use of alginate gel as a secondary phase material in 3D porous β-tricalcium phosphate scaffolds to improve cell seeding and provide controlled release of growth factors for bone tissue engineering. Cells were seeded in scaffolds in three ways: conventional seeding (CS), alginate gel-assisted seeding (GS), and alginate GS with bone morphogenetic protein-2 (BMP-2, GSB). In vitro study with MG-63 cells showed that cell seeding efficiency and cell population 1 week after seeding were significantly elevated in GS and GSB samples compared to CS samples. The GSB system demonstrated a sustained, steady release of BMP-2 over 2 weeks. In vivo, scaffolds seeded with rat mesenchymal stem cells were implanted ectopically into Sprague-Dawley rats for 8 weeks. GS and GSB samples exhibited improved osteogenic activity, with the GSB samples inducing the greatest osteocalcin and osteoid deposition. This study suggests that the alginate gel-assisted cell seeding increases seeding efficiency and allows for sustained release of growth factors. The use of the secondary phase polymer bolsters bone formation in vivo and has the potential for improving outcome in other tissue engineering applications.

Effect of ZrO2 addition on the mechanical properties of porous TiO2 bone scaffolds

This study aimed at the investigation of the effect of zirconium dioxide (ZrO2) addition on the mechanical properties of titanium dioxide (TiO2) bone scaffolds. The highly biocompatible TiO2 has been identified as a promising material for bone scaffolds, whereas the more bioinert ZrO2 is known for its excellent mechanical properties. Ultra-porous TiO2 scaffolds (>89% porosity) were produced using polymer sponge replication with 0–40wt.% of the TiO2 raw material substituted with ZrO2. Microstructure, chemical composition, and pore architectural features of the prepared ceramic foams were characterised and related to their mechanical strength. Addition of 1wt.% of ZrO2 led to 16% increase in the mean compressive strength without significant changes in the pore architectural parameters of TiO2 scaffolds. Further ZrO2 additions resulted in reduction of compressive strength in comparison to containing no ZrO2. The appearance of zirconium titanate (ZrTiO4) phase was found to hinder the densification of the ceramic material during sintering resulting in poor intergranular connections and thus significantly reducing the compressive strength of the highly porous ceramic foam scaffolds.

Biological Properties of Solid Free Form Designed Ceramic Scaffolds with BMP-2: In Vitro and In Vivo Evaluation

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.

Reverse freeze casting: A new method for fabricating highly porous titanium scaffolds with aligned large pores

Highly porous titanium with aligned large pores up to 500μm in size, which is suitable for scaffold applications, was successfully fabricated using the reverse freeze casting method. In this process we have newly developed, the Ti powders migrated spontaneously along the pre-aligned camphene boundaries at a temperature of 45.5°C and formed a titanium–camphene mixture with an aligned structure; this was followed by freeze drying and sintering. As the casting time increased from 24 to 48h, the initial columnar structures turned into lamellar structures, with the porosity decreasing from 69 to 51%. This reduction in porosity caused the compressive yield strength to increase from 121 to 302MPa, with an elastic modulus of the samples being in the range of 2–5GPa. In addition, it was demonstrated that reverse freeze casting can also be successfully applied to various other raw powders, suggesting that the method developed in this work opens up new avenues for the production of a range of porous metallic and ceramic scaffolds with highly aligned pores.

Mineralized Tissue Formation by Bone Morphogenetic Protein-7–transfected Pulp Stem Cells

Introduction Recombinant human bone morphogenetic protein-7 (BMP-7) is a well-established agent to induce bone and dentin formation. Little is understood until now whether BMP-7 could be used to genetically modify human dental pulp stem cells for tissue engineering applications. Methods This study was to determine the feasibility of mineralized tissue formation from human dental pulp-derived stem cells (DPSCs) transfected with adenoviral-mediated human BMP-7 gene through in vitro and in vivo evaluations. Results In vitro results of alkaline phosphatase, calcium content, and real-time polymerase chain reaction showed that BMP-7–transfected cells had the ability to differentiate towards the odontoblast phenotype and produce a calcified extracellular matrix. Transfected cell were seeded onto a porous ceramic scaffold and implanted subcutaneously in mice. Samples were retrieved after 4 and 8 weeks for histology evaluation. The results indicated that only the cultures with BMP-7 gene transfection showed obvious hard-tissue generation. Conclusions Our data suggest that adenovirus-mediated BMP-7 expression can induce odontogenic differentiation of human DPSCs and show effectively mineralized tissue formation in vivo, which may provide support for gene therapy candidate of BMP-7 in dental tissue engineering.

Preparation and characterization of porous Tri-Calcium Phosphate ceramic scaffold

Objective Bio-ceramics has been widely utilized as a common type of bone repair material.The research was designed to focus on preparation and characterization of porous TCP.Methods By using hydroxyapatite (HA) as the raw materials,and adding calcium phosphate bio-glass to adjust the ratio between calcium and phosphate,in order to achieve making porous TCP bio-ceramic scaffolds during high temperature heating process.Results Test results showed that by applying this method,TCP scaffolds had significantly higher mechanical strength then those were made from powdered TCP,and its compressive strength reached 9.11 MPa.Conclusion Tri-Calcium Phosphate (TCP) has excellent biocompatibility and it is also biodegradable.In the physiological environment,it is able to be gradually decomposed,absorbed and to promote bone growth ultimately,it is capable of replacing the damaged bone components with brand new bone tissues. Key words: Bioactive glass; Hydroxapatite; Tri-Calcium Phosphate; Scaffold

Fabrication of Porous Ceramic Scaffolds via Polymeric Sponge Method Using Sol-Gel Derived Strontium Doped Hydroxyapatite

Recently, development of porous calcium phosphates ceramics has raised considerable interest. A porous structure promotes cell attachment, proliferation and provides pathways for biofluids. Therefore, a high porosity with interconnected pore structure generally favors tissue regeneration. In this work, replication of 0, 2, 5, 10 and 15% SrHA (strontium-doped hydroxyapatite) porous scaffolds via polymeric sponge method has been employed using the sol-gel derived SrHA powders. To prepare the porous samples, the synthesized SrHA powders were mixed with distilled water and dispersing agent followed by drying in the ambient air and specific sintering process. Morphological evaluation by FESEM measurement revealed that the SrHA scaffolds were characterized by macro-micro interconnected porosity, which replicates the morphology of the cancellous bone. Compression test on the porous scaffolds revealed that doping 10 mol% of strontium in HA has increased the compressive strength by a factor of two compared to the undoped HA with 1.81±0.26 MPa at 41% porosity.

Fabrication of Porous Hydroxyapatite Ceramic Scaffolds with High Flexural Strength Through the Double Slip‐Casting Method Using Fine Powders

The formation of sufficiently large three‐dimensional nano/macroporosity in bone scaffolds remains a challenge despite the numerous fabrication methods reported in literature. The presence of such porosity is required for tissue ingrowth and for the concurrent degradation of implanted structure. A new technique of combining the slip casting and polymer sponge methods using the slips with different powders was introduced in the study to prepare hydroxyapatite scaffolds with bimodal pore sizes. This technique provided better control over the microstructures of scaffolds and enhanced their mechanical properties compared to traditional methods. With this technique, we were able to produce scaffolds with mechanical and structural properties that could not be attained by the polymer sponge or slip‐casting method alone. The scaffolds were prepared with an open, uniform, and interconnected porous structure with a bimodal pore size of 100–300 μm. The bimodal porous hydroxyapatite scaffold sintered at 1200°C had a large flexural strength of 73.3 MPa and a porosity of 52.5 vol%. These bioscaffolds hold promise for applications in hard tissue engineering.

Direct ink writing of highly porous and strong glass scaffolds for load-bearing bone defects repair and regeneration

The quest for synthetic materials to repair load-bearing bone lost because of trauma, cancer, or congenital bone defects requires the development of porous, high-performance scaffolds with exceptional mechanical strength. However, the low mechanical strength of porous bioactive ceramic and glass scaffolds, compared with that of human cortical bone, has limited their use for these applications. In the present work bioactive 6P53B glass scaffolds with superior mechanical strength were fabricated using a direct ink writing technique. The rheological properties of Pluronic® F-127 (referred to hereafter simply as F-127) hydrogel-based inks were optimized for the printing of features as fine as 30 μm and of three-dimensional scaffolds. The mechanical strength and in vitro degradation of the scaffolds were assessed in a simulated body fluid (SBF). The sintered glass scaffolds showed a compressive strength (136 ± 22 MPa) comparable with that of human cortical bone (100–150 MPa), while the porosity (60%) was in the range of that of trabecular bone (50–90%). The strength is ∼100-times that of polymer scaffolds and 4–5-times that of ceramic and glass scaffolds with comparable porosities. Despite the strength decrease resulting from weight loss during immersion in SBF, the value (77 MPa) is still far above that of trabecular bone after 3 weeks. The ability to create both porous and strong structures opens a new avenue for fabricating scaffolds for load-bearing bone defect repair and regeneration.

A 3D in vitro bone organ model using human progenitor cells

Three-dimensional (3D) organotypic culture models based on human cells may reduce the use of complex and costly animal models, while gaining clinical relevance. This study aimed at developing a 3D osteoblastic-osteoclastic-endothelial cell co-culture system, as an in vitro model to mimic the process of bone turnover. Osteoprogenitor and endothelial lineage cells were isolated from the stromal vascular fraction (SVF) of human adipose tissue, whereas CD14+ osteoclast progenitors were derived from human peripheral blood. Cells were co-cultured within 3D porous ceramic scaffolds using a perfusion-based bioreactor device, in the presence of typical osteoclastogenic factors. After 3 weeks, the scaffolds contained cells with endothelial (2.0±0.3%), pre/osteoclastic (14.0±1.4%) and mesenchymal/osteoblastic (44.0±8.4%) phenotypes, along with tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells in contact with deposited bone-like matrix. Supernatant analysis demonstrated sustained matrix deposition (by C-terminus procollagen-I propeptides), resorption (by N-terminus collagen-I telopeptides and phosphate levels) and osteoclastic activity (by TRAP-5b) only when SVF and CD14+ cells were co-cultured. Scanning electron microscopy and magnetic resonance imaging confirmed the pattern of matrix deposition and resorption. The effectiveness of Vitamin D in replacing osteoclastogenic factors indicated a functional osteoblast-osteoclast coupling in the system. The formation of human-origin bone-like tissue, blood vessels and osteoclasts upon ectopic implantation validated the functionality of the developed cell types. The 3D co-culture system and the associated non-invasive analytical tools can be used as an advanced model to capture some aspects of the functional coupling of bone-like matrix deposition and resorption and could be exploited toward the engineering of multi-functional bone substitute implants.

Reinforcing of Porous Hydroxyapatite Ceramics with Hydroxyapatite Fibres for Enhanced Bone Tissue Engineering

Porous hydroxyapatite (HA) ceramic implants have attracted attention in bone tissue engineering due to their excellent bioactivity and biocompatibility due to their chemical similarity with the mineral component of natural bone. Unfortunately, HA when is formed into porous structures exhibits very low compression strength. In this study, fabrication of porous HA ceramic scaffolds containing HA fibers is presented. The primary aim of the study is to improve mechanical properties of the scaffold by introducing the fiber with uniform component relative to the scaffold. Scanning electron microscopy was used to observe the surface morphology and pore size of the scaffold. X-ray diffraction (XRD) was used to detect the phase composition and crystallinity of the scaffold. The compressive strength was determined using a universal material test machine. The results and the characterizations demonstrate the addition of HA fiber could enhance the uniformity of mechanical properties among samples and also the strength for a given open porosity.

TBA-based freeze/gel casting of porous hydroxyapatite scaffolds

Porous ceramic scaffolds with a controlled “designer” pore structure have been prepared by the freeze/gel casting route using a TBA-based hydroxyapatite slurry system with 20–40 wt.% solid content. The products were characterized in terms of sintered microstructure, together with physical and mechanical properties. After sintering at 1050–1250 °C, the advantages of freeze casting and gel casting appeared in the pore structure and compressive strength of the ceramics, i.e., unidirectional aligned macro-pore channels developed by controlling the solidification direction of the TBA solvent used in the freeze casting together with small diameter (micron sized) isolated pores formed in the dense outer walls of the pore channels when processed by gel casting. The sintered porosity and pore size generally resulted in a high solid loading giving low porosity and small pore size, this leading to higher compressive strengths. The scaffolds obtained exhibited an average porosity and compressive strength in the range 41.9–79.3% and 35.1–2.7 MPa, respectively, depending on the processing conditions used.

Comparison of TCP and TCP/HA Hybrid Scaffolds for Osteoconductive Activity

Two types of porous ceramic scaffolds were prepared, consisting of β-tricalcium phosphate (TCP) or the mixed powder of TCP and hydroxyapatite (HA) at a 2:1 mass ratio. A variety of methods have been used to fabricate bone scaffolds, while the sintering approach was adopted in this work. An extremely high temperature was used on sintering that proposed to consolidate the ceramic particles. As revealed by SEM, a well opened pore structure was developed within the scaffolds. The θ-values were measured to be of 73.3° and 6.5° for the composite scaffold and TCP sample, respectively. According to XRD patterns, the existence of grains coalescence and partial bonding between HA and TCP powders was demonstrated. Scaffold mechanical property in the term of flexural strength was also determined. The result showed decreasing of the strength by HA supplement, suggesting the more brittle characteristic of HA in comparison with TCP. By soaking the composite scaffold in PBS for a period of 2 weeks, transformation from particles to flank-like crystalline was clearly observed. Such change was found to be favorable for cell attachment, migration, and growth. By implanting cell-seeded scaffolds into nude mice, an abundant osseous extracellular matrix was identified for the composite implants. In contrast, the matrix was minimally detected in TCP implanted samples. Thus, the composite scaffold was found superior for hard tissue regeneration.

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 26.0-71.9vol% with pore diameters of approximately 340μm. Compressive strength of the specimens (1.3-27.6MPa) 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 60wt% hydroxyapatite and 26.0vol% 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.

Generating Porous Ceramic Scaffolds: Processing and Properties

For tissue regeneration in medicine three-dimensional scaffolds with specific characteristics are required. A very important property is a high, interconnecting porosity to enable tissue ingrowth into the scaffold. Pore size distribution and pore geometry should be adapted to the respective tissue. Additionally, the scaffolds should have a basic stability for handling during implantation, which is provided by ceramic scaffolds. Various methods to produce such ceramic 3D scaffolds exist. In this paper conventional and new fabrication techniques are reviewed. Conventional methods cover the replica of synthetic and natural templates, the use of sacrificial templates and direct foaming. Rapid prototyping techniques are the new methods listed in this work. They include fused deposition modelling, robocasting and dispense-plotting, ink jet printing, stereolithography, 3D-printing, selective laser sintering/melting and a negative mould technique also involving rapid prototyping. The various fabrication methods are described and the characteristics of the resulting scaffolds are pointed out. Finally, the techniques are compared to find out their disadvantages and advantages.

Effects of cell-seeding methods of human osteoblast culture on biomechanical properties of porous bioceramic scaffold

Many studies have been performed to accelerate osteoinduction and osteoconduction into porous ceramic scaffolds by seeding them with cells. In this study, we compared available cell-seeding methods on a porous β-tricalcium phosphate (β-TCP) scaffold and evaluated the effects of cell-seeding on the mechanical properties of the porous β-TCP scaffold. Three types of porous bioceramic scaffolds were used: dry scaffold, scaffold wetted with media, and scaffold cultivated with normal human osteoblasts (NHOs). Cell-seeding into the porous β-TCP scaffolds was performed by conventional, centrifuge, high-density, and vacuum methods. After confirming cell proliferation with MTT assay and cell staining, a compressive test was performed after 2 and 4 weeks of cell culture. The vacuum method based on the high-density cell culture inserted effectively NHOs into the β-TCP scaffolds. The compressive elastic modulus of wetted β-TCP scaffolds decreased significantly (p < 0.05) about 20∼30% after 2 and 4 weeks of incubation in comparison with that of the dry scaffold. However, the compressive strength of the scaffolds cultivated with NHOs for 3 weeks was significantly (p < 0.05) higher than that of scaffolds without NHOs. The vacuum with the high-density of cell-seeding seems to be a suitable method for seeding cells into complex porous ceramic scaffolds. Cell proliferation and uniform distribution in the scaffolds can change the initial mechanical properties of porous ceramic scaffolds.

Maxillary sinus floor elevation using a tissue engineered bone complex with BMP-2 gene modified bMSCs and a novel porous ceramic scaffold in rabbits

Objectives To study the effects of maxillary sinus floor elevation by a tissue engineered bone complex with bone morphogenetic protein-2 (BMP-2) gene modified bone marrow stromal cells (bMSCs) and a novel porous ceramic scaffold (OsteoBone™) in rabbits. Materials and methods bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with adenovirus AdBMP-2 and with AdEGFP gene (without BMP-2 gene sequence) as a control, respectively, in vitro. These bMSCs were then combined with OsteoBone™ scaffold at a concentration of 2 × 10 7 cells/ml and used to elevate the maxillary sinus floor in rabbits. Eight rabbits were randomly allocated into groups and sacrificed at weeks 2 and 4. For each time point, 8 maxillary sinus floor elevation surgeries were made bilaterally in 4 rabbits for the two groups ( n = 4 per group): group A (AdBMP-2-bMSCs/material) and group B (AdEGFP-bMSCs/material). All samples were evaluated by histologic and histomorphometric analysis. Results The augmented maxillary sinus height was maintained for both groups over the entire experimental period, while new bone area increased over time for group A. At week 4 after operation, bone area in group A was significantly more than that in group B ( P < 0.05), and was more obviously detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed in the EGFP group, which suggests that those implanted bMSCs had contributed to the new bone formation. Conclusion bMSCs modified with AdBMP-2 gene can promote new bone formation in elevating the rabbit maxillary sinus. OsteoBone™ scaffold could be an ideal carrier for gene enhanced bone tissue engineering.

The development of tissue-engineered bone of different origin through endochondral and intramembranous ossification following the implantation of mesenchymal stem cells and osteoblasts in a murine model

The study of host cell recruitment by implanted exogenous cells is one of the novel challenges in tissue engineering. We previously reported the development of tissue-engineered bone deposited by host cells in porous ceramic scaffolds seeded with murine mesenchymal stem cells (MSC) and implanted in immunocompromised mice. To better highlight the contribution of host cells to the development of the engineered tissue and to investigate whether the capacity to recruit host cells was dependent on the donor cell commitment, we implanted ceramic scaffolds seeded with either murine GFP labeled MSC or GFP labeled osteoblasts (OB) into immunocompromised mice. Although we observed formation of bone in all scaffolds, the origin of bone cells and the ossification type were strictly dependent on the nature and commitment of the seeded cells. MSC implants led to formation of bone of host origin through the activation of an endochondral ossification process while an intramembranous ossification directly performed by the seeded cells was observed in OB implants. Moreover, we observed an increased vascularization in MSC implants due to the higher capacity of MSC to recruit host CD31+ endothelial cells. The relationship between this enhanced vascularization and the type of ossification is discussed.

Osteoblast and Osteoclast Differentiation in anIn VitroThree-Dimensional Model of Bone

There is increasing interest in developing new in vitro tissue models using typical tissue engineering approaches. This study was designed to (1) develop a novel three-dimensional (3D) in vitro model of bone by seeding murine primary osteoblasts and osteoclast precursors on a resorbable porous ceramic scaffold based on silicon-stabilized tricalcium phosphate (Skelite), and (2) investigate bone cell interactions in a 3D environment mimicking an in vivo condition and compare it to traditional two-dimensional (2D) cultures. Murine primary osteoblasts from C57Bl6/J mice and osteoclast precursors from C57Bl/6-Tg(ACTB-EGFP)1Osb/J mice were co-cultured on 3D Skelite scaffolds and on standard plastic culture dishes. The differentiation of these cells in both culture conditions was compared by histology (hematoxylin-eosin staining and polarized light analysis), immunohistochemistry (collagen type I), and gene expression analysis by real-time PCR for Runt-related transcription factor 2, osterix, osteocalcin, cathepsin K, and tartrate resistant acid phosphatase. To analyze and compare bone turnover in 3D and 2D co-cultures, we evaluated the modulation of RANKL and OPG mRNA expression. We observed an enhancement of osteoblast differentiation in the 3D mineralized environment that in turn promoted earlier osteoclast differentiation. In this paper, we also report that the increased osteoblast differentiation in the 3D model led to a deposition of extracellular matrix that faithfully reflected the morphology of bone tissue.