Key Engineering Materials Vols. 493-494

Abstract: Body fluid permeability and blood compatibility of biomaterials are especially critical properties for regenerative bone therapy [1, 2]. To have a role in bone repair, biomaterials must have the adsorptive performance of various bone growth factors. The bone-inductive property of rabbit dentin was discovered in 1967 [3]. In our previous study, we have been researching the autograft of human demineralized dentin matrices (DDM) as a clinical study [4]. DDM is an acid-insoluble collagenous material. On the other hand, hydroxyapatite (HAp) is a mineralized material. Commercially available HAp block (APACERAM-AX^®, 85%-porosity with micropore) has been used as the artificial biomaterial in bone therapy [5]. Bone morphogenetic proteins (BMPs) are the strong hard tissue-inducing factors [6]. In this study, we investigated the existence of BMP-2 and -7, among proteins adsorbed to DDM and HAp, using immunoblottong analyses. The DDM granules and HAp blocks (64mm³) were implanted subcutaneously in 8-week-old Wistar rats, and sacrificed at 2 days after the implantation. Explanted DDM and HAp were homogenized by the ultrasonic procedure in phosphate-buffered saline (PBS) and the adsorbed proteins were separated on a 5-20% sodium dodecyl sulphate (SDS) polyacrylamide gradient gel by electrophoresis. For Western blotting, proteins in the gel were transferred to a polyvinylidene difluoride membrane and detected by anti-BMP-2 monoclonal antibody and anti-BMP-7 monoclonal antibody. BMP-2 and BMP-7 were detected as a major band at 50 kDa among proteins collected from the in vivo implanted DDM and HAp. BMP-2 was detected the second major band at 125 kDa in HAp and both BMP-2 and BMP-7 were detected the some minor bands in DDM and HAp. The bands of BMP-2 were stronger than those of BMP-7 in all. The DDM and HAp adsorbed BMP-2 and BMP-7. These results indicate that DDM is a useful bone substitute as much as HAp, adsorbed to the bone-inducing factors, in the bone engineering field.

Abstract: Mg-phosphate ceramics have aroused growing interest as bone replacement materials due to their ability to degrade under physiological conditions. To mimic cancelous bone and to promote tissue repair mechanisms a highly macroporous structure with open cells is desired. In this study trimagnesium phosphate (farringtonite, Mg₃(PO₄)₂) and struvite ((NH₄)Mg(PO₄)·6H₂O) scaffolds were developed as open cell foams using the Schwarzwalder-Somers technique and optimized for pore size and mechanical performance. Polyurethane (PU) foam (20-80 ppi) was used as a template. For the optimization of the farringtonite scaffolds, ppi number of the PU foam as well as the technique that was used to remove excess slurry were varied. Sample characterization was done by SEM, XRD and compression testing. For best results were obtained using 60 ppi PU foams leading to a compressive strength of 58 kPa (90 % porosity). Farringtonite scaffolds were modified by either polymer infiltration or transformation into struvite with an ammonium phosphate solution. The pore macrostructure was retained for both of these processes and a reduction of porosity was observed. The microstructure of struvite foams was significantly altered showing larger and more facetted crystals than farringtonite. Mechanical properties substantially improved by transformation into struvite to 730 kPa (68 % porosity). Cytocompatibility was tested using osteoblasts and fibroblasts. Cell number and cell activity (WST) were tested over a period of 3 to 13 days. Farringtonite foams showed a tendency for higher cell numbers than struvite, while the WST activity was similar. Infiltration of farringtonite with PLGA approximately doubled cell number compared to pure farringtonite. In conclusion macroporous Mg-phosphate foams have been successfully produced. Compressive strength of the foams was drastically improved by optimization of pore fineness, transformation to struvite and infiltration with PLGA. The open porous structure was retained and the materials showed good cytocompatibility.

Abstract: Hydroxyapatite scaffolds have been being produced by a wide range of processes. The optimun material to be used as bone graft has to be partially resorbable, with resorption rates similar to new bone formation ones. The samples must have porosity compatible with tissue ingrowth. Hydroxyapatite and tricalcium phosphate ceramics are good choices for designing such materials. In the present study, polymeric sponges were coated with hydroxyapatite and sintered. The method consists of coating polyurethane sponges substrates in an aqueous solution rich in phosphate (PO₄)^3- and calcium (Ca)²⁺ ions. The solution is composed by 0.5M Ca(OH)₂, 0.3M H₃PO₄ and 1M CH₃CHCO₂HOH (lactic acid) at pH of 3.7. The sponges were immersed in a beaker with the solution and heated up to 80°C to precipitate monetite on the sponge. Continuous and adherent coatings were formed on the surface of sponges interconections. These coatings were characterised by X-ray diffractometry and the only identified phase was monetite. The substrates were converted to hydroxyapatite in an alkali solution.The total conversion from monetite to hydroxyapatite was confirmed by XRD analyses. The struts were heat treated in order to eliminate the organic sponge and sinter the scaffolds. After sintering, hydroxyapatite and tricalcium phosphate were identified on the struts. Optical microscopy revealed the morphology of the struts, while scanning electron microscopy (SEM) showed the precipitates morphology. The method showed to be efficient in the production of porous scaffolds.

Abstract: The application of a hybrid composite consisting of biopolymer and calcium phosphate, similar morphology and properties of natural bone, may be a way to solve the problem of the fragility of ceramics without reducing its mechanical properties, retaining the properties of biocompatibility and high bioactivity. This work aims at the preparation and characterization of three-dimensional scaffolds composite HA / biopolymers (chitosan and gelatin). The freeze-drying technique was employed in this study to obtain these frameworks and partial results showed the effectiveness of this method. This involved the study of structural, chemical and morphological frameworks, in order to direct the research suggested the application. The X Ray Diffraction (XRD) and infrared spectroscopy and Fourier transform (FTIR) results confirmed the formation of hydroxyapatite (HA) phase and the presence of characteristic bands of HA and biopolymers in all compositions. The microstructure of the scaffolds study conducted by Scanning Electron Microscopy (SEM) revealed the formation of longitudinally oriented microchannels with interconnected pores. In all compositions the porous scaffolds showed varying sizes and mostly larger than 100μm, and is therefore considered materials with potential for application in bone tissue engineering.

Abstract: Calcium phosphate cement that foams fully-interconnected porous structure along with its gradual replacement to bone may be ideal for bone defect reconstruction. In the present study, α-tricalcium phosphate (αTCP) microspheres were exposed to acidic calcium phosphate solution. It was found that the αTCP microspheres set in approximately 10 min to form fully-interconnected porous structure. The porosity was approximately 50% and the pore size was 300µm. The surface of the porous body was dicalcium phosphate dihydrate whereas the inside was αTCP.

Abstract: International standard for test method on cell migration into a scaffold is one of the important things to evaluate the scaffold. The "cell migration" ability can divide into two parts. One is infiltration of cell suspension before in vitro cell culture on the scaffold. Another is migration of adherent cells from the edge of scaffold. The latter one could be closely related to cell/tissue migration into the scaffold when it is implanted into bone. Thus, in the present study, the cell migration ability was evaluated toward standardization of in vitro evaluation method for in vivo cell/tissue migration ability using several bioactive ceramics and composites including commercially available materials. The specimen 5 mm in diameter was placed on confluent MG63 cell layer. After 3 days incubation, the specimen was harvested, fixed and divided into two parts. Inside and outside of the scaffold were stained by Giemsa and observed by optical microscopy. In addition, the same specimen was critical point dried and observed with scanning electron microscope (SEM). From microscopic observation, MG63 cells migrated to pore walls of the specimen as well as a sidewall. Maximum migration distances were different among specimens and seemed to depend on pore structure and size as well as porosity. Similar behaviors were observed with SEM.Even relations between this test method and in vivo cell/tissue migration have not been evaluated, this test method is potentially a good method for testing cell migration ability of porous bioactive ceramics as well as other porous scaffold materials.

Abstract: Materials for bone defect filling should have 3D macroporous structure and be flexible to be packed into complex defects with limited entrance space. Tissue engineering scaffolds should also mimic the structure and morphology of the host tissue. Electrospinning is a versatile technique to produce materials with micro/nanofibrous structure, large surface area and high porosity. Electrospun materials are very promising for tissue engineering due to the possibility of mimicking the fibrous structure of natural extra cellular matrix (ECM). Siloxane-containing vaterite (SiV)/poly (_L-lactic acid) (PLLA) hybrids (SiPVH) with controlled silicate and calcium ions releasing ability has been produced in our group. They have also demonstrated good cell infiltration into the electrospun hybrid materials that had fiber diameters greater than 10 μm. However, these electrospun hybrid materials were planar (2D) and are not suitable for large defect regeneration. In this work, the development of a fabrication technique for the production of 3D cotton wool-like structures with fiber diameter in the range of 10 μm was performed. SiPVH cotton wool-like structure containing 0, 30 and 60 wt % SiV were prepared by blowing air in the direction perpendicular to fiber spinning. Si-vaterite particles and small pores were found on the surface of the fibers. The fiber diameter of the samples were found to be in the range of 10 ~ 20 μm. Stretch tests showed more than 50 % extension for the SiPVH cotton wool-like material containing 30 wt % SiV (SiPVH30). This extension was similar to that observed for the PLLA cotton wool-like material. The results suggest that the SiPVH30 cotton wool-like material are good candidates for bone tissue engineering scaffolds.

Abstract: Natural bone constitutes of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). The challenge is to develop a material that can regenerate lost bone tissue with degradation and resorption kinetics compatible with the new bone formation. The aim of this study was to prepare self-organized magnesium and carbonate substituted apatite/collagen scaffolds, cross-linked with glutaraldehyde (GA). Bovine tendon was submitted to alkaline treatment resulting in a negatively charged collagen surface. The scaffolds were prepared by precipitation: simultaneous dropwise addition of solution containing calcium (Ca) and magnesium (Mg) ions and collagen into a buffered solution containing carbonate and phosphate ions in reaction vessel maintained at 37 °C, pH=8. The reaction products were cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and freeze-dried. The samples were characterized by Fourier-transformed infrared spectroscopy (FTIR). In vitro cytotoxicity (based on three parameters assays) and scaffolds degradation in culture medium and osteoblastic cells culture were performed in the cross-linked materials. No cytotoxic effects were observed. The cross-linked samples with the lower GA concentration showed a lower stability when placed in contact with culture medium. Human osteoblasts attached on the scaffolds surface cross-linked with 0.25% GA, forming a continuous layer after 14 days of incubation. These results showed potential application of the designed scaffolds for bone tissue engineering.

Abstract: Calcium alkaline phosphate granulates can be used for substitution of several bone defects but for the reconstruction of large skeletal parts in the maxillofacial and orthopaedic fields fitted scaffolds are preferable. Within the additive manufacturing methods, the 3D printing process offers exciting opportunities to generate defined porous scaffolds. We used a R1 printer from ProMetal Company, USA, for producing scaffolds directly from a ceramic powder. For this direct free form fabrication technology the powder has to possess a lot of specific properties both for the generation of a stable green body and also for the subsequent sintering preparation. For this printing process we prepared different granules in a fluidized bed process containing Ca₂KNa(PO₄)₂ as main crystalline phase. Granules were characterized by different methods and several sieve fractions were used for preparing disc like and cylindrical parts. The suitability of granules for this printing process was determined by porosity and strength of produced bodies. Next to granules’ performance both of these properties can be directly influenced by 3D printing process parameters. With knowledge of suitable process parameters scaffolds with different porosity in a respective desired design can be created. In this study, cylindrical scaffolds with graded porosity were produced for bone regeneration of segmental defects in maxillofacial surgery and dental implantology by tissue engineering.

Abstract: A highly porous (~90%) interconnected hydroxyapatite/wollastonite (HA/WS) scaffolds were prepared by polymeric sponge replica method using a slurry containing HA:Calcium silicate in the weight ratio of 50:50 and sintered at 1300 °C. The phase purity of the scaffolds were analyzed by using XRD. The pore size, pore structure, microstructure and elemental analysis of the scaffolds before and after SBF soaking were analyzed using SEM and EDS. In-vitro bioactivity and bioresorbability confirmed the feasibility of the developed scaffolds. The HA/WS scaffold shows two fold increase in the compressive strength compared to pure HA scaffold.