Kazuyo Igawa

Tailor-made tricalcium phosphate bone implant directly fabricated by a three-dimensional ink-jet printer

Rapid prototyping (RP) is a molding technique that builds a three-dimensional (3D) model from computer-aided design (CAD) data. We fabricated new tailor-made bone implants (TIs) from α-tricalcium phosphate powder using an RP ink-jet printer based on computed tomography (CT) data, and evaluated their safety and efficacy. CT data of the skulls of seven beagle dogs were obtained and converted to CAD data, and bone defects were virtually made in the skull bilaterally. TIs were designed to fit the defects and were fabricated using the 3D ink-jet printer with six horizontal cylindrical holes running through the implants, designed for possible facilitation of vascular invasion and bone regeneration. As a control, hydroxyapatite implants (HIs) were cut manually from porous hydroxyapatite blocks. Then, craniectomy was performed to create real skull defects, and TIs and HIs were implanted. After implantation, CT was performed regularly, and the animals were euthanized at 24 weeks. No major side effects were observed. CT analysis showed narrowing of the cylindrical holes; bony bridging between the implants and the temporal bone was observed only for TIs. Histological analysis revealed substantial new bone formation inside the cylindrical holes in the TIs, while mainly connective tissues invaded the porous structures in HIs. Bone marrow was observed only in TIs. Osteoclasts were seen to resorb regenerated bone from inside the cylindrical holes and to invade and probably resorb the TIs. These data suggest that TIs are a safe and effective bone substitute, possessing osteoconductivity comparable with that of HIs.

Maxillofacial reconstruction using custom-made artificial bones fabricated by inkjet printing technology

Ideally, artificial bones should be dimensionally compatible with deformities, and be biodegradable and osteoconductive; however, there are no artificial bones developed to date that satisfy these requirements. We fabricated novel custom-made artificial bones from α-tricalcium phosphate powder using an inkjet printer and implanted them in ten patients with maxillofacial deformities. The artificial bones had dimensional compatibility in all the patients. The operation time was reduced due to minimal need for size adjustment and fixing manipulation. The postsurgical computed tomography analysis detected partial union between the artificial bones and host bone tissues. There were no serious adverse reactions. These findings provide support for further clinical studies of the inkjet-printed custom-made artificial bones.

Raloxifene and alendronate containing thin mesoporous titanium oxide films improve implant fixation to bone

This study tested the hypothesis that osteoporosis drug-loaded mesoporous TiO2 implant coatings can be used to improve bone-implant integration. Two osteoporosis drugs, Alendronate (ALN) and Raloxifene (RLX), were immobilized in nanoporous oxide films prepared on Ti screws and evaluated in vivo in rat tibia. The drug release kinetics were monitored in vitro by quartz crystal microbalance with dissipation and showed sustained release of both drugs. The osteogenic response after 28days of implantation was evaluated by quantitative polymerase chain reaction (qPCR), removal torque, histomorphometry and ultrastructural interface analysis. The drug-loaded implants showed significantly improved bone fixation. In the case of RLX, stronger bone-remodelling activity was observed compared with controls and ALN-loaded implants. The ultrastructural interface analysis revealed enhanced apatite formation inside the RLX coating and increased bone density outside the ALN coating. Thus, this novel combination of a thin mesoporous TiO2 carrier matrix and appropriate drugs can be used to accelerate implant fixation in trabecular bone.

Hydroxyapatite coating affects the Wnt signaling pathway during peri-implant healing in vivo.

Owing to its bio- and osteoconductivity, hydroxyapatite (HA) is a widely used implant material, but its osteogenic properties are only partly evaluated in vitro and in vivo. The present study focused on bone healing adjacent to HA-coated titanium (Ti) implants, with or without incorporated lithium ions (Li(+)). Special attention was given to the Wnt signaling pathway. The implants were inserted into rat tibia for 7 or 28 days and analyzed ex vivo, mainly by histomorphometry and quantitative real-time polymerase chain reaction (qPCR). HA-coated implants showed, irrespective of Li(+) content, bone-implant contact (BIC) and removal torque values significantly higher than those of reference Ti. Further, the expression of OCN, CTSK, COL1A1, LRP5/6 and WISP1 was significantly higher in implant-adherent cells of HA-coated implants, with or without Li(+). Significantly higher β-catenin expression and significantly lower COL2A1 expression were observed in peri-implant bone cells from HA with 14 ng cm(-2) released Li(+). Interestingly, Ti implants showed a significantly larger bone area (BA) in the threads than HA with 39 ng cm(-2) released Li(+), but had a lower BIC than any HA-coated implant. This study shows that HA, with or without Li(+), is a strong activator of the Wnt signaling pathway, and may to some degree explain its high bone induction capacity.

Molecular and structural patterns of bone regeneration in surgically created defects containing bone substitutes

Several biomaterials have been introduced for bone augmentation. However, information is lacking about the mechanisms of bone regeneration and/or integration of these materials in the recipient bone. This study aimed to determine the molecular and structural events in bone defects after augmentation with synthetic tetrapod-shaped calcium phosphate (Tetrabone; TetraB) compared with natural deproteinized bovine bone (DBB). Defects were created in the epiphyses of rat femurs and filled with TetraB or DBB or left empty (Sham). After 3, 6, 14 and 28 d, samples were harvested for histology, histomorphometry, ultrastructure and gene expression analyses. At 3 d, higher expressions of bone formation (ALP and OC) and remodeling (CatK) genes were detected in TetraB compared with DBB and Sham. Downregulation of bone remodeling genes (TRAP and CatK) was detected in DBB as compared to Sham after 14 d. Histomorphometry at 6 and 14 d demonstrated greater bone contact with the granules in TetraB. At 28 d, a larger bone area per defect was found in TetraB. The present experiments show that a synthetic substitute, consisting of α-tricalcium and octacalcium phosphates, induces early osteogenic and osteoclastic activities and promotes bone formation in trabecular bone defects.

Development of high-throughput screening system for osteogenic drugs using a cell-based sensor

To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.

Bone regeneration within a tailor-made tricalcium phosphate bone implant with both horizontal and vertical cylindrical holes transplanted into the skull of dogs

A new tailor-made bone implant (TI) with six horizontal cylindrical holes fabricated from α-tricalcium phosphate powder, as described in our previous report, was modified to include five additional vertical holes (TI-v) in an attempt to accelerate the bone regeneration through the holes. This TI-v implant and hydroxyapatite implants (HI) as controls were transplanted into experimental skull defects in dogs. Computed tomography (CT) was performed immediately after the surgery and then every 4 weeks. The dogs were killed for histological analysis at 24 weeks of implantation. On CT, bone bridging between the implant and the skull was observed in the TI-v group from 8 weeks of implantation, whereas a clear bone bridge was not formed in the HI group after 24 weeks of implantation. Histological analysis revealed collagen tissues and new bone formation in the horizontal cylindrical holes in most of the TI-v group, whereas mainly connective tissues invaded the porous structures in the HI group. In the Ti-v group, at the middle of the horizontal holes where they crossed the vertical holes, fibrous collagen tissues and muscular tissue filled up the hole and new bone formation seemed to be blocked. However, in the TI-v group more collagen and bone tissues were formed than in the HI group; when compared with the data in our previous report, however, the total volume of regenerated bone in the horizontal cylindrical holes in the TI-v seemed to be less than that in the TI. Thus, the addition of vertical cylindrical holes in the TI-v was not effective in promoting the faster stabilization of the TI-v in the skull of the dog.

Identification of oxytetracycline as a chondrogenic compound using a cell-based screening system.

To effectively treat degenerative joint diseases including osteoarthritis (OA), small chemical compounds need to be developed that can potently induce chondrogenic differentiation without promoting terminal differentiation. For this purpose, we screened natural and synthetic compound libraries using a Col2GFP-ATDC5 system and identified oxytetracycline (Oxy) as a chondrogenic compound. Oxy induced cartilaginous matrix synthesis and mRNA expressions of chondrocyte markers in ATDC5 cells. In addition, Oxy suppressed mineralization and mRNA expressions of terminal chondrocyte differentiation markers in ATDC5 cells, primary chondrocytes, and cultured metatarsal bones. Oxy’s induction of Col2 mRNA expression was decreased by the addition of Noggin and was increased by the addition of BMP2. Furthermore, Oxy increased mRNA expression of Id1, Bmp2, Bmp4, and Bmp6. These data suggest that Oxy induces chondrogenic differentiation in a BMP-dependent manner and suppresses terminal differentiation. Oxy may be useful for treatment of OA and also for regeneration of cartilage tissue.

Clinical application of artificial bone in the maxillofacial region

Hard tissue reconstruction is very useful for bony defects of the maxillofacial region. Autogenous bone, allogeneic bone, and artificial bone have been used to reconstruct maxillofacial bone; however, the use of autogenous bone involves high surgical invasiveness because of the need to harvest the bone. The use of allogeneic bone is associated with infections, raises ethical concerns, and is not widely used in Japan. Artificial bone has several advantages, including no need for bone harvesting, excellent biocompatibility, and a relatively easy surgical procedure. Use of artificial bone avoids the much greater invasiveness of harvesting bone, and several types of artificial bone have been developed. Design requirements for artificial bone include surgical manipulability, structural compatibility with the defective area, support properties, and the ability to induce bone regeneration; however, no artificial bone meeting all these requirements has yet been developed. Artificial bone is used in many patients in our medical center, and we have been active in developing the next generation of artificial bone with better properties. In this article, we present a case history and discuss the future development of artificial bone for use in maxillofacial reconstruction.

Healing of complement activating Ti implants compared with non-activating Ti in rat tibia

Recent studies have revealed that ozone ultraviolet (UVO) illumination of titanium (Ti) implants improves bone-implant anchorage by altering the physico-chemical and immune activating properties of the titanium dioxide (TiO(2)) layer. In the present rat tibia model, the authors compared the early events of inflammation and bone formation around UVO-treated Ti and complement activating immunoglobin g (IgG)-coated Ti. Machined Ti and machined Ti coated with a physical vapour-deposited Ti layer were used as references. Screw-shaped test and reference implants were implanted into rat tibia and harvested after 1, 7 and 28 days. Messenger RNA expression of implant adhered cells and peri-implant tissue ~250 μm from the surface were subsequently analysed with regard to IL-1β, TNF-α, osteocalcin, cathepsin K, BMP-2 and PDGF. Separate implants were retrieved after 7 and 28 days for removal torque measurements, and histological staining and histomorphometric analysis of bone area and bone-to-implant contact. While enhanced expression of inflammatory markers, TNF-α and IL-1β, was observed on IgG-coated surfaces throughout the observation time, UVO-treated surfaces indicated a significantly lower early inflammatory response. In the early phases (1 and 7 days), the UVO-treated surfaces displayed a significantly higher expression of osteoblast markers BMP-2 and osteocalcin. In summary, complement activating Ti implants elicited a stronger inflammatory response than UVO-treated Ti, with low complement activation during the first week of healing. In spite of this, the UVO-treated Ti induced only marginally more bone growth outside the implants.