**4. Discussion**

(**Figure 4a**). In contrast, no bone tissue was observed on the implant surface in the control group of animals (**Figure 4b**). Representative histological specimens of un-decalcified tissues with Villanueva bone staining are shown (**Figure 5a–d**). In the BMP-treated group, the implant was covered by a new bone that was continuous with the original ilium, and the new bone was found to have entered pores inside the implant (**Figure 5c**). The dough material pasted on the implant surface was not observed in histological specimens obtained 12 weeks postoper‐ atively. This indicates biodegradation during the experimental period. No inflammatory

**Figure 5.** Histological sections at 12 weeks after surgery [lower (\*20) and higher magnification (\*200), *scale bar* indicates 100μm, 8μm in each magnification]. (a, b) Boxed area of reconstructed axial CT image shows a histological picture of (c) (BMP-treated group) and (d) (control group), respectively. (c) In the BMP-treated group, the implant was covered by a new bone that was continuous with the original ilium, and the new bone was observed in the pore of the implant. d. In the control group, no bone tissue was observed. (arrowhead: new bone, asterisk: IP-CHA implant, cross: ilium,

In the BMP-treated group, the volume of the calcified new bone tissue around the implant on CT slice images of the pelvis peaked 3 weeks postoperatively, then decreased over time, and

arrow: fibrous tissue, circle: osteoid, box: calcified bone).

**3.4. Changes over time in the amount of the new bone formed**

response was observed around the implant over the experimental period.

150 New Trends in 3D Printing

BMPs are established osteoinductive proteins, and BMP-2 and BMP-7, in particular, are used in combination with animal-derived collagen as a carrier for spinal fusion, pseudarthrosis treatment, and open fractures of the tibia [6–8]. These cytokine therapies accomplish a high rate of bone union and have reduced the frequency of bone grafting. However, the currently available carrier material of BMP is collagen derived from animals, which carries a possibility of infectious pathogens. We established a new DDS of BMP-2; efficacy of the rhBMP-2 retaining putty for repair of the large bone defect had been presented previously by our group in other bone defect models in beagles and posterolateral spinal fusion models in rabbits. In those experimental studies, a special A-B-A type of block copolymer (molecular weight 9,800, A;-*dl polyethylene glycol* and B; *polylactic acid)* was successfully used as the carrier material for BMP-2. The polymer was originally produced by us in collaboration with a chemical company as an optimal delivery system for rhBMP-2. Because this polymer itself was highly sticky and difficult to handle, an equal weight of biodegradable β-TCP powder was mixed with the polymer to transform it to a slightly sticky putty or dough for easy implantation. Thus far, with respect to the use of this carrier material in experimental animals, no local or systemic adverse events were noted, and no significant inflammatory reaction has been observed on histology. This synthetic carrier material is a candidate for efficacious use of rhBMP-2 to repair or reconstruct the damaged bone after extensive safety check and clinical trials. Regarding the rhBMP-2 dose added to the implant, 100 μg of rhBMP-2 in combination with 400 mg of the delivery system (carrier material) per animal was used in the current study. This formulation was fixed based on our previous experimental results obtained in beagle models. As widely known, a minimal dose of BMP-2 to elicit local new bone formation in *in vivo* condition depends on the animal species, and humans are less sensitive to BMP-2. Therefore, a higher BMP-2 dose to the order of a few mg will be required for bone defect repair as observed in clinical reports of rhBMP-2 used for spinal body fusion, non-union of fracture, and other off-label use of rhBMP-2 in humans.

Recent reports have described successful reconstruction of maxillary bone defects in rabbits and bone union achievement in rats with pseudarthrosis using a combination of BMP genetransfected cells and an artificial material or using sheets of cultured mesenchymal stem cells [34, 35]. However, an expensive facility equipped with a special culture system is required to establish a cell culture system for clinical application in humans. Conversely, BMPs can be conveniently used anywhere, provided that the user has adequate storage expertise. In this study, we accomplished the accurate skeletal reconstruction after resection in a virtual iliac bone tumor model in beagle dogs by combining BMP with a 3D printer, CAD, CAM, and a computer navigation system. In future, skeletal reconstruction will become possible for more specialized skeletal tissue, such as the vertebral body and load-bearing sites.

In wide resection surgery of malignant musculoskeletal tumors, optimal tumor margins and resection margin of the bone and soft tissue would be decided preoperatively on the basis of CT and/or MRI to avoid exposing the tumor tissue to the surgical field and later local recurrence of the tumor. But the accurate resection as planned preoperatively has often proven difficult. In this experimental study, a CAS system of CT-based navigation was used as a guide for bone resection margin, and the results indicated that it is possible to perform bone resection exactly as planned using the CAS system. But planning of the exact resection margin of the soft tissue covering the tumor was not attempted in this series, and similar studies based on CT and/or MRI will be necessary to determine optimal resection margin of the soft tissue.

The 3D configuration of the defect on the left iliac bone after resection of the virtual tumor was depicted on screen by subtraction of the 3D image of the left iliac bone from the 3D mirror image of the right iliac bone. CT data of the defect images were then transferred to the CAM system to drive the system to fabricate an IP-CHA implant to fill and fit the bone defect generated in the left iliac bone. The shape and size of the implants thus fabricated were acceptable and matched the defects well. However, the IP-CHA implant alone did not repair the bone defect with a new bone, as observed in control animals, in spite of its high osteoin‐ ductive capacity due to its fully interconnected porous structure. To overcome this disadvant‐ age of the IP-CHA implant and to enhance regenerative potential of the implant, rhBMP-2 with its potent bone-inducing capacity in combination with a specific delivery system (carrier material for BMP-2) was added to the surface of the IP-CHA implant. This successfully elicited new bone formation on the surface of the implant and restored normal anatomical surface contour of the iliac bone in 12 weeks using the IP-CHA non-degradable block within the new bone mass. Replacement of the non-biodegradable IP-CHA block by a porous β-TCP implant might have resulted in complete regeneration of the bone defect without remnant biomaterials. However, because of the highly fragile nature of the porous TCP and easy breakage of the implant during fabricating and surgical procedures, the IP-CHA implant was used in this study, and the bone mass with stable connection to the original iliac bone and porous implant was created.

Recent developments in 3D printing have expanded its range of application to include production of implants, scaffolds for tissue engineering, and instruments for DDSs in addition to visualization of surgical sites preoperatively and casting of surgical tools on the basis of the additive manufacturing technology used in the present study. In addition, it has become possible to directly print biological materials onto solvent-free, aqueous-based 3D scaffolds for tissue transplantation [36–38]. Using CAD/CAM techniques, spatially defined implants can be constructed by attaching functional living cells, physiological substances, and bioactive factors in layers to organ scaffolds created on the basis of MRI or CT. For this bioprinting technique, three major modes are currently available: inkjet, micro-extrusion, and laserassisted bioprinting. These modes differ in various properties, such as the range of material viscosity accommodated, gelling method, viability, and concentration of cells to be handled. Skin and cartilage regeneration by the inkjet method, creating heart valves and blood vessels by the micro-extrusion method, and creating skin containing the heart tissue or cells by the laser-assisted method have been reported [37, 38]. We expect that for cases involving extensive tissue defects or requiring organ transplantation, printing technologies, scaffold development, cell surgical techniques, and the use of growth factors will advance further and the scope of clinical application will be further broadened if necessary tissues can be created in a shorter period of time in the future.
