**2.1. Methods**

**1. Introduction**

144 New Trends in 3D Printing

bone [2–4].

[5].

is not required) [6–8].

Extensive bone defects can occur after severe trauma, infection, or bone tumor resection, and in some cases require bone tissue reconstruction. Therefore, auto/allografts and artificial materi‐ als are implanted. Materials currently used for bone tissue reconstruction include autologous bone tissue, such as the ilium and fibula, and allogeneic materials, such as cryopreserved bone, titanium alloys, and bioactive ceramics [1]. Each of these has distinct advantages, but they also

The autologous bone is the most effective material for small bone defects and is characterized by strong bone-forming ability, accompanied by the capability of bone union and remodeling capacity. However, it has disadvantages, including limitations in the collectable quantity and complications after collection, such as pain, infection, fracture, deformation, and risk of damage to major nerves or blood vessels. The use of allogeneic bone is associated with low bone-forming ability; potential transmission of infectious agents; and cost problems, including cleanliness management. Disadvantages of the heterologous bone include possible transmis‐ sion of animal infections and immunological rejection in addition to those listed for allogeneic

Although artificial materials offer the advantages of easy access and processing, they are usually incapable of practical bone formation and thus are ineffective for bone regeneration in large bone defects. It is difficult to reconstruct relatively large bone defects in a shape that

Scaffolds, growth factors, and cells represent three key elements in regenerative medicine. An ideal approach for bone repair with regenerative medicine technology should have the abilities of osteogenesis, osteoconduction, osteoinduction, and osteointegration to resolve the disad‐ vantages of currently available graft materials. Biocompatible and biodegradable scaffolds include those made of biological materials, such as type 1 collagen and demineralized bone, and of synthetic (artificial) materials, such as porous metals, bioactive glass, synthetic poly‐ mers, and calcium phosphate ceramics [hydroxyapatite (HA) and tricalcium phosphate (TCP)]

Among bone morphogenetic proteins (BMP), recombinant human BMP (rhBMP)-2 and -7 are potent osteoinductive cytokines and are clinically used as graft materials in spinal fusion, pseudarthrosis after long bone fractures, repeated posterolateral fusion, and treatment of open fractures of the tibia in some countries, such as European countries and the United States. We expect that they will find increasing application in bone-regenerating medicine because of their high rates of bone union and their simplicity and minimal invasiveness (i.e., bone harvesting

Recent advances in computer-assisted techniques have led to computer-assisted preoperative planning, custom production of surgical implants using patient data, and use of navigation systems in orthopedics. Examples include three-dimensional (3D) printing-based preoperative planning, in which 3D printing of the relevant bone is performed before a trauma patient undergoes surgery and plates to be used are templated with the reference to the simulated

have various disadvantages or problems that remain to be solved.

is anatomically similar to the normal structure.

## *2.1.1. Preoperative planning*

In beagle dogs (male, 10-month-old, body weight: 9–11 kg) scheduled for surgery, pelvic CTs were performed a few days before the surgery, and the acquired data [digital imaging and communication in medicine (DICOM)] were used for preoperative planning with CAD software (Mimics, Magics, Materialise, NV, USA). 3D images created from CT data after DICOM data are transferred to CAD and converted into a standard triangulated language format that can be separated or combined in any way with CAD. On the 3D images of the pelvis obtained in this manner, a virtual bone tumor of 15 mm in diameter was created in the left iliac wing, and a bone resection model was created by establishing the bone resection line, 10 mm distant from the tumor (**Figure 1a** and **b**). An implant was designed to compensate for the defect occurring after resection by subtracting the bone defect part from a mirror image of the right ilium (**Figure 2a**).

**Figure 1.** (a) A virtual spherical bone tumor of 15 mm in diameter (color: orange) was created in the left iliac wing of the canine pelvis with preoperative planning on computer-aided design (CAD) software. The bone resection line was set 10 mm distant from the bone tumor on the CAD image (color: green). (b) A bone resection model was established on CAD image.

**Figure 2.** (a) CAD image of defected bone generated by subtracting the left iliac bone from the mirror image of the right iliac bone. (b) Fabricated interconnected porous calcium hydroxyapatite (IP-CHA) block by using a 3D drilling machine based on CAD data. Multiple dimples were made to facilitate retention of the BMP-retaining dough material. (c) BMP-retaining biodegradable dough material.

#### *2.1.2. Implant preparation*

We selected an artificial bone made of interconnected porous calcium HA (IP-CHA, pore size: 150 μm, porosity: 72–78%, MMT Co., Ltd., Osaka, Japan) as a material to fill the defect because of its high osteoconductive capacity [14]. CT data of the bone defect as described above were transferred to a 3D cutting machine (CAM; Modela player, Roland DG Co., Shizuoka, Japan), and an IP-CHA block (cuboid, 40 × 20 × 10 mm) was processed with a 3D drilling machine (MDZ-20, Roland DG Co.). The processed HA block was then machined with a high-speed surgical drill to create a large number of dimples facilitating the application of a BMPcontaining dough bone-forming material (described below) (**Figure 2b**). To confirm the size of the defect at the bone resection site during surgery, CAD design data were migrated to a 3D printer (ZPrinter Z-310, Toyotsu machinery, Aichi, Japan) and printed with gypsum. These were autoclaved or gas sterilized so that they could be used during surgery.

### *2.1.3. Dough bone-forming material*

**Figure 1.** (a) A virtual spherical bone tumor of 15 mm in diameter (color: orange) was created in the left iliac wing of the canine pelvis with preoperative planning on computer-aided design (CAD) software. The bone resection line was set 10 mm distant from the bone tumor on the CAD image (color: green). (b) A bone resection model was established

**Figure 2.** (a) CAD image of defected bone generated by subtracting the left iliac bone from the mirror image of the right iliac bone. (b) Fabricated interconnected porous calcium hydroxyapatite (IP-CHA) block by using a 3D drilling machine based on CAD data. Multiple dimples were made to facilitate retention of the BMP-retaining dough material.

We selected an artificial bone made of interconnected porous calcium HA (IP-CHA, pore size: 150 μm, porosity: 72–78%, MMT Co., Ltd., Osaka, Japan) as a material to fill the defect because of its high osteoconductive capacity [14]. CT data of the bone defect as described above were transferred to a 3D cutting machine (CAM; Modela player, Roland DG Co., Shizuoka, Japan), and an IP-CHA block (cuboid, 40 × 20 × 10 mm) was processed with a 3D drilling machine (MDZ-20, Roland DG Co.). The processed HA block was then machined with a high-speed surgical drill to create a large number of dimples facilitating the application of a BMPcontaining dough bone-forming material (described below) (**Figure 2b**). To confirm the size

on CAD image.

146 New Trends in 3D Printing

(c) BMP-retaining biodegradable dough material.

*2.1.2. Implant preparation*

Because BMP administered alone rapidly diffuses in vivo, it cannot maintain the local concentration at a level required for bone formation and thus does not induce bone formation. Therefore, an appropriate slow-release carrier (drug delivery system, DDS) is necessary to accomplish effective bone formation with BMP. Bovine type 1 collagen is currently used as a DDS for BMP, but it has the disadvantages of requiring a large amount of expensive BMP and the potential transmission of pathogens, such as variant Creutzfeldt-Jakob disease, which cannot be completely excluded [4].

To solve these problems, our group has developed a poly-d, l-lactic acid-polyethylene glycol block copolymer (PLA-PEG, Taki Chemical Co., Ltd., Kakogawa, Japan), which is a fully synthetic artificial DDS material that reduces the BMP dose required for bone formation [15– 25]. Given that the PLA-PEG polymer is viscous at room temperature, it is difficult to process manually but can be transformed into a dough-like material by the addition of powdery β-TCP, which can be readily processed. By adding BMP to this mixture, we devised a doughlike bone-forming material (**Figure 2c**) [26–32]. rhBMP-2 was synthesized by *Escherichia coli* transfected with the rhBMP-2 gene. After inactive monomer rhBMP-2 molecules were produced, they were dimerized to active rhBMP-2 by biochemical processing. The BMP-2 delivery system for one IP-CHA implant was generated by mixing 100 μg of rhBMP-2 (Osteopharma Inc., Osaka, Japan), 200 mg of powdery β-TCP (particle size <100 μm in diameter, Olympus Biomaterial Corp., Tokyo, Japan), and 200 mg of the PLA-PEG polymer [33]. Physiochemical characteristics of this dough bone-forming material have been reported previously. Dough bone-forming material preparations devoid of rhBMP-2 were prepared as the control group. The samples were stored at −30°C until use during surgery.

#### **2.2. Bone resection with the navigation system and implantation of BMP-added IP-CHA implant**

Data of the bone resection model created with CAD software were converted into the DICOM format. Converted DICOM data were transferred to a CT-based computer navigation system (Stealth Station Tria, Medtronic Navigation, Louisville, Co, USA). Anesthesia, ketamine (10 mg/kg) and xylazine (1.2 mg/kg) was injected intramuscularly and maintained with a contin‐ uous pentobarbital (25 mg/kg) intravenously. The left ilium was opened at the acetabular margin in beagle dogs in the right lateral decubitus position. A reference frame was secured to the iliac wing with two threaded pins and matched with navigation by point and surface registration. A Surgairtome equipped with a passive pointer was used for bone resection. Bone resection was performed as preoperatively planned according to the navigation system. The accuracy of bone resection was evaluated by testing the fit of the 3D printer-produced gypsum implant. The dough bone-forming material was then pasted evenly on the surface of the preoperatively designed and processed HA implant, and the coated implant was secured with two or three 0.6 mm wires (Synthes Co., Ltd., Tokyo, Japan). The implant was carefully sutured in place to ensure contact with the surrounding muscle. A prophylactic antibiotic agent (10 mg/kg body weight) was administered to animals before and after the surgery. This animal experiment was performed after institutional approval was obtained.

#### **2.3. Evaluation of efficacy of computer-assisted bone defect repairing system**

CT slice images of the iliac bone were obtained immediately and 3, 6, 9, and 12 weeks post‐ operatively to observe the bone repair reaction at the bone defect site and reconstructed into slice and 3D images as desired using the reconstruction software (Aze, Tokyo, Japan). CT images of the mid-axial slice of the implant and the next slice 1 mm from the center were analyzed. A high-density area surrounding the implant was considered to be a new bone area and quantified with ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Results were statistically analyzed by the Mann-Whitney U test with a significance level of P < 0.05. The ilium was harvested 12 weeks postoperatively and fixed in 70% ethanol. From un-decalcified specimens, 10 μm sections were prepared and subjected to Villanueva bone staining for histological evaluation. Specimens were macroscopically evaluated.
