3D printing of calcium phosphate bioceramic with tailored biodegradation rate for skull bone tissue reconstruction
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The bone regenerative scaffold with the tailored degradation rate matching with the growth rate of the new bone is essential for adolescent bone repair. To satisfy these requirement, we proposed bone tissue scaffolds with controlled degradation rate using osteoinductive materials (Ca-P bioceramics), which is expected to present a controllable biodegradation rate for patients who need bone regeneration. Physicochemical properties, porosity, compressive strength and degradation properties of the scaffolds were studied. 3D printed Ca-P scaffold (3DS), gas foaming Ca-P scaffold (FS) and autogenous bone (AB) were used in vivo for personalized beagle skull defect repair. Histological results indicated that the 3DS was highly vascularized and well combined with surrounding tissues. FS showed obvious newly formed bone tissues. AB showed the best repair effect, but it was found that AB scaffolds were partially absorbed and degraded. This study indicated that the 3D printed Ca-P bioceramics with tailored biodegradation rate is a promising candidate for personalized skull bone tissue reconstruction.
Keywords3D printing Skull repair Calcium phosphate ceramics Tailored biodegradation rate Bone reconstruction
The skull is a highly personalized bone tissue. Ideal skull repair and reconstruction implants are needed further explored. For children or adolescents, the inert implants used by adults are not suitable for this condition since their bone tissues are growing up. Osteoinductive biodegradable implants may be a good solution for this case. With the improvement of people’s health level, patients are currently not satisfied with the use of traditional bone orthopedic implants to restore basic shape restoration but to reconstruction of their skulls. This requires new generation of biomaterials with accurate shape to match the defect and tailored biodegradation rate to support bone regeneration . Calcium phosphate (Ca-P) shows good osteoconductive and osteoinductive and is a promising bone repair biomaterial owing to their similar composition to normal bone [2, 3, 4, 5].
Ca-P ceramics with suitable pore structure and composition were found to be osteoinductive under certain circumstances. Zhang et al.  reported that their synthetic porous Ca-P ceramics induced osteogenesis in a non-bone environment, and later explored the mechanism of induced osteogenesis. At present, the international academic community has reached a consensus on the osteoinductivity of Ca-P ceramics, which promotes the application of Ca-P ceramics in clinical medicine. Ca-P with different compounds has been successfully synthesized in the laboratory. As the calcium phosphate ratio (Ca/P) decreases, the solubility and hydrolysis rate of calcium phosphate salt increased; therefore, only optimized Ca/P composition ceramic is suitable for implantation . Hydroxyapatite (HA) is very similar in composition to the inorganic matter of human bones, but it dissolves very slowly in the body [6, 8]. Ripamonti et al. reported that the porous HA ceramics using coral reefs and implanted into the muscles of the iliac crest observed bone induction phenomenon . Tricalcium phosphate (TCP) has two crystal phases: α- and β-tricalcium phosphate (TCP) . β-TCP showed better biological activity and become the popular bone substitute biomaterials. β-TCP possesses a Ca/P ratio of 1.5, which is more soluble than HA [11, 12]. Although β-TCP shows a higher dissolution rate, it is difficult to retain a temporary mechanical support for the desired duration of implantation. Yuan et al. prepared α-TCP and β-TCP ceramics with the same macroscopic structure and microstructure, and implanted them into the dorsal muscle of the dog. It was found that only β-TCP ceramic formed new bones inside the scaffolds . HA has good biological activity, but it showed poor biodegradation rate in vivo. β-TCP has good degradability, but it showed poor mechanical properties. Therefore, a large number of studies have been investigated combining β-TCP and HA together, i.e., the biphasic calcium phosphate ceramics (BCP), which have better biological properties than HA or β-TCP alone [14, 15, 16, 17, 18]. By controlling the composition ratio and preparation conditions of the material, BCP with suitable degradation rate can be tailored. The degradation rate of BCP depends on the ratio of HA and β-TCP. The higher the ratio of β-TCP, the better the degradability.
In bone tissue engineering, the bone repair materials only need to mimic the chemical composition of the natural bone, but it is also needed to achieve the desired degradation rate. The chemical composition and the porous structure of the scaffolds determined the bioactivity of the material [8, 19, 20, 21, 22, 23, 24]. Complex porous structures scaffolds are difficult to obtain via conventional fabrication process . In the past two decades, a lot of porous scaffolds fabrication methods have been reported; for example, freeze-casting [26, 27], addition of porogen [28, 29], gas foaming method [30, 31, 32] and directional crystallization method  were widely studied. The above techniques showed some shortcomings; for example, it is difficult to customize internal pore structure. It is difficult to prepare a transplant with a complex shape. Three-dimensional (3D) printing technologies show better ability to control the porosity of the scaffolds [34, 35]. By using the 3D printing technologies, it is very easy to control the composition and the porous structure of varied bone tissue scaffolds. It was reported that this 3D printing technologies have been successfully used to make different types of bone tissue implants [3, 36, 37, 38].
It is clear that a biodegradable scaffold after implantation is intimately connected with its new bone regeneration and biodegradation. Therefore, it is crucial to explore a new type of controllable degradation rate scaffold to reconstruct the adolescent bone repair. In this study, we proposed calcium phosphate scaffolds with tailored biodegradation rate by using 3D printing technology. The degradation rate of the calcium phosphate scaffold is controlled by adjusting the composition and porous structures of the scaffold, which are expected to achieve a balance between biodegradation of scaffold and new tissue growth. A simple animal model was proved on the beagle skull repair. It indicated that the 3D printed Ca-P bioceramics with tailored biodegradation rate is a promising candidate for personalized skull bone tissue reconstruction.
Calcium phosphate powders (HA, β-TCP, BCP) were purchased from National Engineering Research Center for biomaterials (Chengdu, China). PVB (polyvinyl butyral, Mn = 70,000–90,000), ethanol (AR), PVA (polyvinyl alcohol) and PEG (polyethylene glycol PEG-2000) were purchased from sigma. The printing ink was prepared by mixing PVB, calcium phosphate powders and ethanol in a ratio of 3.6:32:32. The ink was continuously stirred in open environment to maintain powders distribution and increase the viscosity by solvent evaporation until ideal viscosity for 3D printing.
Tailored Ca-P bioceramic biodegradation rate by 3D printing
Post-treatments and sintering
After the specimens were printed, the specimens were dried for 4 h at temperature of 60 °C; then, the specimens were sintered in muffle furnace. Briefly, specimens were sintered by two-step sintering process. Firstly, specimens were heated by rate of 5 °C/min to 600 °C and hold for 2 h to evaporate polymer and form micropores. Then, specimens were heated to 1100 °C with same heating rate and hold time. Finally, specimens were cooled down to room temperature in the furnace.
Characterization of the tailored biodegradable Ca-P bioceramics
The morphology of the printed specimens was evaluated by scanning electron microscope (SEM, JSE-5900LV, Japan). The specimens and powders were sputter-coated with gold before observation. Phase composition of the scaffold was analyzed using XRD (Philips X’Pert 1 X-ray diffractometer, Netherlands) with CuKα radiation at a current of 20 mA and voltage 30 kV. Scans were performed with 2θ values from 20° to 60° at a rate of 0.05°/s. The obtained peaks were compared with standard references in the JCPDS file available in the software for HA (09-0432) and β-TCP (09-0169). The mechanical properties of the printed specimens were tested with electronic universal testing machine. Standard test specimens which have different porosities were fabricated as cylinder with φ7 × 12 mm. Stress–strain curve of the scaffolds was performed at a crosshead speed of 1 mm/min, and compressive strength was generate by stress–strain curve. The force was loaded along the vertical direction of the pores. The shrinkage after sintering was measured by Vernier caliper.
In vivo experiments
Animal skull defect models
Beagles were obtained from the Sichuan University Laboratory Animal Center (body weight: 10–11 kg; gender: male). All the experiments were approved by the Animal Care and Use Committee of Sichuan University. The animals were anesthetized by intraperitoneally injecting 0.02 g/ml pentobarbital sodium before experimentation (40 mg/kg body weight; Sigma Chemical, St. Louis, MO, USA). The three-dimensional data of the skull of experimental animals were obtained by computed tomography (CT, Optima CT680, GE Medical, USA). Then, the 3D model of the skull was established by computer. And two round defects with a diameter of 15 mm of the skull were generated by modeling software to mimic a freeform bone defect. The shapes of defects were remolded by Boolean operation through Magics 22 (Materialise, Belgium).
The animals were anesthetized by intraperitoneally injecting 0.02 g/ml pentobarbital sodium before implantation (40 mg/kg body weight; Sigma Chemical, St. Louis, MO, USA). Then, two pre-designed bone defects on the skull of the experimental animal were prepared by surgeon team. The autologous bone (AB) taken from the defect site was used as a new repair material. The experiment consisted of two groups of beagles: one group was implanted with 3DS and FS scaffolds, and the other group was implanted with autologous bone (AB) and 3DS scaffolds. Triple parallel specimens were tested for each implantation. After implantation, the beagles were continuously injected with penicillin at 105 U/kg per day for 3 days to prevent infection. The specimens were harvested after 5 months of implantation by killing the beagles. The implanted specimens were extracted from the surrounding tissues and washed with a phosphate-buffered saline (PBS). The attached tissues were washed with a mixture of PBS (90 wt%) and pepsin (10 wt%). The harvested implants were then fixed in 10% formaldehyde in PBS solution for 2 weeks and then dehydration treatment. The dehydrated process was taken with a gradient of ethanol solutions (60%, 80%, 96%, 100%, 100%, ethanol/technovit 7200 VLC: 70/30, ethanol/technovit 7200 VLC: 50/50, ethanol/technovit 7200 VLC: 30/70, and technovit 7200 VLC: 100%) 1 day for each solutions, and in 100% technovit 7200 VLC for 1 week. The dehydrated samples were embedded in technovit 7200 VLC and light curing (Technovit 7200 VLC, Kulzer-Exakt, Wehrheim, Germany), and then mounted with resin onto sample holders suitable for an automated brushing machine (SD Mechatronik GmbH, Feldkirchen-Westerham, Germany). The embedded specimens were cut into 5 μm histological slides by using Leica Polycut (Leica, SM 2500E, Germany) and transferred to glass slides. The sections were stained with hematoxylin and eosin (HE) for histological analysis.
Composition and phase analysis of 3D printed Ca-P ceramics
Morphology of tailored biodegradable Ca-P bioceramic
The mechanical property and shrinkage
Phase composition and biodegradation rate
Ca/P ratios and the aqueous solubility of several Ca-P phases
Beagle skull regeneration
The AB and 3DS were observed for 8 months after implantation. The model with the reconstruction of the CT data is shown in Fig. 5b. By the time of repair to 8 months, the autologous bone was absorbed obviously, and the gap between the tissue and the surrounding tissue continuously increased. In contrast, 3DS showed better repair ability.
Histological observation and biological functions
Vascularization in 3DS and FS
Effect of the composition and structures on the degradation of 3D printing Ca-P ceramics
Personalized repair requires precise dimension implants. At present, direct 3D printing technology can only be used to prepare ceramic green bodies, which actually will shrink after sintering and resulting in an error between the design models and the printed scaffolds. Therefore, the design models should be scaled up according to the shrinkage before and after sintering of the ceramic to prepare the required scaffolds. After a large number of measurements, the shrinkage rate of calcium–phosphorus ceramic in this study was about 22% in linear direction. It should be noted that the horizontal shrinkage is slightly greater than that of longitudinal direction, which may be because of the slight compression of the material under the action of gravity during the printing process.
Calcium phosphate shows excellent osteoconductive and osteoinductive and is a promising bone repair biomaterial. In this paper, the degradation rate of the scaffold was controlled by adjusting the porous structure and the material composition, which may tailor the biodegradation rate to match the growth rate of new bone regeneration. In previous studies, it was proved that idea degradation rate of the implants helped bone repair. 3D printing technology can accurately control the pore structure and material composition of the scaffolds, so that it can design and tailor the degradation rate of the scaffold. Smaller the diameter of the filament inside the scaffold caused faster degradation rate of the scaffold. In this study, we have prepared different porosity scaffolds, and the scaffolds with larger pores show higher porosity (Fig. 3), which theoretically corresponds a faster degradation rate. However, increase in porosity led to decrease in mechanical properties. We have determined the relationship between porosity and compressive strength, when the porosity was 60%, 70% and 80%; the compressive strength was 5.5 MPa, 3.0 MPa and 1.0 MPa, respectively. The compressive strength is much lower than the compact bone, which is just close to the mechanical requirements of cancellous bones. The increased compressive strength is limited for the porous BCP bioceramics by direct 3D printing technology. 3D printing technology is used as a forming technology to design and prepare the macroscopic structure of materials, but the final mechanical properties of ceramics depend on the property and inherent properties of the printing material. Although calcium phosphate ceramics show excellent bioactivity and osteoinductive properties, their mechanical strength and fracture toughness are relatively low. So, this type of material cannot be used for load-bearing bone defects. It is mainly used as a filler and coating. 3D printing strategy provides the possibility to design a porous scaffold with different degradation rates according to different bone repair requirements. A scaffold with a gradient pore structure can be designed to realize the gradient degradation of scaffold to meet gradient degradation application as shown in Fig. 1b.
Ca-P with different phase compositions showed different solubility. Hydroxyapatite has lowest solubility and hardly degrades in vivo, while the degradation rate of tricalcium phosphate is too fast to retain a temporary mechanical support for implantation duration. Therefore, a composite mixture consisting of HA and TCP was able to achieve an optimum solubility. From the phase composition and solubility study, as well as the comparison of osteoinductivity, it founded that a higher solubility of Ca-P ceramics results in higher osteoinductivity. These results are consistent with previous literature reports; the trend of osteoinductivity is BCP > β-TCP > HA > α-TCP [5, 39, 40]. Therefore, this research provided a 3D printing strategy to regulate the degradation rate of bioceramic by regulating different its components. Various formulations of calcium phosphate, i.e., different proportions of HA and TCP, have been successfully used in clinic; it depends on what kind of their degrade rate is needed [39, 40, 41]. Although the clinically used BCP is usually composed of nearly 60–70% HA and 30–40% β-TCP, the BCP consisting of 30% HA and 70% β-TCP promoted higher expression of bone morphogenetic protein-2 (BMP-2) and showed stronger osteoinductivity in mice than BCP (70% HA and 30% b-TCP), pure b-TCP and HA . The BCP (70% β-TCP and 30% HA) degraded faster than the BCP consisting of 30% β-TCP and 70% HA, and is more suitable for defect that requires rapid degradation rate of the implants. This study only provides a way to regulate the degradation rate of calcium phosphate ceramics through 3D printing method. The specific formulation of calcium phosphate ceramics depends on the requirements of clinical application.
Regeneration of the skull
Although calcium phosphate ceramics show excellent bioactivity and osteoinductive properties, their mechanical strength and fracture toughness are relatively low. This type of material cannot be used for load-bearing bone defects. It is mainly used as a filler and coating. This research actually simulates the surgical procedure and proves its feasibility application in craniofacial and maxillofacial regeneration, which is conducive to the promotion and application of 3D printed ceramics. The skull model of the target animal was first obtained by CT, and a circular defect was created. Then according to the defect shape, the corresponding implant model was designed by computer and the ceramic scaffolds were prepared by 3D printing technology. Therefore, the 3DS was closely combined with the surrounding tissue after implantation and was more suitable for the original skull curve. It satisfied the aesthetic requirements of the craniofacial repair. The new bone tissue grew along the surface of the scaffold to the center; new bones were found at outer perimeter of the 3DS. There was isolated new bone tissue inside the scaffold. This part of the bone tissue was not combined with the surrounding bone tissue. It may be the induced new bones. The circular pores of the gas-foamed ceramic (FS) were full of new bone tissues.
In addition to the influence of the phase composition of the material, the porous structure of the scaffold also plays a key role in biological activity. Ripamonti et al. made a concave pore with a diameter of 1.6 mm and a depth of 0.8 mm on the surface of the HA. After implanting it into the baboon muscle for 90 days, only new bone formation in the concave pore of the implant was found . It is indicated that the concave pore facilitates the formation of new bone. The pore structure affects the aggregation state of the cells, which in turn affects the activity of the material. The proposed scaffolds have spherical concave porous structure which may be beneficial to the growth of new bone. In addition, the pore size of natural bone tissue ranges from millimeters to nanometers. The scaffolds with a series of hierarchical porosity within the micron range could mimic the hierarchically porous structures of natural bones and promote regeneration of new bone. In bone tissue engineering, the vascularization is one of the most important of components. A larger number of new blood vessels were found in 3DS (Fig. 7), indicating that a highly interconnect pore structure possibly facilitates the growth of blood vessels. These vessels may help in regeneration of new bones.
Although autologous bone is the best active material for bone repair, autologous bone still has limitations, and it must be in close contact with the original bone tissue to be successfully repaired. In this surgery study, it may be due to the excessive gap of the original implanted autologous bone and surrounding tissue, and the autologous bone repair group showed poor reconstruction results. 3D printing technology can print the scaffolds with accurate dimension matched with the defect sizes. In addition, 3D printing can be used for fabrication of macroporous structures of scaffolds, but 3D printing technology is still unable to obtain microstructures smaller than 10 μm due to the print nozzle accuracy. Therefore, the final hierarchical porosity of ceramic depends on the optimization of the material composition design and post-sintering process.
3D printing technology provides a versatile strategy to fabricate Ca-P ceramics with accurate shape to match the defect and tailored biodegradation rate to support bone regeneration. The raw materials used in this study are the Ca-P powders with good bone regeneration ability; by using the 3D printing technology, the porous structures and phase composition of the scaffold can be well controlled and tailor the degradation rate. The 3D printed Ca-P ceramic with required geometry and size is good for developing patient-tailored implants. The mechanical properties of the scaffolds can meet requirements for cancellous bone implantation. Based on clinical surgical cases, a personalized skull defect repair was conducted and proved the possibility of regeneration of skull. Vascularization phenomena were observed in the 3DS scaffolds, which promoted bone regeneration in the later stage. This research provided a 3D printing strategy to regulate the degradation rate of bioceramic by regulating scaffolds composition and porous structures. It indicated that 3D printed Ca-P bioceramics with tailored biodegradation rate is a promising candidate for personalized skull bone tissue reconstruction.
This work was supported by the National Key Research and Development Program of China (No. 18YFB1105600, 2018YFC1106800), National Natural Science Foundation of China (51875518), Sichuan Province Science & Technology Department Projects (2016CZYD0004, 2017SZ0001, 2018GZ0142, 2019YFH0079), Research Foundation for Young Teachers of Sichuan University (2018SCUH0017) and The “111” Project (No. B16033).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
The animal experiments were approved by the Animal Care and Use Committee of Sichuan University. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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