An HA/PEEK scaffold with modified crystallinity via 3D-bioprinting for multiple applications in hard tissue engineering

Hard tissues, including bones, teeth and cartilage, provide support and protection for other organs of the human body, and their capacity to new-renew is limited [1]. Large scale of bone defect, as well as tooth loss is irreversible and always needs autologous or alloplastic materials for reconstruction. Although autologous bone grafts are always viewed as an ideal way, their clinical application is much limited for the strict accessibility, unpredictable resorption rate and poor plasticity [2, 3]. Hard tissue engineering provides another promising strategy to reconstruct hard tissue defect [4, 5]. The porous scaffolds provide support for the defect and platform for cell growth. The bioactive factors of the scaffold promote the regeneration [6]. Hard tissue engineering puts forward higher requirements for mechanical properties of scaffolds than soft tissues, especially in terms of bones and teeth which are highly mineralized, that the scaffolds need enough stiffness to withstand pressure and weight [7]. But inappropriate stiffness of scaffolds like metal materials represented by titanium alloy causes stress-shielding and further leads to bone fracture [8]. Due to the pressure from the muscle movement and mastication, the hard tissue engineering scaffolds for bones and teeth are required both stiffness and ductility to avoid breaking itself and bone fracture. Polyether ether ketone (PEEK) as a common-used implants in spine surgery, orthopedic surgery and maxillo-facial surgery, has excellent physical and chemical properties [9, 10]. However, its bio-inertia makes it difficult to produce effective combination with tissues and easy to form fiber wrapping as an implant material [11].

Hydroxyapatite (HA) is a commonly used bioactive material for its osteogenic induction ability [12, 13]. Previous studies have found that mingling HA with the PEEK tissue engineering scaffolds can enhance their osteogenic activity and tissue binding ability [14]. HA/PEEK composites seem a potential material for ideal hard tissue engineering scaffolds. However, composite HA will affect the mechanical properties of PEEK itself, bringing adverse influence to the application of PEEK scaffolds [15]. In latest studies trying to improve the biological activity of PEEK by mixing HA, it was found that the modified PEEK scaffolds tend to be more brittle and have a higher risk of fracture [16, 17]. Several efforts have been made to balance the weakening of mechanical properties of PEEK scaffolds caused by the addition of HA. Yu et al ameliorated the HA/PEEK composites by adjusting the content of nano HA and showed that the tensile, compression, bending and impact strength of the composites were improved compared with pure PEEK [18]. Converse et al found that there was a reinforced interface between PEEK matrix and HA whisker by using powder processing and molding technology, and its elastic modulus and tensile strength were similar to those of human cortical bone [19]. Even so, HA/PEEK mixtures prepared by different preparation processes have great uncertainty and poor repeatability. To achieve the balance between the mechanical and biological properties of HA/PEEK scaffolds would be one of the breakthroughs in constructing an ideal hard tissue engineering scaffold based on PEEK.

Additive manufacturing (AM) is a promising strategy to solve the problem of the poor repeatability of HA/PEEK composites and is potential to accurately control the mechanical properties and bioactivity. Fused deposition melting (FDM) is a representative technology of three-dimension (3D) printing [20]. In terms of macro morphology, it can customize the corresponding clinical samples of different defect models. As for microstructure, it is expected to accurately control the internal structure of the material by adjusting the printing parameters [21], and affect the physical and chemical properties of the material based on the change of the internal structure of the material, that is, the crystallinity [22].

Crystallization is that the molecular chains of materials are arranged according to the ordered structure, and the crystallinity is the proportion of the crystalline region in the polymer to the total material. The degree of crystallinity directly affects the physical and chemical properties of the material [23, 24]. The properties of HA/PEEK composites constructed by different preparation processes are obviously different due to different internal crystallinity. The crystallinity of HA/PEEK composites prepared by traditional manufacturing process is affected by preparation temperature, forming pressure, auxiliary nucleating agent and other factors and is uneasy to control, which is manifested in unstable material properties, uneven internal structure and poor repeatability between batches. By adjusting the printing parameters, 3D printing technology can accurately regulate the crystallinity of HA/PEEK composites to achieve the purpose of regulating their physical and chemical properties [25].

We put forward a strategy to fabricate an HA/PEEK scaffold whose mechanical properties were precisely controlled by modified internal crystallinity and biological properties by HA mix ratio. The HA/PEEK hard tissue engineering scaffolds were further applied in three chosen sites in beagles, which were rib, mandible and tooth, to prove the practical performance (figure 1).

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2.1. Fabrication and characterization of HA/PEEK scaffolds

2.2. Cytocompatibility of HA/PEEK scaffolds

2.3. Osteogenic differentiation induction of HA/PEEK scaffolds in vitro

2.4. Biological evaluation of HA/PEEK scaffolds in vivo

2.5. Statistic analysis

3.1. Characterization of 3D-HA/PEEK scaffolds

As well known, HA could give PEEK scaffolds a great improvement of bioactivity. But the optimal ratio is still controversial. In scanning electron microscope (SEM) images, we saw that the HA powders were basically sphericity (figure S3). Using two typical printing modes named as high/low crystallinity printing modes, the HA/PEEK scaffolds with different concentration of HA could demonstrate different crystallinity. TG-DSC proved that the two printing modes did endow PEEK, 10%HA/PEEK, 20%HA/PEEK, 30%HA/PEEK scaffolds with different crystallinity (figures 2(a) and (b)). Using XPS, we found that the mixture of HA and the change of crystallinity had little influence on the element composition of the PEEK scaffolds, with the C, O, N element in PEEK, 10%HA/PEEK, 20%HA/PEEK, 30%HA/PEEK scaffolds with both high and low crystallinity all at the same level (figures 2(c)–(f)).

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3.2. Evaluation of mechanical properties

3.3. Evaluation of cytotoxicity and cell proliferation

Based on the results of the mechanical evaluation, we found that scaffolds with low crystallinity were more suitable. Since titanium and its alloy is still the usual material for the bone reconstruction and dental implant, we used titanium alloys (Ti) as the positive control group. BMMSCs were seeded on the Ti, PEEK, 10%HA/PEEK, 20%HA/PEEK and 30%HA/PEEK scaffolds and cultured for 24 h. We could see from the SEM that the cells adhered to the surface of different materials and unfolded themselves especially on the 20%HA/PEEK scaffold (figure S4). Live/dead staining showed that most of cells in all five groups were live (green), and few cells were dead (red) (figure 3(a)). The quantitative analysis also showed that there was no significant difference in cell survival rate between groups (figure 3(b)), which proved that HA/PEEK materials printed with low crystallinity have no obvious cytotoxicity, as well as Ti and pure PEEK scaffolds. CCK8 and Ki67 staining were performed using the extraction liquid of the five scaffolds. BMMSCs were cultured for 1, 3, 5, 7, 9 d. CCK8 proliferation curves showed that the addition of HA increased the proliferation of BMMSCs after 7 d (figure 3(e)), which was proved by the results of Ki67 staining as well (figures 3(c) and (d)).

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3.4. Evaluation of cell migration

The results of cell scratch test showed that the cell scratch closure speed in HA/PEEK groups was faster, suggesting stronger cell migration ability (figure 4(a)). The statistical results showed that after 24 h, a significant reduction of the scratch width began to appear in 20%HA/PEEK and 30%HA/PEEK groups (figure 4(c)) (p < 0.05). We further used the Transwell plates to elucidate the attraction of HA/PEEK scaffolds to BMMSCs by putting the materials on the lower chambers and BMMSCs on the upper chambers. BMMSCs could move through the 8 μm pores of the filter membrane in all groups, while the number of cells was much larger in 10%HA/PEEK, 20%HA/PEEK and 30%HA/PEEK groups (figure 4(d)). By adding HA to the PEEK scaffold, the composites showed a proved ability to attract cells and promote the cell migration, which was obvious when the HA mixture ratio was 20% and 30% (w/w).

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3.5. Evaluation of osteogenic differentiation in vitro

3.6. Evaluation of tissue integration of subcutaneous implantation in SD rats

To evaluate soft tissue integration, we implanted Ti, PEEK and 20%HA/PEEK scaffolds with low crystallinity into the subcutaneous areas of 8 week male SD rats (figure 5(a)). We found that the rats all kept in good health with stable bonding of scaffolds and no ulcers on the back skin after 4 weeks and 8 weeks. The tissue sections showed continuous soft tissue–material interfaces in all three groups after implanted for 4 weeks (figure 5(b)) and 8 weeks (figure 5(c)). And there seemed to be a trend for the soft tissue to grow into the inner lacunae of 20%HA/PEEK scaffold with low crystallinity which was reserved in the bioprinting process for better tissue integration (figure 5(b)).

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3.7. Reconstruction of tooth loss in beagles

To imitate the tooth implantation to see the scaffolds' capacity as dental implants to reconstruct the tooth loss, we implanted the designed frustum scaffolds (figure S5) into trimmed alveolar sockets in beagles. After 4 weeks, the wounds of Ti scaffolds and 20%HA/PEEK scaffolds with low crystallinity all closed well, while PEEK scaffolds with low crystallinity (figure 6(b)) and one of the Ti scaffolds (figure S6) appeared to extrude out of the gingiva after 8 weeks. Two of three PEEK scaffolds with low crystallinity had an unusual mobility and one had already fall out after 8 weeks. The success rate was shown in table 1, with Ti scaffolds and 20%HA/PEEK scaffolds sharing a 100% success rate. The tissue sections demonstrated that both Ti and 20%HA/PEEK scaffolds could achieve a tight combination with the surrounding tissue, while PEEK scaffolds tended to have gaps between the tissue (figure 6(c)). There were fibers similar to periodontal ligament between Ti scaffolds and alveolar bone, but the orientation of fibers was parallel to the surface of Ti scaffolds, unlike the vertical structure as Sharpey's fiber (figure 6(c)). New-born blood vessels could be seen in the connective tissue formed between 20%HA/PEEK scaffolds and the alveolar bone. Unsurprisingly, compared with pure-PEEK scaffolds, adding HA in an appropriate proportion in PEEK scaffolds could activate the scaffolds, so that they could better combine with soft/hard tissues. Moreover, 3D printing created regular pores in the scaffolds allowing the tissues to grow into and obtain higher implant stability than titanium alloy scaffolds. The 20%HA/PEEK scaffolds seemed to be a better dental implant material than titanium alloy.

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Table 1. Prognosis of alveolar socket implantation in beagles. The prognosis of Ti, PEEK and 20%HA/PEEK scaffolds were assessed according to whether they had mobility or dislocated from the alveolar socket.

Alveolar sockets implants	Total number	Prognosis
		Mobility	Dislocation
Ti	3	2	1
PEEK	3	0	0
20%HA/PEEK	3	0	0

3.8. Reconstruction of large-scale bone defects in beagles

Considering that the fixture of rib bone fracture similar to dentofacial bone defect was largely affected by external force due to daily breathe movement and mastication, we chose two sites in beagles, mandible and rib, to build defect models and conduct the implantation. After 12 weeks of implantation in vivo, we photographed computed tomography (CT) and performed 3D model reconstruction of mandible (figure 7(b)) and rib (figure 7(f)) according to the images. The 3D model showed us the location of the defect. Two sites were selected in the anterior and posterior parts of the mandible defect area as examples. When observing the CT images from the sagittal, coronal and axial directions, it was found that the bone in the mandible defect area was continuous and not absorbed (figures 7(c) and (k)), demonstrating that 20%HA/PEEK scaffold with low crystallinity successfully endured the complex external forces without any harm to both surrounding tissue and the scaffold itself, largely due to the flexible mechanical characteristics. We also saw from the tissue sections that the scaffolds could combine to the surrounding tissue closely and showed little inflammation (figure 7(d)), which was inseparable from the excellent biological performance brought by the reasonable addition of HA.

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The need to participate in breathing activities requires the ribs a physiological mobility caused by external force. To realize the function, the ribs are flexible and not easy to fold, which also conforms to the mechanical properties of 20%HA/PEEK scaffolds with low crystallinity. The two broken ends of the rib defect were selected as the two observation sites, and the CT image showed that the scaffold was closely combined with the bone tissue (figure 7(g)) and the density maintained the same as the normal bones (figure 7 (l)). We could also observe normal lung tissue from CT images, which meant that the scaffold will not affect adjacent organs (figure 7(h)). From tissue sections, we observed that the inner and outer surfaces of the tune-shaped scaffold were connected with the outer fibrous connective tissue (figure 7(I)) and the inner bone tissue (figure 7(j)) respectively. These results illustrated that 20%HA/PEEK scaffolds could be an ideal bone tissue engineering material and had a great potential to be used in the reconstruction of other hard tissue defect.

The injury of hard tissue, especially those of high mineralized tissues like bones and teeth, often requires an exogenous material for reconstruction. An ideal type of this bone or tooth repair material is expected to display good mechanical properties which allow it endurable under the complex forces during mastication, and proper bioactivities for regeneration. The elastic modulus of PEEK is close to that of cortical bone, which is conducive to solving the problem of stress shielding caused by the inapt elastic modulus of titanium [26, 27]. In addition, PEEK can be used as a raw material for 3D printing, which greatly broadens its application in the medical field, especially in regenerative medicine. However, the bio-inertia of PEEK makes it hard to produce effective combination with the surrounding tissue, let alone to promote osteogenesis. Two major methods have come forward to activate PEEK material. One is to blend other biomaterials into it, which would do damage to its mechanical properties [12, 14]. The other option is to construct an activated surface on PEEK without affecting its inherent properties, but the surface modifying is unstable [11, 28, 29]. We have not yet found previous studies succeeding to achieve the balance between the mechanical properties and biological activation of PEEK scaffolds.

In this study, we successfully constructed a composite of HA and PEEK by changing the internal crystallinity of the material, taking into account both mechanical and biological properties. The micro crystal structure of polymer materials affects the macro physical and chemical properties. The heat treatment conditions during 3D-bioprinting can change the crystallinity. In the process of high-temperature printing, the printed samples need to gradually recover from the high-temperature state to room temperature, and the internal molecular chain structure is gradually arranged to form a crystal structure in the process of slow temperature decline. If the external temperature drops suddenly, the arrangement process of molecular chains will be limited, making it difficult to form a neat and regular crystal structure and resulting in the decrease of crystallinity. The cooling rate of the polymer changes with the external temperature followed by the change of the crystallinity, which is easily affected by the characteristics of heterogeneous materials. Therefore, it is difficult to accurately control the thermal conditions in the preparation process. FDM as a common 3D-bioprinting technology, can create complex thermal conditions with the accurate control of cooling rate thus to control the thermal properties of the polymer materials during the melting and extruding processes. Geng et al [30] used the air-force cooling in FDM and succeeded to improve the mechanical properties of the 3D printed polyphenylene sulfide, which also gave us the idea to apply this principle to PEEK. And in this study, we designed two printing modes corresponding to low/high crystallinity based on FDM, which was proved by TG-DSC (figure 2(a)).

The addition of HA endows PEEK scaffolds with good biological properties and enables the polymer scaffolds to better integrate with natural tissues and even promote regeneration, but at the same time this composite structure brings mechanical properties different from pure PEEK. As we can see from figure 2(g), the mixture of HA did change the mechanical properties compared with pure PEEK material, mainly in the reduction of bending stress and compressive stress and little change of corresponding modulus, but the relationship between this change and the mixing ratio is not clear. However, changes in mechanical properties brought about by HA mixing could be completely masked by those by changes in crystallinity. The 20%HA/PEEK fabricated by high and low crystallinity printing mode both met the national standard for PEEK for surgical implants application, but the impact resistance and elastic deformation ability of HA/PEEK prepared by printing with low crystallinity were more suitable as bone/dental restorative materials, considering that the material especially those to repair teeth loss or facial bone defect would be subjected to various directions and magnitudes of force transmitted to normal bone tissue when performing its function [3]. Following in vitro studies confirmed that different mixture ratio of HA was associated with the proliferation, adherence and differentiation of stem cells represented by BMMSCs (figures 3 and 4). The osteogenesis effect brought by mixing HA into PEEK is very significant, and the crystallinity inside the mixed material is adjusted by FDM bio-printing, which can obtain stable mechanical characteristics of strength that meets the requirements of clinical use and toughness and elasticity suitable for bone conduction.

To optimize the clinical application strategy based on the mechanical properties of different materials, it is required that the materials have better elasticity, so as to resist external force through their own deformation without brittle fracture. Therefore, in this study, 20% HA/PEEK composites with low crystallinity printing with better strength and hardness, elasticity, toughness, impact resistance and fatigue resistance were selected as the experimental group for the evaluation in vivo. The ability of 20%HA/PEEK to bind to natural tissues was evaluated using a rat subcutaneous model. Whether it is the implant material for teeth loss or the restoration material for maxillofacial bone defects [31, 32], titanium alloys are very commonly used, thus titanium alloys were chosen as the positive control group. And we found that 20%HA/PEEK could achieve similar tissue integration to titanium alloys as dental implants, and better tissue ingrowth could be observed inside 20%HA/PEEK thanks in part to the porous structure produced by 3D printing. In order to further simulate the repair of clinical bone defects, we prepared an irreversible bone defect in the mandibular premolar region of beagles, and implanted a 3D printed 20%HA/PEEK scaffold strictly designed corresponding to the size of the defect, showing tight binding to surrounding bone tissue and no general health hazard. In addition to the field of stomatology, PEEK materials have been used in many medical researches [33–38], including many animal experiments and clinical application on various hard tissues such as tibia, femur, rib and so on [18, 39–42]. However, few animal experiments are conducted on the HA/PEEK scaffolds [28, 43]. As shown in figures 7, 20%HA/PEEK also showed excellent biological properties in the rib transplantation of beagles, which demonstrated that this scaffold could be used in the repair of different bone tissues. Both the effect of dental and bone reconstruction of 20%HA/PEEK scaffold with low crystallinity demonstrated it was versatile in the field of hard tissue engineering. However, the limitation of this study is that we were unable to analyze the force of PEEK implanted in animals due to technical reasons.

In this study, we designed a 20%HA/PEEK scaffold with low crystallinity by FDM for multiple applications in hard tissue engineering. The low crystallinity printing mode realized the regulation of the internal crystallinity of HA/PEEK composites and endow them with excellent mechanical properties. The 20%HA/PEEK scaffolds showed the best ability to promote cell adhesion, migration, proliferation and osteogenesis compared to pure PEEK, 10% HA/PEEK and 30% HA/PEEK. The 20%HA/PEEK scaffolds with low crystallinity further showed good biocompatibility in animal implantation experiments, and its osteointegration ability was comparable to that of titanium alloys and better than pure PEEK. And the scaffolds fabricated under this strategy did succeed to reconstruct different bone/tooth defects in beagles.

All data that support the findings of this study are included within the article (and any supplementary files).

This study was supported by Nature Science Foundation of China (31971281), Innovative Talents Program of Sichuan Province (2022JDRC0043), Research and Develop Program, West China Hospital of Stomatology Sichuan University (RD-03-202106), the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (20HXJS007), Miaozi Project in Science and Technology Innovation Program of Sichuan Province (2022081) and National Natural Science Foundation of China (82270958).

W G and X M guided and supervised the total progress of this study including the research design, experiment implementation, and manuscript editing. J C, J X and X H performed the experiments. J C and J X analyzed the data. All authors have read and approved this final manuscript.

All animal procedures were in compliance with Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People's Republic of China. All experimental animal procedures were performed in strict accordance with the Guidelines for Care and Use of Laboratory Animals of Sichuan University. The animal experiments were approved by the Research Ethics Committee of West China Hospital of Stomatology, Sichuan University (Permit No. WCHSIRB-D-2021-048). All efforts were devoted to minimize animal suffering.