Characterization of scaffolds
According to the protocols, the Ti6Al4V porous scaffolds were prepared by 3D printing technology successfully. In order to detect that whether the parameters of the scaffolds were in line with the pre-design, Micro-CT was used to measure the porosity and SEM was also carried out to observe the pore size distribution. The results demonstrated that the porosity of the porous scaffolds was 69.2 ± 0.9 %. SEM image of the scaffold was displayed in Fig. 1A, and then quantitative analysis by Image J software indicated the average pore size of the scaffolds was 593.4 ± 16.9 µm. In general, the parameters (porosity and pore size) of the printed scaffolds are consistent with the pre-design model (70% and 600 µm, respectively).
The osseointegration ability of the bone tissue engineering scaffolds not only depends on the bone conductivity of the material itself, but also is associated with the pore distribution, porosity and pore size of the implants. Increasing the porosity and surface area of the implants to a certain extent can increase the initial stability and coefficient of friction between the bone and scaffolds, thereby reducing micro-motion, inducing bone ingrowth, and accelerating osseointegration after implantation . For an ideal orthopedic porous implant, the porosity should be higher than 50%, especially in the range of 65–75%, and its structure and machinery should be similar to human trabecular bone. As for pore size, it should be between 300-700-µm, this range is conducive to the adhesion, proliferation and differentiation of osteoblasts [28, 29]. Therefore, as a bone tissue engineering implant, the porosity and pore size of the porous Ti6Al4V scaffolds in this study were considered to have ideal osteoinductive parameters.
In addition to ideal porosity and pore size, the structure of the scaffold's pores also has an important impact on osteogenesis and vascularization after implantation. The structure of internal interconnection of the porous implants were also in favor of oxygen and nutrient exchange . Moreover, the interconnected porous structure with internal perforations facilitates mesenchymal stem cells adhesion, proliferation, and differentiation, and provides a rich interface bonding area for blood vessel formation, bone ingrowth and osseointegration. Finally, a long-term stable outcome is achieved, with the implant and bone forming a biologically fixation [31, 32]. Regarding the range of porosity that is conducive to blood vessel formation, previous studies believed that the porosity of 60%-70% is ideal, which can promote the exchange of oxygen, nutrients and metabolite exchange, and benefit cell penetration, and can better induce vascularization [33, 34]. Previous researches have demonstrated that porous scaffolds with a pore size > 300µm could benefit oxygen and nutrients penetrate into the internal pores of the scaffolds and induce angiogenesis [33, 35]. Herein, the interconnected 3D printed porous Ti6Al4V implants with the average porosity and pore size were 69.2 ± 0.9 % and 593.4 ± 16.9 µm, respectively, was sufficient to induce bone ingrowth and promote vascularization. Generally, 3D printing technology offers a meaningful approach for manufacturing high-precision customized implants with pre-determined parameters of porosity and pore size.
Schematic diagram of poloxamer 407 hydrogel incorporated scaffolds was displayed in Fig. 1B. After permeation with the hydrogel, the original pores of the Ti6Al4V scaffolds were filled with interconnected hydrogel networks ranging from 100 µm to 300 µm (Fig. 1C), which showed that the composite implant system was constructed successfully.
Biocompatibility and cell viability
The biocompatibility of the BMSC and EPC co-cultured with eTi scaffolds and hTi scaffolds were detected by CCK-8 (Fig. 2A and B). BMSC and EPC showed a good proliferation trend when co-cultured in different scaffold samples at all time points. The cell proliferation rates of the BMSC and EPC cultured with eTi and hTi scaffolds were nearly indistinguishable from those of the Con group on the 3rd and 5th day after culture. These results indicated that both porous scaffolds and hydrogel incorporated porous scaffolds have good biocompatibility to cells.
Furthermore, BMSC and/or EPC loaded hydrogel was filled in the porous scaffolds to evaluate the proliferation of these cells. The schematic diagram of the cell-loaded hydrogel incorporated scaffolds is shown in Fig. 2C. As shown in Fig. 2D, with the extension of culture time, the cells increased significantly from day 1 to day 5 within hTi scaffolds. They were displayed simultaneously in BMSC/hTi, EPC/hTi, and Dual/hTi groups, that the number of cells on the 3rd and 5th day was significantly higher than that on the 1st day (p < 0.01), and the cell proliferation on the 5th day was also significantly increased than that on the 3rd day (p < 0.05). Different cells seeded within hTi scaffolds were stained by Calcein AM/PI to study the cell survival. As displayed in Fig. 2E, the fluorescence images clearly demonstrated that the cells in each group maintained an excellent viability. After 3 days, the viability of BMSC/hTi, EPC/hTi, and Dual/hTi groups were 95.24 ± 1.48 %, 94.99 ± 1.156 %, and 92.79 ± 1.27 %, respectively (Fig. 2F). These results indicated that the hydrogel incorporated Ti6Al4V scaffolds were not toxic to the cells, and maintained BMSC and EPC proliferation.
Due to lack of sufficient interface biological activity, it is difficult for 3D printed Ti6Al4V scaffolds to adhere enough cells for in vivo transplantation. However, this strategy of encapsulating cells in hydrogel, a three-dimensional cell culture system, and then filling into the pores of porous scaffolds provides a potential solution for 3D printing technology combined with cell therapy for bone defect repair.
Evaluation of new bone formation and ingrowth
Bone regeneration and ingrowth into the porous implants was evaluated by Micro-CT. Figure 3A displayed the 3D reconstructed pictures of the scaffolds and surrounding bone tissues. The spatial distribution of the regenerated bone indicated that bone regeneration of the eTi Group and hTi Group were limited, EPC/hTi Group and BMSC/hTi Group were medium, and Dual/hTi Group was abundant. Subsequently, parameters of BV/TV, Tb.Th, Tb.N, and Tb.Sp were used to quantitatively analyze the quality of regenerated bone, according to the Micro-CT results. BV/TV values have been widely applied to quantitatively evaluate the regenerated bone mass . In this study, the BV/TV values of eTi Group, hTi Group, EPC/hTi Group, BMSC/hTi Group, and Dual/hTi Group were 12.92 ± 1.81 %, 13.0 ± 1.52 %, 18.05 ± 1.62 %, 20.07 ± 2.51 %, and 26.46 ± 2.14 %, respectively (Fig. 3B). The BV/TV results were consistent with 3D reconstruction, namely, EPC/hTi Group and BMSC/hTi Group showed more bone formation than eTi Group and hTi Group (p < 0.05), and Dual/hTi Group achieved the most superior bone regeneration outcome compared to the other four groups (p < 0.05 or p < 0.01). Similarly, Dual/hTi Group was provided with the highest Tb.Th and Tb.N values (Fig. 3C and D) and the lowest Tb.Sp among these five experimental groups (Fig. 3E). In general, according to the Micro-CT results, EPC-loaded or BMSC-loaded scaffolds significantly enhanced bone regeneration compared with the cell free scaffolds, especially for scaffolds loaded with EPC and BMSC, simultaneously.
Osteointegration between implants and surrounding host bone is critical for bone repair, which including a series of complex biological processes. A functional prosthesis should firmly integrate and combine with the surrounding host bone tissue, thereby reducing the risk of prosthesis displacement and loosening after surgery, and reconstructing the function of bone and limb [37, 38]. In order to observe the osteointegration effect, the undecalcified bone sections were prepared and stained. The results of histological examination were consistent with that of Micro-CT scanning. Representative images were exhibited in Fig. 3F. There was just rare bone regeneration around the scaffolds in eTi Group, hTi Group. However, in EPC/hTi Group and BMSC/hTi Group, more bone tissue was formed and combined with the micropores on the surface of the scaffolds. This phenomenon of bone ingrowth was more obvious in the Dual/hTi Group, regenerated bone tissue not only almost completely integrated with the surface pores of the scaffolds, but also grew into the internal pores.
Sufficient bone formation and ideal osseointegration between implants and surrounding bone is crucial for stability during bone remodeling, which is closely associated with the differentiation, maturation, and mineralization of BMSC [2, 39]. During the healing process, local transplantation BMSC could improve the osteogenic microenvironment in the defects, thus, to promote the progression of bone formation . Therefore, BMSC loaded scaffolds are regarded as a promising tissue engineering strategy to promote repair of bone defects .
Evaluation of neovascularization
When cells were loaded into the hydrogels, the nanochannels in the hydrogels have a positive effect on the exchange of oxygen and nutrients, thereupon then promoting cell growth and communication, and further benefiting bone repair [30, 41]. However, there are limited research has focused on hydrogels as cell carriers or drug delivery systems to incorporate into 3D printed porous titanium implants to form a composite implant. In spite of BMSCs, exogenous BMP-2, as well as VEGF were also incorporated into porous implants to achieve a long-term survival of prosthesis [42, 43]. However, these studies unilaterally promoted bone regeneration but limited to the comprehensive consideration of the synergistic role of BMSC and EPC in osteogenesis and vascularization, therefore they could not obtain the optimal effect of osseointegration.
In this study, neovascularization was observed after implanting vasculogenic cells, EPC, loaded composite scaffolds into bone defects. After microangiography, blood vessels around the scaffolds were detected by Micro-CT. As indicated in Fig. 4A, the blood vessels formation in the EPC/hTi Group and Dual/hTi Group was increased visibly. And then, quantitative analysis of BVV/TV was carried out according to the collected images. The BVV/TV value in the EPC/hTi Group (17.24 ± 2.17 %) was A significantly higher than that in the eTi Group (12.55 ± 1.60 %, p < 0.05) and hTi Group (12.08 ± 2.08 %, p < 0.05), respectively. In addition, the BVV/TV value was 22.65 ± 1.92 % in the Dual/hTi Group, which was higher when compared with that in the eTi Group (p < 0.01) and hTi Group (p < 0.01), EPC/hTi Group (p < 0.05) and BMSC/hTi Group (15.52 ± 1.61 %, p < 0.01), respectively (Fig. 4B). Generally, the microangiography detection revealed that the EPC-loaded porous scaffolds could promote neovascularization surrounding the scaffolds, which may achieve more angiogenesis inside the scaffolds. When BMSC and EPC were transplanted simultaneously, the efficiency of inducing angiogenesis was more obvious. This may be due to the synergistic effect of BMSC. BMSC have autocrine and paracrine functions, and can secrete many soluble growth factors and cytokines, as well as exosomes, which can benefit to EPC proliferation, migration and formation of blood vessels [44, 45].
Despite numerous types of 3D printed scaffolds for bone repair have been prepared and researched in vitro and in vivo, it is still challenging to design the bone tissue engineering scaffolds to induce vascularization during new bone regeneration . In this study, we encapsulated BMSC and EPC in hydrogels, and then implanted them into bone defects with 3D printed porous scaffolds. Hydrogel can effectively prevent the loaded-cells from being cleaned up at the target tissue, thus ensuring the concentrated and effective distribution of the loaded-cells at the defects, and giving full play to the dual functions of osteogenesis and angiogenesis. These characteristics are of great significance for cell therapy and tissue regeneration.
Angiogenesis- and osteogenesis-related gene expression in Bone
The expression of osteogenesis-related genes ALP and OCN, and angiogenesis-related genes HIF-1α and VEGF during the in vivo repair process was investigated by PCR. ALP is evaluated to indicate the osteogenic differentiation, and the activation of ALP activity and up-regulated expression of ALP gene is a critical event happening in early osteogenesis, which indicates the beginning of osteogenic differentiation . The levels of ALP transcription were significantly higher in the BMSC/hTi Group and Dual/hTi Group compared with the eTi Group, hTi Group, and EPC/hTi Group (p < 0.05, Fig. 5A). OCN is always regarded as a marker of late osteogenesis to evaluate osteogenic maturation and bone formation, which is synthesized by osteoblasts . The level of OCN expression was higher in the EPC/hTi Group, BMSC/hTi Group, and Dual/hTi Group than the eTi Group and hTi Group at 12 weeks after implantation (p < 0.05). In addition, the expression of OCN in the Dual/hTi Group was also significant up-regulated than EPC/hTi Group and BMSC/hTi Group, exhibited by Fig. 5B.
It is known that HIF-1α and VEGF is an effective factor to improve the scaffold vascularization via angiogenesis [15, 49]. The levels of angiogenesis-related gene, HIF-1α, was increased significantly in the EPC/hTi Group compared with eTi Group, hTi Group, and BMSC/hTi Group (p < 0.05). In the Dual/hTi Group, the level of HIF-1α was 1.52-fold, 1.43-fold, and 1.25-fold higher than the eTi Group, hTi Group, and BMSC/hTi Group (p < 0.05). In addition, the expression level of another angiogenesis-related gene, VEGF, in these groups was similar with the HIF-1α, namely, EPC/hTi Group and Dual/hTi Group indicated an abundant gene expression.