The magnetic bioassembler “Bioprinter Organ.Aut” was delivered to the ISS Russian segment on the Soyuz MS-11 spacecraft. The use of magnetic fields to form structures of various nature (tissue constructs, protein crystals, bacterial conglomerates and scaffolds) is a platform technology, based on which a series of experiments were performed during the ISS expeditions: 57, 60/61, 61/62, 63 (Fig. 1a).
Figure 1b shows the scheme of the proposed space experiment during the ISS expedition 61/62. The raw materials were developed and delivered to the Russian segment of the ISS by the rocket “Soyuz-FG” and the spacecraft “Soyuz MS-15” (Supporting Information 1–2). All sessions of magnetic levitational assembly during expeditions were performed by Oleg Skripochka. In particular, in the ISS experiments, the scientific equipment used, consists of hermetically sealed cuvettes and a magnetic bioassembler “Bioprinter Organ.Aut” (Fig. 2a-c, Supporting Information 3). The magnetic bioprinter is independent of the interfaces provided by ISS class I. “Bioprinter Organ.Aut” generates an inhomogeneous magnetic field (Fig. 2d) with a high gradient and a "magnetic pit" in the center of the working area, which allows both the assembly of constructs and the recrystallization of chemical elements. It is a self-contained compact “central aisle payload” interfacing uniquely with an electric power source available for portable loads. Additionally, the autonomic video camera was applied to film the assembling process of bone graft formation through the transparent observation window (Supporting Information 3–4).
The experimental equipment and the scheme of cuvette filling are shown in Fig. 2e. The special cuvette consists of three assembled cases to provide three levels of biosafety. Functionally, the cuvette is comprised of two containers for reagents and a working chamber in which all three solutions can be mixed. The following steps were taken to prepare cuvettes for the experiment (Fig. 2e): 1) buffer # 1 was placed in container 1; 2) buffer # 2 was placed in container 2; 3) a working chamber was filled with CP particles (α-tricalcium phosphate (α-TCP, Ca3(PO4)2)) in distilled water. The use of distilled water avoided the formation of air bubbles during the experiment. The cuvettes presented in Fig. 2b provide delivery, activation of the process, fixation, and return to the Earth of the obtained experimental samples (Supporting Information 5).
In detail, the astronaut pushed the first button on the cuvettes, and the suspension of CP particles was introduced into buffer # 1, the weight ratio of particles to the solution was 1/100. The pH value of the admixed solution increased from 4.5 to 4.75 because of the decline of CH3COONa concentration. The cuvettes were placed in the magnetic bioassembler, and CP particles started to assemble into the 3D levitating scaffold under the action of magnetic forces (S3) and simultaneously started to transform into dicalcium phosphate dihydrate (DCPD, CaHPO4×2H2O) due to interaction with buffer #1 according to scheme 1:
CH3COONa + Ca3(PO4)2 + H3PO4 + 6H2O → 3CaHPO4×2H2O (1)
The first stage of levitation in buffer # 1 into magnetic force action occurred during the 48 h.
In the second stage, the astronaut push button 2 and buffer # 2 were introduced in the cuvets with levitating scaffolds. The weight ratio of scaffolds to solution became 1/200, pH value increased up to 8.2. The transformation of the DCPD into octacalcium phosphate (OCP, Ca8H2(PO4)6×5H2O) passed during the additional 48 h according to scheme 2:
10CaHPO4×2H2O ↔ Ca8H2(PO4)6×5H2O + 2Ca2+ + 4H2PO4−+15H2O (2)
A video camera incorporated into a magnetic assembler was used for recording the magnetic levitational assembly process. The translation of the processes of ISS magnetic levitational assembly is presented in the supplementary materials (Supporting Information 4).
The same processes were conducted on the Earth, the control 3D tissue engineering scaffolds were obtained under the same conditions excluding the Earth's gravitation.
The chemical composition analysis of the final product indicated that the concentration of the Gd3+ in the ISS sample was lower than 0.001 wt. %. On the other hand, the Earth samples were characterized by 0.85 wt. % of Gd3+ (Table 1.). The calculation of (Ca + Gd)/P ratio evaluated the more complete processes of ISS samples, which was characterized by 1.33 for the stoichiometric octacalcium phosphate (OCP, Ca8H2(PO4)6×5H2O) phase. The presence of the Gd3+ in the Earth samples could be linked with gravitation forces and materials morphology, resulting in the more noticeable adsorption of the surface.
Table 1
Chemical compositions ISS and Earth samples.
Sample code | Sample | Ca concentration, wt. % | Gd concentration, wt. % | P concentration, wt. % | Ca concentration, mol. % | Gd concentration, mol. % | P concentration, mol. % | (Ca + Gd)/P |
ISS | Ca8(HPO4)2(PO4)4×5H2O | 29.70 | < 0.001 | 18.11 | 7.29 | 0.00 | 5.74 | 1.33 |
Earth | Ca8(HPO4)2(PO4)4×5H2O | 27.21 | 0.850 | 19.79 | 6.68 | 0.05 | 6.27 | 1.07 |
According to XRD data (Fig. 3a), the materials obtained by magnetic levitation assembly in the ISS conditions are characterized by the predomination formation of the OCP phase (ICDD card no. 026-1056) up to 90 wt. % with minor preservation of dicalcium phosphate anhydrous (DCPA, ICDD card no. 070–0359) and DCPD (ICDD card no. 11–293), which could be saved in the center of the materials. The initial α-TCP (ICDD card no. 09-0348) phase was not observed. 3D tissue engineering scaffolds obtained on the Earth demonstrated the presence of up to 50 wt. % of the impurity phases. This is due to the low concentration of the Gd3+ salts compared to our previous investigation 11 – 2M compared to 3M at the close conditions of the transformation. The materials obtained on the ISS were characterized by low crystallinity degree and significant texture formation. Thus, peaks of [020] and [110] are characterized by the increased values of the relative intensity of 28 and 30 concerning the 100 peak of [010]. At the same time, the Earth sample pattern with values of these peaks of 17 and 16 was close to the theoretical ones (15 and 13, correspondingly). Also, for the ISS samples, peaks of [260] and [2–41] demonstrated decreased intensity – 14 and 15, compared to 33 and 32 presented in the ICDD 026-1056 card. Additionally, the halo in the range of 12 to 22° 2Θ was observed, linked with the peculiar texture character of the OCP crystals formation of the ISS samples. Thus, the microgravitation conditions resulted in a higher transformation degree linked with an active free-motion under the influence of the magnetic forces in the buffer solutions of the initial α-TCP and DCPD particles but influenced the crystal growth formation with the obtaining of clear textured materials.
A specific crystallization process of the main phases of ISS samples was confirmed by FTIR results (Fig. 3b). The spectra are characterized by two times lower intensity of all peaks in the case of ISS samples, compared with Earth ones. The wide band in the range of 3000 − 3700 cm− 1 was assigned as H-O-H crystalline water of OCP vibration mode and a slight peak at 1652 cm− 1 as bending mode 17. The OCP HPO43− groups were presented by P–OH bending mode observed at 1198 cm− 1, and P–OH stretching mode at 914 and 876 cm− 1. The P–OH bending at 1296 cm− 1 characterized for OCP was not detected 18. It could be linked with distorted crystal formation in the ISS conditions. The P–O in HPO42− and PO43− groups were recorded at 1123, 1074, 1024, 967,611, 578, 560, and 536 cm− 1. The bands in the range between 1540 and 1400 cm− 1 were assigned as atmospheric CO3 − 19,20.
The Earth samples were characterized by the absence of 1198 cm− 1 P–OH bending mode, and the disappearance of the P–O peaks at 1123, 967, and 611 cm− 1, which indicated the lower degree of OCP formation. At the same time, O–H stretching of water at approximately 3544, 3491, 3290, and 3163 cm− 1 appropriate for DCPD was not registered 17, pointing predominately to DCPA preservation in materials21. The peak at 1569 cm− 1 could be linked with С=N of the glutamine acid. It should be noted, that in the ISS samples a similar peak was not detected, which could be linked to the lower absorbance capacity of the formed crystals or more complicated processes of the glutamine acid interaction with α-TCP 22.
Raman’s spectra of the Earth and ISS samples are presented in Fig. 3c. The spectra demonstrated the presence of PO43− groups for both materials at 959 cm− 1 assigned as symmetric stretching mode (ν1), double (ν2), and triple (ν4) degenerate bending modes (O − P−O bond) were observed at 430 and 448 cm− 1 and in the region of 579 to 606 cm− 1 23. The region of 1033–1060 cm− 1 and peak at 1080 cm− 1 were attributed to PO stretching 24. In addition, for the ISS samples, a strong band at 1012 cm− 1, weak bands at 875 and 919 cm− 1, and a shoulder at 410 cm− 1 on the ν2 PO43− the band were assigned to HPO42− vibrational modes of OCP 25. The spectra of the Earth samples are characterized by the significant decrease of HPO42− bands. At the same time, a strong peak at 531 cm− 1 was observed, assign to out-of-plane vibration bands of NH26 linked with residual glucosamine acid confirming the FTIR results.
According to SEM results, the initial α-TCP particles intended for transformation in the 3D assembler in a magnetic field are shown in Figure. 4 and Supporting Information 6. SEM data demonstrated, that the obtained after two-stage transformation 3D tissue engineering scaffolds both on the Earth and in the ISS are characterized by a size of about 5 mm. The bone graft materials of the same size are applied in surgery and dentistry up to date27. The Earth samples were formed as conglomerated spherical clusters with irregular surfaces formed by faceted plate particles with a size of up to 2 µm and a thickness of about 100 nm. The ISS samples were characterized by globular chrysanthemum-like structures, previously described for DCPD – magnesium fluoride coating obtained via a fluoride conversion process followed by electrochemical deposition on the Mg-Ca-Zn alloy28. The observed in our work chrysanthemum structure was formed by petal-like thin crystals with two-three times lower thickness compared with the Earth particles and ragged edges. Materials are characterized by very uniform crystal growth with practically without intersections and rosette-like formation of the particles due to the congenerical loading of microgravity and magnetic forces. The formation of the so-called spherulites looks like the sea urchin was observed for HA (hydroxyapatite, Ca10(PO4)6(OH)2) and OCP crystals obtained in the space conditions during the EURECA Solution Growth Facility (SGF) experiments29. The formation of OCP additionally to HA took place only in the space conditions, and well-crystallized narrow and thin blades, typically about 0.1 mm long particles, which formed spherulites were observed30. A figure of the ISS sample microstructure, which could be observed via 3D glasses (3D -scan red-blue) was shown in Supporting Information 7. The formation of OCP was explained by the lower supersaturation and nucleation rate in the space conditions and estimated faster growth kinetic of OCP compared to HA. A similar tendency of homogeneous crystal growth in the microgravity conditions was observed by N. Bergeon and el. for succinonitrile–camphor alloy formation during the heating in the Bridgman furnace31.
The OCP crystals formation on the DCPD surface was described previously and the formation of the thin blades up to 250 µm32,33 as well as the formation of platy crystals when hydrolysis of DCPD was conducted during 3 months34. The rosettes formed by the plate morphology of OCP were observed for materials, obtained by the direct precipitation method35. Moreover, the crystallization of the long narrow, and thick OCP blades, which formed the spherulites was previously reported for space microgravity conditions 29,30,36, but the OCP was a minor phase and crystallization occurred in the solutions without substrate. The chrysanthemum flower-like formation on the initial TCP – DCPD substrate particles was observed for OCP for the first time to our best knowledge and was achieved due to the combination of the microgravity and magnetic forces conditions.
The data was confirmed by HRTEM results, which is shown on Fig. 5. Thus, the ISS samples were characterized by the formation of crystals with very tiny thickness confirmed by the observation of the bubble-like defects after electron beam irradiation on the HRTEM images. These bubble-like defects were previously presented for HRTEM investigations of OCP synthetic tiny flake-like single crystals with a thickness of only tens of nanometres obtained from the National Institute of Standards and Technology in the USA and their appearance was linked with OCP transformation in the HA structure37. At the same time, we did not observe similar defects in the images of the Earth samples because the crystals were thicker. The crystals, obtained on the ISS, are characterized by the size of 100–500 nm. At the same time, the images of the Earth samples have demonstrated the presence of the two phases – lamellas-like and needle-like. The SAED data indicated that this zone corresponds to the coexistence of both polycrystals of OCP38 and monocalcium phosphate (Ca(H2PO4)2×H2O) phases in the Earth sample. The SAED indication corresponds to the overlapping of [0 0 1] zones axis of Ca(H2PO4)2×H2O and [1 1 0] zones of OCP. The calculated and theoretical interplanar spaces are listed in Supporting Information 8 as well as estimated individual lattice vectors. The ISS samples SAED investigation demonstrated the formation of the quite perfect OCP polycrystal (area 1) and single crystal (area 2) dots diffractograms, without any additional phases, which confirmed the improved transformation stage of the sample. According to diffractGIU calculation, the [-1 -1 0] zone axis of OCP was evaluated and the presence of 001, -111, -110, and − 11 − 1 individual lattice vectors were presented. The calculated interplanar spaces are listed in Supporting Information 8. Additionally, [3 4 8] zone axis of single-crystalline OCP and corresponding vectors are listed in the SAED area 2. The experimental cells were built in the CellViewer application and their similarity to theoretical ones evidenced the formation of the estimated phases – OCP in the case of the ISS samples and both OCP and Ca(H2PO4)2×H2O for the Earth sample.
The possibility of the elaboration of the DCPD and lead monetite monocrystals in space conditions was reported early39. In our work, we observed formations of the thermal unstable and highly soluble OCP with perfect structure.
The microCT investigations demonstrated the formation of consolidated porous scaffolds with a size of up to 5 mm. The microporosity of the scaffolds was formed by interconnected channels heritable from initial α-TCP40 pores modified by transformation into DCPD and OCP with diameters in the range of 20–100 µm. The macroporous with a size up to 500 µm was observed on the whole scaffold’s space and was a result of the magnetic levitation assembling (Fig. 4h-g).
The electron paramagnet resonance data demonstrated the presence of gadolinium in the obtained ISS and Earth samples. The data are presented in Fig. 6. There are no paramagnetic centers in nominally pure OCP samples, hence no EPR signal in materials can be observed41. The rare-earth gadolinium ion Gd3+ with a 4f7 electron configuration (ground state − 8S7/2) is paramagnetic and has an electron spin S = 7/2 with a zero orbital moment L = 0. The non-Kramers Gd2+ ions are not observed in the EPR spectra, therefore, the absorption lines in Fig. 6 should attribute to the impurity Gd3+ center (Fig. 6). The corresponding spectrum contains 2*S = 7 spin transitions with the center of spectrum gravity at g ≈ 2.001 since there is no orbital magnetic moment 42. The effect of the electric crystal field gradient on the Gd3+ ion leads to the formation of a fine structure (zero field splitting –DStrain) the line positions of which depend on the angular orientation of the OCP nanocrystal. The powder EPR spectrum contains contributions from all equally probable positions of the nanocrystal relative to the external magnetic field B0, which leads to an inhomogeneous broadening of each absorption line.
The high-frequency (W – band, ν = 94 GHz) EPR approach allows, firstly, to conduct experiments on several sample particles due to the increased sensitivity of the experimental setup and the reduction of the powder-broadening effects. Secondly, the high spectroscopic resolution makes it possible to identify different paramagnetic particles with approximately close g-factors43. The figure shows that the line width for the Earth sample and one, synthesized on the ISS, is different, where ΔBISS > ΔBEarth 15 times, where the broadening extent is determined by zero-field parameters distribution (DStrain). It can be assumed the samples obtained at the ISS conditions have a higher ordering (degree of crystallinity). The values of the crystal field action on Gd3+ increase and the lines become wider. The manifestation of zero-field splitting effects indicates the localization of the Gd3+ ion in the crystal lattice of the sample44. Figure 6b shows the additional positive influence of the magnetic field on the OCP nanocrystal's growth. Narrow EPR lines and the presence of their weak angular dependence at α = 0° (α is the angle between crystallographic axis c and B0 direction) and 20° indicate the formation of ordered bulk-like particles with higher crystallinity. The Earth sample does not depend on the rotation of the sample in the goniometer, indicating a disordered powder structure of the materials. Therefore, the absence of a gravitational with the existence of a magnetic field has a favorable effect on the quality of synthesized samples. The spectrometer's sensitivity allows observing the signal from gadolinium ions with a sufficiently low concentration and estimates the number of spins ≈ 1014. Comparing the integral intensity of the EPR spectrum with the reference sample, the Gd3+ mass concentration can be estimated as x < 0.001.
The study of the local nuclear environment was carried out by the method of double electron-nuclear resonance, the spectrum of which is shown in the Fig. 6. Thanks to structural hydrogens in the OCP crystal lattice, we can register a hyperfine (electron-nuclear) interaction between the impurity centers and the surrounding 1H ligands45. Following the assumption of anisotropic dipole-dipole interaction with hyperfine constant value Ad−d = 1.77 MHz, the Gd3+ - 1H distance can be calculated rd-d = 3.5 Å, which is the average intermolecular range. Consequently, the presence in the EPR spectra of the fine (zero-field) structure and the ENDOR signal from the surrounding gadolinium ion hydrogens unambiguously establish the presence of Gd3+ in the crystal structure.
It follows from the EPR and ENDOR results that the gadolinium ions with low concentration have a valence of 3+, with localization in the crystal lattice of the OCP sample and not on the internodes or the surface. The absence of gravitational forces and the presence of a magnetic field during synthesis leads to an improvement in the quality of the material with an increase in the degree of crystallinity, creating a specifically directed orientation for the growth of particles with significant texture formation on the surface, confirming the SAED data.
ISS samples were returned to Earth and were investigated in vivo in comparison with Earth samples (Fig. 7 and Supporting Information 9). After 4 weeks of ISS and Earth samples implantation, the histological investigations demonstrated the formation of well-vascularized connective tissue with immured bone grafts in the area of the defect. At the border of the particles and connective tissue cells of foreign bodies are visualized. The Earth sample demonstrated the same picture after 12 weeks, but inside some particles, the newly formed bone tissue are noted. The defect zone of the ISS samples was filled with vascularized connective tissue with particles embedded in it. Around individual particles are rims of newly formed bone tissue. In the space of individual particles, there are the newly formed bone tissue. The most significant difference was observed after 5 months of the implantation. For the Earth samples, the amount of newly formed bone tissue slightly increases: bone islands inside the particles, thin rims of bone tissue around the particles, and small areas of newly formed bone tissue between the particles in the connective tissue were observed (Fig. 7e). For the ISS samples, already more than half of the OCP particles are surrounded by wide rims of bone tissue (Fig. 7f). The results of a pathohistological examination of the area of the bone defect were also confirmed by microCT data performed after 4, 12, and 20 weeks of the experiment (Fig. 7c,d). Thus, the osteoconductive and osteoinductive properties of the bone grafts were dramatically improved, when samples were formed in the microgravity conditions on the ISS.
The application of the 3D printing approaches for personalized medicine has great potential. This is due to the allowance of the creation of 3D objects with high accuracy, repeatability, and reliability under Computer-Aided Design/ComputerAided Manufacturing data obtained by the individual patient requirements by the results of the computer tomography46. The magnetic levitation approach allows the fabricating material into space to form 3D objects and these objects can be moved and rotated to any angle without the need for a support structure47. This free motion led it possible to perform the chemical and structure transformation, as well as the formation of the scaffolds, which contained inorganic components (biomaterials), biophysical and chemical factors, and live cells following the conception of the bone tissue engineering 48. At the same time, the development of magnetic levitational assembly is associated with a big challenge – the toxicity of gadolinium salts. This problem becomes more pronounced in the case of inorganic compounds, characterized by higher density, compared to organic 49 and cell-contained hydrogel-based36,50 3D objects, because they required higher gadolinium solution concentration. To overcome these disadvantages, different strategies could be chosen, including the application of strong magnetic fields, generated by super magnetics or due to a combination of different forces, for example, magneto-acoustic or laser magneto-acoustic approaches. In our investigations, we have demonstrated the influence of microgravity conditions on the fast formation of OCP scaffolds. The original composition of the buffer solution including low-concentrated gadobutrol and glutamine acid was developed and a comparison of the Space and Earth samples was conducted. Microgravity conditions resulted in the formation firstly observed chrysanthemum-like OCP microstructure with vanishingly low gadolinium concentration, the obtained crystals were quite perfect, which has confirmed simultaneously SAED, EPR, and ENDOR methods, also according to XRD, the textural structure was evaluated. Previously, the formation of the OCP as a minor phase in the space conditions was observed together with HA formation during the 5-month precipitation processes and the possibility of the high crystalized structure was demonstrated 29,30,36. In our work, we deliberately created the conditions optimal for OCP crystallization and could obtain the material in a very short time – 48 h. The combination of microgravity and magnetic forces resulted in obtaining the scaffolds with the predicted geometrical properties, phase composition, and homogeneous structure. The micro and macroporosity of scaffolds provided adsorption of the cells on the scaffold surface. It was demonstrated, then the cells-contained solution was added in the space of the magnetic assembler. This led to improvement in osteoconductive and osteoinductive properties, which were demonstrated in the calvarial critical-sized defects model. Moreover, a preliminary experiment to produce OCP-cell constructions (tissue engineering constructs) during the magnetic levitation processes in microgravity conditions was performed by us at ISS (Supporting Information 10). Thus, for the first time, calcium-phosphate-based cells impregnated tissue constructs were obtained in space conditions.