ZIF-8 induced hydroxyapatite-like crystals enabled superior osteogenic ability of MEW printing PCL scaffolds

doi:10.21203/rs.3.rs-1990250/v1

Download PDF

Research article

ZIF-8 induced hydroxyapatite-like crystals enabled superior osteogenic ability of MEW printing PCL scaffolds

https://doi.org/10.21203/rs.3.rs-1990250/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

In general, ZIF-8 tends to undergo ion responsive degradation in ionic solutions, which makes it difficult to maintain its original structure, thus restricting its direct application in biological settings.

Methods

ZIF-8 was synthesized using one-pot method and the ZIF-8/PCL scaffolds were built by a round mould or printed by melt electrowrittten (MEW) technology. Mineralization performance was evaluated by SEM with EDS-mapping and micro-CT scanning after soaking in simulated body fluid (SBF). The osteogenic activity in vivo or in vitro was assessed using different methods including micro-CT scanning, Alizarin red staining, and immunohistochemical staining.

Results

Herein, an abnormal phenomenon is reported that ZIF-8 can form large hydroxyapatite-like crystals when getting immersed directly in multi-ion simulated body fluid. The abnormal crystals showed the continuation of rapid growth in 14 days, with its volume increasing by more than ten times. As suggested by the release of Zn2+ and the emergence of new XRD diffraction peaks, ZIF-8 particles might gradually collapse and congregate through competitive coordination and re-nucleation. The above phenomenon is also observable on the surface of ZIF-8/PCL composite materials and MEW printing ZIF-8/PCL scaffolds. ZIF-8 increased the roughness of PCL by altering its surface topography, which significantly improved its biocompatibility and osteoinductivity both in vitro and in vivo. Notably, the capability of pro-biomineralization makes ZIF-8 also applicable in polylactic acid.

Conclusions

To sum up, the results shown in this study demonstrate that ZIF-8 can serve as a bioactive additive that enables the surface modification to synthetic polymers, which suggests its potential of application for in-situ bone regeneration.

metal-organic framework

polycaprolactone

biomimetic mineralization

melt electrowritten printing

bone regeneration

melt electrowrittten (MEW)

Zeolite imidazolate framework-8 (ZIF-8) is a subclass of metal-organic framework (MOF) materials that are composed of zinc metal ions (Zn²⁺) and organic ligands of imidazole derivatives. ^[1] Due to its exceptional chemical characteristics, such as low cytotoxicity, stable structure, and large specific surface area, ZIF-8 is of interest in various biomedical areas, ^[2–4] including in bio-catalysis, ^{[5, 6]} sensing, ^[7] and drug delivery ^{[8, 9]} among others. While the tetrahedral structure of ZIF-8, which is formed between Zn atoms and imidazolate linkers, has a hydrolytic stability, the biological milieu can be quite complex and, for in vivo or in vitro studies, several aqueous buffered systems are routinely used.^[10] In phosphate buffered saline (PBS), ZIF-8 decomposes and collapses. Miriam et al. revealed the mechanism involved in the degradation of ZIF-8 in PBS. They reported that phosphates have a high affinity for Lewis’s metal clusters, which changes the coordination equilibrium to formation of insoluble zinc phosphates, thereby promoting the release of 2-methylimidazole (2-HmIM). ^[11] Moreover, competitive binding may also mediate anion exchange with ZIF-8 in other media solutions that are rich in metallic cations and inorganic anions. ^[12]

Calcium phosphate (CAP), the biomineral that is most relevant for our day-to-day lives, provides a favorable environment for bone mesenchymal stem cells (rBMSCs) during bone regeneration. ^{[13, 14]} Therefore, the CAP coating technology was developed to improve the properties of bone tissue engineering scaffolds. Coating of CAP using simulated body fluid (SBF) is a promising approach as it provides a suitable temperature, concentration of ions, and pH for cells that is similar to that of human blood plasma. ^[15] Compared to PBS buffer solutions, the SBF solution has a high concentration of phosphates and more active metal cations. Attached crystals and charged regions of the substrate in SBF act as nucleation sites for crystal growth by converting the high levels of Ca²⁺, PO₄³⁻, and amorphous calcium phosphate into ordered carbonated apatite, similar to the apatite found in native bones. ^[16] There is a need to develop a strategy for enhancing the formation of a uniform apatite deposition of sufficient thickness on polymer materials lacking these charged regions or functional groups, which could coordinate ions to mineralize.

In advanced bone grafting-techniques, printed polymer scaffolds have been successfully identified as temporary extracellular matrix substitutes in bone tissue engineering. ^[17] Polycaprolactone (PCL) is an aliphatic and semi-crystalline polymer that exhibits relatively slow biodegradation, reasonable elastic properties, low inflammatory responses, and a low melting point of around 60°C. ^[18] Due to its thermoplastic and semi-crystalline properties, PCL can easily be mixed with other materials and extruded as filaments for melt electrowritten (MEW) printing. However, PCL is associated with low bioactivities, which compromises cell attachment, osteoconduction, and osteoinduction. ^[19] In some studies, the offset and gradient pore size-structured MEW printed scaffolds provided dense structures with mechanical integrity and high porosity for better cell infiltration in bone tissue engineering, ^[20–22] however, osteogenesis has not been fully accomplished. Thus, there is an urgent need for MEW printed PCL scaffold modifications for excellent cell compatibility and bone regeneration.

With regards to biomimetic mineralization for bone regeneration, we hypothesized that interactions between ZIF-8 degradation and competitive binding of metallic cations/inorganic anions in SBF enables biomimetic PCL mineralization. Therefore, through the MEW printing technology, we synthesized ZIF-8/PCL scaffolds for bone regeneration (Fig. 1). In this study, the components and morphologies of MEW printed scaffolds were characterized, and their biocompatibility as well as osteoconductive properties assessed in vitro using rBMSCs cultures. A rat skull critical-size bone defect model was also established to evaluate bone repair effects of ZIF-8/PCL scaffolds. Collectively, ZIF-8 exhibited a great potential in providing active sites to low biological activity materials and endowing them with an effective approach to promote osteogenesis in bone tissue engineering.

1. Synthesis of ZIF-8 particles

We synthesize ZIF-8 according to previously reported studies with a little modification.^[23] Zinc nitrate hexahydrate (1.68 g) in 20 ml methanol solution was slowly added to 4 g of 2-methylimidazole dissolved in 40 ml of methanol solution. The mixture of two solutions was placed on a magnetic agitator and stirred at room temperature (RT) for 2 h (650 r·min ^-1). The reaction solution with molar composition of Zn²⁺: 2- HmIM: methanol=1:10:30. Then, ZIF-8 powders were isolated by centrifugation at 8500 rpm, washed twice in methanol, and dried at 60 °C for 24 h.

2. ZIF-8-induced biomimetic apatite deposition

In-vitro biomimetic mineralization was assessed by immersing ZIF-8 particles in SBF at 37 °C in a thermostated container for 12 h, 1 d, 4 d, 7 d, 14 d, and 21 d. The SBF solution was replenished every other day. Then, after being washed twice using deionized water, the solid was dried using a lyophilizer. The SBF solution was prepared by dissolving NaCl, NaHCO₃, KCl, K₂HPO₄•3H₂O, MgCl₂•6H₂O, CaCl₂, Na₂SO₄ in ddH₂O and buffering to pH 7.40 at 36.5 ℃ with Tris (hydroxymethyl) aminomethane and aqueous 1 M HCl solution.

3. Characterization of the mineralized ZIF-8

3.1 TEM and EDS-mapping. Transmission electron microscopy (TEM) imaging and energy dispersive spectrometer analysis (EDS-Element mapping) were performed on Talos F200X (FEI, Netherlands) operated at an accelerated voltage of 200 kV. After being dispersed in ethanol and sonicated for 5 min (2 mg/ml), the resultant powders were deposited on the TEM grid.

3.2 XRD and FT-IR evaluation. Crystalline phases of the resultant powders were characterized using an X-ray diffract meter (XRD) at a scanning rate of 4.7°/min over a scanning range of 2θ of 5 – 40 degrees. Fourier Transform Infrared (FT-IR) spectra were collected by an FT-IR spectrophotometer (Thermo Nicolet, USA).

3.3 Determination of the concentrations of Zn²⁺, Ca²⁺, and 2-methylimidazole. After 15 mg ZIF-8 had been immersed in 15 ml SBF for 1 h, 3 h, 6 h, 12 h, and 24 h, the supernatants were obtained as mother liquors. The concentrations of Zn in mother liquors were quantified via axially viewed ICP-OES (Agilent 720, Agilent Technologies, Inc. USA). The Zn (II) 213.856 nm emission line was used and the mother liquor was analyzed by GC-MS (7890A/5975C, Agilent, USA). Injection of 1 µl of equal solution in split mode was done at an injection port temperature of 250°C. Separation was performed on the pole at a temperature gradient starting from 60°C (1 min), rising to 240°C at a temperature gradient of 15°C min^-1. With helium as the carrier, the downline speed was 35cm/s, in constant current mode. The quadrupole was operated in scan mode at a scan range of 50-150 amu and at a scan speed of 5 times per second. The interface temperature was set to 230 ° C while the ion source temperature was set to 200 °C. A two-point external standard method was used to establish the corresponding correction curve. Each sample was analyzed in triplicates.

3.4 Particle size and zeta-potential evaluation. Particle sizes and zeta-potentials of ZIF-8 and mineralized ZIF-8 were determined by a dynamic light scattering (DLS) plus ZETA system (Malvern Zetasizer Nano S, UK).

4. In vitro assessment of mineralized ZIF-8-induced osteogenic differentiation

4.1 Isolation of rat rBMSCs and in vitro cultures: Rat bone marrow stromal cells (r-rBMSCs) were isolated and harvested as previously described.^[24] Briefly, newborn Sprague Dawley rats (3-5 days old) were euthanized by cervical dislocation and soaked in 75% alcohol for 30 min. Femurs and tibias were separated from attached muscles and soft tissues. Then, cartilages at both ends of bones were cut off, followed by repeated washing of cavities using the culture medium until the cavities appeared white. The fresh bone marrow tissues were seeded in 10 cm culture dishes supplemented with 7 mL of culture medium (L-DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin). Culture dishes were incubated (Heracell150i, Thermo Scientific, USA) in a 5% CO₂ environment at 37 ℃. The culture medium was changed every 3 days. When the attached r-rBMSCs were confluent, they were passaged.

4.2 Alizarin Red S Staining. The rBMSCs were seeded in 6-well plates at a density of 1.5×10⁵ for 24 h or grown until 60% confluent. Then, after co-culturing with mineralized ZIF-8 (1 mg/ml) for 14 days in a culture medium without osteogenic induction solution, they were fixed in 4% paraformaldehyde for 30 min. After removal of the fixation liquid, Alizarin Red was added to indicate ECM mineralization. Excess Alizarin dye was removed using DI water. The Nikon Eclipse Ts2R-FL microscope was finally used for imaging.

4.3 Osteogenic gene expressions. For Real-Time PCR, after co-culturing with mineralized ZIF-8 for either 5 or 14 days, rBMSCs were treated with Trizol-up for mRNA extraction, followed by qPCR analysis using a SYBR Green qRT-PCR kit (ELK Biotechnology, Wuhan, China) for assessing mRNA expressions of Alp, Runx2, Ocn, Opn. β-actin was the internal reference.

4.4 Evaluation of osteogenic protein levels. For immunodetection of cell proteins, western blot (WB) analysis was performed to determine osteogenic protein levels in rBMSCs. Cellular proteins were isolated using the Total Protein Extraction Reagent (Aspen Biological, Wuhan, China) on ice. After denaturization at 99 °C for 8 min in a loading buffer, equal amounts of protein extracts were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, USA). The PVDF membranes were incubated in the presence of specific primary antibodies: anti-RUNX2 (ab236639, 1:1000, Abcam, UK), anti-ALP (LS-C780836, 1:1000, LSBio, USA) antibodies for samples at day 5, and anti-OCN (sc-390877, 1:500, Santa Cruz, USA), anti-OPN (ab63856, 1:1000, Abcam, UK) antibodies for samples at day 14. After incubation in the presence of secondary antibodies (1:10000), membranes were detected by chemiluminescence (ECL) (AS1059, Aspen, China). The proteins were visualized by a ChemiDoc Touch chemiluminescent system (Biorad, USA). Protein levels of osteogenic markers were normalized to GAPDH (ab181602, Abcam, UK). Relative target protein levels were determined by gray value analysis using Image J version 1.8.0. (NIH, USA).

5. Preparation of ZIF-8/PCL and MEW printed ZIF-8/PCL

The molten mixture of PCL and ZIF-8 (10% wt.) was blended on a 120 ℃-heating plate for 30 min. The PCL and ZIF-8/PCL compacts (10 mm in diameter and 2 mm high) were obtained after pressing. Melt electrowritten PCL and melt electrowritten ZIF-8/PCL were designed and fabricated using the MEW printing machine (QINGZI NANO, China). Then, 3.6 g PCL particles and 400 mg ZIF-8 powders were evenly mixed, placed in the feeding tank, and heated to 120 ℃ for 30 min. The printed MEW parameters were: atmospheric pressure 0.18 Mpa, interval 1 mm, square wave 20 times, speed 8 mm/s, and acceleration 48 mm/s². Threshold voltage between negative 5 and 7 kV were applied to create the charged polymer fiber. The obtained samples were sliced into round sheets of 5 mm diameter and 2 mm height.

6. Biomimetic apatite deposition on PCL, ZIF-8/PCL and MEW printed PCL, ZIF-8/PCL

To accelerate biomimetic apatite deposition, samples were immersed in CaCl₂ and K₂HPO₄ solutions. Then, scaffolds were immersed in 20 mL 0.2 M CaCl₂ solution for 3 min and soaked in 30 mL ddH₂O for 10 s, followed by soaking in 20 mL 0.2 M K₂HPO₄ solution for 3 min and in 30 mL ddH₂O for another 10 s. The entire pretreatment assay was repeated thrice. These alternately soaked samples were subsequently immersed in SBF for biomimetic apatite deposition (30 mL of SBF was poured into one 50 ml centrifuge tube containing six alternately soaked samples). Samples were kept at 37 ℃ for 1 day, 7 days, 14 days, and the SBF renewed every day to sustain a consistent ionic strength throughout the assay. The samples were removed from the SBF, gently washed with ddH₂O, and subsequently lyophilized at -50 ℃ for 24 h under a vacuum.

7. Characterization of PCL, ZIF-8/PCL, and MEW printed PCL, ZIF-8/PCL

7.1 SEM and EDS-mapping. Scanning electron microscopy (SEM) images and energy dispersive spectrometer analysis (EDS-Element mapping) were obtained on Gemini 300 (Zeiss, Germany). Mold-made PCL, ZIF-8/PCL, and MEW printed PCL, ZIF-8/PCL samples were sprayed with gold for 15 s and scanned at 5 kV.

7.2 Micro-CT analysis. To assess biomimetic apatite deposition on samples, a Micro-CT scanner (SkyScan 1176; Broker) was used. Scanning parameters were: 58 kV, 385 mA, and 18 μm slice thickness. Corresponding 3D images were reconstructed using a VG studio software (Volume Graphics GmbH, Heidelberg, Germany). Apatite volume (AV) and percentage of apatite volume, relative to total volume (AV/TV) were determined by a micro-CT assistant software, CTAn (Scanco Medical, Zurich, Switzerland).

7.3 Calcium ions levels assay. This assay was performed by dissolving the mineralized samples in 0.4 mL 0.5 M acetic acid overnight followed by quantification using a calcium assay kit (BioAssay Systems, USA), as instructed by the manufacturer.

7.4 Mechanical properties. Elastic modulus was tested using an Electronic Universal Testing Machine (AG-IC 100kN, Shimadzu, Japan) to evaluate the mechanical properties at a loading rate of 1 mm/min until 80% compression of the samples was achieved. The slope of the initial linear portion of the stress-strain curves were calculated to obtain the elastic modulus.

7.5 Water contact angle. Hydrophobicity was measured using a contact angle goniometer (LSA100, LAUDA Scientific, Germany). The angle between a liquid droplet and the solid surface was measured using a CCD video camera and lens mounted on a viewing stage.

7.6 Morphological characterization. Microscopic features of PCL, ZIF-8/PCL samples were evaluated by atomic force microscopy (AFM) (SPM9700, Shimadzu, Japan). Microscopic features of Melt electrowritten PCL, ZIF-8/PCL samples were assessed by laser scanning confocal microscopy (LSCM) (OLS5000, Olympus, Japan).

8. Cellular responses to MEW printed PCL and ZIF-8/PCL in vitro

8.1 Biocompatibility assay. Cell proliferation and cytotoxicity effects of the MEW printed PCL, ZIF-8/PCL scaffolds were analyzed by Cell Counting Kit-8 (CCK-8) assays. After ultraviolet radiation and ozone sterilization, scaffolds were immersed in the cell culture medium and incubated overnight. Then, rBMSCs were cultured in the presence of the above-mentioned leaching liquor. For cell proliferations, 3000 rBMSCs were seeded in 96-well plates, while 1.6×10⁵ rBMSCs were used for cytotoxicity assays. At different time points (day 1, 2, 3, and 7), 1 mL of fresh culture medium containing 10% CCK-8 solution (Dojindo, Japan) was added to each well and incubated for 2 h. To quantify cell proliferations, absorbance of the solution at 450 nm was measured using a microplate reader (Thermo, USA).

Live/dead staining was performed using Calcein AM (live)/PI (dead) for cell viability assays. Live cells were stained using Calcein AM (green) while nuclei of dead cells were stained using ethidium homodimer-1 (red). Cells (2 × 10⁵ cells/well) were seeded on the surface of each porous scaffold (n=6). After an initial attachment period of 2 h, all scaffolds were transferred into a new 24-well plate, and 1 mL fresh culture medium added into each well. Plates with scaffolds were incubated in a 5% CO₂ atmosphere at 37 ℃. After incubation for 3 and 7 days, stained cells were imaged using LSCM. Cell adhesions to the scaffold surface were observed by LSCM (A1R, Nikon, Japan). After incubation for 3 h, 12 h, 3 d, and 7 d, scaffolds were washed thrice using PBS and fixed in 4% paraformaldehyde for 30 min at RT. Cells were permeabilized using 0.5% Triton X-100 for 5 min and washed twice using PBS. Then, F-actin and nuclei were respectively stained with Phalloidin and DAPI. The area, perimeter, and Feret’s diameter of cells were quantified by an image analyzer (ImageJ, NIH, Bethesda, ML).

8.2 Osteogenic differentiation assay. After 24 h, the culture medium was replaced with an osteogenic induction media containing 0.2 mM ascorbic acid, 10 mM β-glycerol phosphates, and 10^-7 M dexamethasone. The osteoinductive medium was refreshed every 2 days. In vitro osteogenesis was analyzed after 5 and 14 days of cell culture in the medium. Osteogenic gene expressions of rBMSCs and osteogenic protein expressions on scaffolds were quantified by qRT-PCR and WB. For the extracellular matrix mineralization assay, VonKossa staining was performed. Scaffolds and cells cultured with the leaching liquor were washed and fixed in 4% paraformaldehyde (PFA) for 15 min at RT and incubated in the presence of 1% silver nitrate solution under UV light for 20 min.

9. Animal responses to MEW printed PCL and ZIF-8/PCL scaffolds in vivo

9.1 Rat critical calvarial defect models and scaffold implantations. To assess the abilities of mineralized ZIF-8/PCL implants to enhance new bone growth in vivo, 24 male adult SD rats (age: 6 weeks, weight: 190-230 g; supplied by the animal center of Tongji medical college, Huazhong University of Science and Technology) were randomized into three groups: i. Blank; ii. PCL; iii. ZIF-8/PCL (n=8 rats per group). Prior to implantation, MEW printed scaffolds were immersed in SBF for 7 days and ethylene oxide–sterilized. For surgery, rats were anesthetized using isoflurane and subcutaneous buprenorphine injection, then a 2.0 cm sagittal incision was made on the middle of the scalp. Two parallel 5.0 mm critical cranial defects were drilled into each rat using a 5.0 mm diameter trephine (Nouvag AG, Goldach, Switzerland). After implantation of the scaffolds into defect areas, the incision was closed.

9.2 Micro-CT analysis. Animals were sacrificed at 4 and 12 weeks post-operation, their skulls collected and fixed in 10% formalin (n=4 rats per group). Bone tissues in defect areas (diameter: 5 mm; height: 2 mm) were evaluated by Micro-CT scanning. 3D images (mean threshold value = 226) of samples were reconstructed by the VG studio software and quantitative morphometric analyses performed using CTAn to determine bone volume (BV), bone volume/tissue volume (BV/TV), and bone mineral density (BMD).

9.3 Histological and immunohistochemical analysis. All samples were decalcified in EDTA for 4 weeks, paraffin-embedded, dehydrated through graded ethanol series and sliced into 3 μm thick sections. Then, Hematoxylin and eosin staining (H&E, Beyotime, China) as well as Masson’s trichrome staining were performed. OCN expressions were assessed via immunohistochemistry using appropriate antibodies (ab13420, Abcam, USA). Histological and immunohistochemical images were obtained by microscopy (Eclipse Ni-E; Tokyo, Japan).

10. Statistical analysis

Data are presented as mean ± SD. Comparisons of means between groups was performed by the Student’s t-test while one-way analysis of variance (ANOVA) combined with the Students’ Newman-Keuls (SNK) post hoc test were used to compare means among groups. p ≤ 0.05 was set as the threshold for statistical significance.

1. Biomimetic apatite deposition and osteogenic differentiation induced by ZIF-8 degradation in vitro

ZIF-8 nanoparticles were synthesized by one-pot method. Crystals of synthesized ZIF-8 were typical rhombic dodecahedral in shape, with a uniform size of 50 nm (Fig. 2a). Under physiological conditions, ZIF-8 nanoparticles were incubated in SBF solution for 12 h, 1 d, 4 d, 7 d, 14 d, and 21 d. After incubation, the solids collected were analyzed by TEM. The initial rhombic dodecahedron morphology was transformed into clusters of spherical-shaped nanoparticles and started to agglomerate and progressively enlarge over time. Further EDS mapping revealed that with increasing volume, a certain proportion of Ca, P atoms were included in the homogeneous agglomerate (Fig. 2e). Atomic fractions of Ca, P, and Zn in the mapping image showed the change in distributions of each element (Fig. 2b). Then, upon their treatment in SBF, the XRD technique was used to evaluate the relative crystallinity of ZIF-8 particles (Fig. 2c). The intensity of the peak arising from the (011) plane of sod-ZIF-8 crystal decreased with soaking time in SBF, revealing that the crystal structure of ZIF-8 was defective. And the appearance of new diffraction peaks indicated the formation of new crystal structures. The GC-MS and ICP-OES assays revealed comparable findings (Fig. 2h, i). GC-MS was performed to measure MeIm concentrations, which was fast protonated and released in mother liquors. ICP-OES was used to determine zinc release and calcium deposition over time.

Upon exposure to SBF, FTIR spectroscopy was used to assess changes in atomic connectivity of ZIF-8 (Fig. 2d). The spectra showed a progressive decrease in peak intensities of vibration modes related to (ν_C=N, 1584 cm^− 1; ν_ring, 1500–1350 cm^− 1), and a progressive reduction in the intensity of the band attributed to the Zn–N stretching mode (421 cm^− 1). The broad bands at 1160–900 cm^− 1 may be attributed to antisymmetric stretching modes of PO₄³⁻, and the 660–530 cm^− 1 is engendered by bending PO₄³⁻ groups. Given the high affinity of phosphate groups for polyvalent cations and the above presented results, we postulated that the new bands are due to biomimetic apatite deposition and formation of fewer zinc phosphates as degradation by-products of ZIF-8. These findings indicate that ZIF-8 particles were mineralized during their degradation.

DLS was used to measure their sizes and zeta potentials (Fig. 2f, g). After biomimetic mineralization, the volume of the mineralized mixture increased. The zeta potential of ZIF-8 was negative, while that of mineralized ZIF-8 was positive.

2. In vitro osteogenic differentiation of mineralized ZIF-8

After Alizarin Red S staining, optical microscopy revealed that rBMSCs cultured with mineralized ZIF-8 in the culture medium without the osteogenic induction solution exhibited mineral nodules after 14 days. However, the control had no mineral nodules (Fig. 3a). rBMSCs incubated with mineralized ZIF-8 were obtained to assess their responses to expressions of osteogenic differentiation-related genes and proteins. Osteogenic markers were measured after 5 and 14 days. Compared to controls, mRNA expressions of alkaline phosphatase (Alp), Runt-related transcription factor 2 (Runx2), osteocalcin (Ocn), and osteopontin (Opn) were upregulated (Fig. 3b). Besides, expressions of the corresponding osteogenic differentiation-related proteins in the mineralized ZIF-8 group were elevated (Fig. 3c, d). These results indicate that mineralized ZIF-8 has the ability to induce osteogenic differentiation in the culture medium without osteogenic induction components.

3. Fabrication and characterization of PCL, ZIF-8/PCL and MEW printed PCL, ZIF-8/PCL scaffolds

After determining the mineralization properties and mechanisms of ZIF-8, we fabricated flake-like PCL, ZIF-8/PCL via melt-mixing and compression molding synthesis. After 7 and 14 days of soaking in SBF, SEM imaging revealed a large number of crystal deposits on the surface of ZIF-8/PCL (Fig. 4a). EDS-mapping confirmed that the crystal deposits are composed of calcium, phosphorus, and zinc elements (Fig. 4b). Cross-section and coronal-section images obtained from micro-CT scanning demonstrated the attachment of apatites to the surfaces over time (Fig. 4c). Quantitative analysis of apatite volume (AV) confirmed that apatites were deposited on ZIF-8/PCL, while PCL is almost free of these deposits (Fig. 4d).

The compression test was performed to evaluate the mechanical properties of PCL and ZIF-8/PCL (Fig. 4e, Figure S1). Young’s modulus of PCL was 170.5 ± 14.92 MPa while that of ZIF-8/PCL was 144.1 ± 9.68 MPa. These findings imply that as the ZIF-8 incorporated, the elastic modulus became smaller while elastic deformation under the same load became larger. Water contact angle measurements confirmed the hydrophilic and hydrophobic degrees. Differences in water contact angle between PCL and mineralized PCL group (70.7 ± 0.46° for PCL and 71.3 ± 0.46° for ZIF-8/PCL) were insignificant. Due to addition of ZIF-8 and its biomimetic functions, water contact angle of ZIF-8/PCL was 74.5 ± 0.75°, while that of mineralized ZIF-8 was 80.7 ± 1.45°. (Fig. 4f). AFM imaging of the sample surface indicated surface roughness in nanoscales. The results confirmed that ZIF-8-induced apatite deposition increases the surface roughness of PCL by a factor of two (Fig. 4g). Thus, ZIF-8 combined with PCL greatly enhanced biomimetic mineralization of PCL materials that could not independently induce apatite depositions.

Then, we synthesized PCL, ZIF-8/PCL scaffolds via MEW printing (Fig. 5a). Morphologies of porous scaffolds were assessed by SEM (Fig. 5b). Microporosity was created after printing, and the addition of ZIF-8 did not augment or reduce the microporous structures of the scaffold. At a high-power field (10000x), rough surface of ZIF-8/PCL scaffolds exhibited abundant crystalline, which differs from the smooth surface of PCL. The EDS mapping of calcium, phosphate, and zinc element distributions confirmed uniform and homogeneous apatite deposition in scaffolds (Fig. 5c). The 3D reconstruction images as well as uniform distribution of the apatite layer were visualized and analyzed by Micro-CT (Fig. 5d). Apatite deposition grew along scaffold fiber surfaces, with a gradual increase over time. Quantitative analysis of apatite volume (AV) and calcium content verified this outcome (Fig. 5e, f). More apatite was deposited on MEW printed ZIF-8/PCL scaffolds, relative to flake-like ZIF-8/PCL materials.

Assessment of surface topography and three-dimensional (3D) micrographs by LSCM revealed altered surface morphologies (Fig. 5g). Under LED light, the color of the mineralized ZIF-8 group significantly differed from the other three groups, appearing whiter and brighter due to apatite deposits. To determine roughness, average roughness (Sa) and root mean square roughness (Sq) were calculated. On micron scales, corresponding Sa and Sq of PCL scaffolds in the original state were 1.112 and 1.614 µm, while those of the ZIF-8/PCL scaffold were 1.284 and 1.642 µm. After soaking in SBF for 7 days, PCL scaffold roughness showed little or no change. In the ZIF-8/PCL scaffolds group, surface roughness tended to decrease as micropores on the surface were constantly being filled by the sediment (Sa = 0.807, Sq = 1.010).

4. Biocompatibility of printed ZIF-8/PCL scaffolds in vitro

Having determined material characteristics of MEW printed ZIF-8/PCL scaffolds, we evaluated cell adhesion morphology and cell viability on different scaffolds. Cell viabilities were evaluated by Live/Dead staining, phalloidin, and DAPI staining. rBMSCs seeded on PCL and ZIF-8/PCL scaffolds were further stained with Calcein-AM and PI for CLSM imaging (Fig. 6a). Cells in the ZIF-8/PCL scaffold were adherent and demonstrated rapid, as well as even distributions, growth and proliferation. Living cells were mainly arranged along orthogonally printed fibers. With regards to PCL scaffold group, cells exhibited little adhesions and proliferations on scaffolds. CCK8 assays of cell proliferation confirmed this result (Fig. 6b), that BMSCs showed better cell viability in ZIF-8/PCL scaffold. Potential toxicities were evaluated by CCK8 assays. As shown in Fig. 6d, both scaffolds had no significant effect on the viabilities of rBMSCs.

The morphologies of cells adhering to the scaffold were assessed by CLSM, phalloidin, and DAPI staining. Actin cytoskeleton in rBMSCs seeding on the scaffold was visualized after culturing for 3 and 7 d (Fig. 6c). The ZIF-8/PCL scaffold exhibited better biocompatibility, compared to the PCL scaffold. Further, images of single BMSC morphology were obtained after cells had been cultured for 3 and 24 h. After 3 h of incubation, cells on the ZIF-8/PCL scaffold surfaces were larger than those on PCL scaffold surfaces (Fig. 6e). After 24 h of incubation, BMSC morphology on the ZIF-8/PCL scaffold surface was widely spread, exhibiting abundantly visible actin filaments with distinct cytoplasmic extensions. Cytomorphometric parameters, including cell area, perimeter, and Feret’s diameter revealed significant differences between these two scaffolds, supporting these qualitative observations (Fig. 6g, h, i). Image-based densitometry showed that actin expressions were markedly upregulated (1.5 times at 3 h and 1.8 times at 24 h) for cells cultured on ZIF-8/PCL scaffold surfaces at both 3 and 24 h (Fig. 6f).

5. Osteogenic differentiation of printed ZIF-8/PCL scaffolds in vitro

Having shown the cytocompatibility of the MEW printed ZIF-8/PCL scaffold, we assessed the scaffold’s ability to promote osteogenesis. For better observation, osteogenic induction components were supplemented in the cell culture medium. Extracellular matrix calcium deposits on the scaffold for mineralized nodule formation were stained by von Kossa, with calcified nodules appearing in black. The cells seeded on the ZIF-8/PCL scaffold exhibited enhanced mineral deposition, compared to control cells on day 14 (Fig. 7a). Comparable findings were obtained from rBMSCs incubated with a leaching solution of scaffolds. At the genetic level, expressions of osteogenesis-associated genes were evaluated by qRT-PCR. Compared to control and PCL groups, the ZIF-8/PCL scaffold group exhibited significantly higher levels of osteogenesis-associated gene expressions, including Alp, Runx2, Ocn, and Opn, which increased by 2.2-, 2.6-, 1.4, and 3.4- fold, respectively (Fig. 7b). Besides, expressions of the corresponding osteogenic differentiation-related proteins were increased in the ZIF-8/PCL scaffold group, compared to PCL groups (Fig. 7c, d). These results indicated that the ZIF-8/PCL scaffold enhanced the osteogenesis of rBMSCs in vitro, which was mainly ascribed to the addition of ZIF-8.

6. Abilities of printed ZIF-8/PCL scaffolds to repair critical-sized bone defects in vivo

Having demonstrated in vitro osteogenesis of MEW printed ZIF-8/PCL scaffolds, we assessed their in vivo osteogenic bioactivities in rat calvarial defect models (diameter: 5 mm) (Fig. 8a). Rats were euthanized at 4 and 12 weeks post-surgery, and their defect sites harvested to assess bone repair by micro-CT and histological analyses. Micro-CT scanning images were rebuilt to show the newly formed bones within the bone defects (Fig. 8b). 3D reconstruction images revealed that compared to the blank group, new bone formations were better in any other scaffold group, and the amounts of newly formed bones were highest in the ZIF-8/PCL scaffold group. Moreover, small islands of new bones were present in the center of defects. Fundamental parameters, including bone volume, relative ratio of bone volume to tissue volume (BV/TV), bone mineral density (BMD), were quantitatively analyzed (Fig. 8c-e). During osteogenesis evaluation, the ZIF-8/PCL scaffold induced a higher BV, BV/TV ratio, and BMD, leading to higher growth kinetics of newborn osseous tissue volume and improved average bone density in the circular calvarial defect, compared to the primitive PCL scaffold.

Histological assessment based on H&E, Masson’s Trichrome, and immunohistochemical staining of decalcified bone samples verified the micro-CT findings (Fig. 9a, b). After 4 weeks of implantation, the new bone growing from defect margins and the space between struts of scaffolds in the ZIF-8/PCL scaffold group indicated osteoconductive abilities of scaffolds. After implantation for 12 weeks in the ZIF-8/PCL scaffold group, the defects were almost entirely closed. Immunohistochemical staining showed that the positive expression area of OCN was largest in the ZIF-8/PCL scaffold group, compared to other groups (Fig. 9c). Histology analyses of major organs (cerebrum, liver, and kidneys) stained with H&E were performed to evaluate potential toxicities. There were no pathological changes in the examined organs (Figure S2).

In addition, ZIF-8 was immersed in SBF after compounded with polymer material polylactic acid(PLA)by electrostatic spinning technology, and it was found that ZIF-8/PLA also had the ability to induce mineral deposition (Figure S3). In parallel, several other MOFs were attempted. The results showing that no similar mineralization effect was seen in MIL-100/PCL, UIO-66/PCL (Figure S4).

We investigated ZIF-8 degradation-induced biomimetic apatite deposition in the humoral milieu, and the materials with poor biological activity could biomimetic mineralization effect can be produced on the surface of using this feature. Therefore, we presented a ZIF-8 incorporating porous scaffolds through MEW printing technology. During ZIF-8 degradation, zinc ions were released while calcium ions competitively bound phosphate ions, leading to apatite deposition. This property of ZIF-8 plays a good role when mixed with PCL materials, which have poor biological activities but good mechanical properties. ZIF-8/PCL materials were proven to actively induce apatite deposition and support osteogenesis.

Due to its excellent chemical and thermal stability as well as pH sensitivity with negligible cytotoxicity, ZIF-8 is an excellent candidate for bone regeneration. Apart from serving as a carrier for drug delivery in studies on biological effects in bone tissue engineering ^{[8, 9]}, ZIF-8 has been reported to promote osteogenesis through the release of zinc ions by degradation in the humoral environment ^[25–27]. While the role of zinc ions release has been elucidated, the products formed by interactions between ZIF-8 degradation and other ions in solution have not been conclusively determined. The mechanism of ZIF-8 degradation in PBS, which involves coordination equilibrium between Zn²⁺ ions and HmIM in solution is altered by the presence of phosphate species. Such phosphates have a high affinity for phosphate groups for polyvalent cations shifting the equilibrium towards the formation of insoluble inorganic by-products ^[11]. Compared to PBS, the balance of ion environments is more complex in SBF. SBF solutions contain active metal ion calcium, which has the potential to produce calcium and phosphorus deposition by competitive combination in solutions. In this study, we confirmed that ZIF-8 degrades and induces apatite deposition in SBF, and finally becomes "mineralized pellets". We hypothesized that hydrogen bonds and zinc–nitrogen bonds in ZIF-8 gradually breakdown due to calcium ions attack from the SBF solution, resulting into the formation of an intermediate composed of zinc and calcium ions boned with a single 2-HmIM ligand. Then, H₂O and HPO₄⁻ acts as hydrogen donors for fast protonation of 2-HmIM, leading to abundant zinc hydroxy phosphate and calcium hydroxy phosphate formation. This protonation also results in partial release of zinc ions. Moreover, since SBF replaces calcium ions daily, calcium ion levels remain relatively stable at a high level, and the precipitation equilibrium constantly shifts in the calcium hydroxyphosphate direction. The "mineralized pellets" continuously agglomerated by covalent bonding or electrostatic adsorption. In the process, we detected the release of Zn²⁺ ions and the consumption of Ca²⁺ in the reaction system, along with the release of a large number of protonated HmIM, which could further promote the change of soluble hydrogen phosphate in SBF to precipitation-prone phosphate ions, leading to apatite deposition. Hydroxyl groups were also detected in both non-mineralized and mineralized ZIF-8 by FTIR, which could promote crystal growth, morphological, as well as aggregation processes by electrostatic interactions ^[28]. As shown in EDS mapping images, Zinc was uniformly distributed in mineralized ZIF-8. This could have been because, before the bond was broken, during degradation, a part of the residue of ZIF-8 was wrapped by "mineralized pellets"; A small fraction of insoluble zinc phosphate was also produced during ion competitive binding, although calcium ions are more reactive than zinc ions. Under the premise that zinc and apatite could promote bone regeneration through enhanced osteogenesis ^[29–32], positive results of osteogenic experiments have been explained.

Surface compositions of scaffolds affects their response and integration ^[33]. PCL is a common biomedical polymer material with good toughness, excellent biocompatibility, and minimal cytotoxicity, as supporting materials of the scaffolds ^[34]. However, PCL-only scaffolds have no active coating on the surface and lack recognition sites for bone binding abilities, which prolongs the healing time of bone repair ^[35].

We have put forward that ZIF-8 is suitable for PCL in improvement of biomimetic mineralization properties and preserved mechanical properties. We prepared flake-like ZIF-8/PCL by melt mixing molding first. After immersing in SBF for several days, there were mineralized crystallization depositions on the surface. Bone regeneration is inseparable from the balance between scaffold structures and mechanical properties. Scaffolds need to ensure sufficient porosity, provide conditions for penetration of vasogenic cells, growth of surrounding tissues and make necessary preparations for the formation of mechanical strength similar to natural bone, so as to ensure the therapeutic effects of implants ^[36]. The MEW technique can effectively control scaffold pore sizes and porosity ^[21]. Previous studies have shown that the optimal stent aperture for hard tissue regeneration is between 200 µm and 500 µm ^[37]. Thus, by adjusting the parameters of the MEW machine, we prepared the scaffold with a diameter of about 150 µm and an aperture between the fibers of 200–300 µm, which maintained the mechanical strength but also facilitated the growth of tissue blood vessels. Compared to flake-like ZIF-8/PCL, MEW printed ZIF-8/PCL scaffold exhibited more deposited apatite for the same immersion times. This could have been because the MEW printing technology provided a Three-dimensional (3D) scaffold that enhances the surface area to volume ratio and allows more apatite deposition. In the extrusion process, the melt produces a microporosity structure, which further increases the surface area and biological mineralization sites of the scaffold.

Anchoring dependent cells do not divide without prior extension on growth substrates, and even undergo apoptosis ^[38]. Therefore, in the first stage of cell expansion, the expansion area size is positively correlated with cell proliferation activities. In this study, the ZIF-8/PCL scaffold evoked favorable cellular responses in terms of cell attachment, adhesion, proliferation. Specifically, cell spreading, morphology, and actin organization were outperformed the PCL scaffold group. Outstanding cell activities of the ZIF-8/PCL scaffold were attributed to various aspects. Most of the surface of ZIF-8 particles were negatively charged, and the fibers were negatively charged through the MEW printing process. Hence, the surface of ZIF-8/PCL is generally negatively charged, which is very favorable for apatite nucleation ^[39]. During mineralization, positively charged apatite layers were gradually formed, the zeta potential of ZIF-8 particles changed from negative to positive, resulting in a positive charge on the surface of ZIF-8/PCL scaffold. There is better cell adhesion to positively charged surfaces than to negatively charged surfaces. The reason is that cell adhesion-mediating ECM molecules are negatively charged, therefore, they preferentially adsorb to positively charged surfaces ^[40–42]. The offset and gradient pore size-structured MEW printed scaffolds provided dense structures that have the advantages of mechanical integrity and high porosity for better cell infiltrations in bone tissue engineering ^[21]. The other aspect was surface roughness. In the process of biomimetic mineralization in microroughness, the ZIF-8/PCL surface became rough. Increased surface roughness of orthopedic implants results in enhanced apatite deposition, initial cell attachment, proliferation, and differentiation ^{[43, 44]}.

Moreover, the influence of MEW printed ZIF-8/PCL scaffolds on rBMSCs osteogenic differentiation in co-culture were marked. In experiments on osteogenic induction without osteogenic induction solution of mineralized ZIF-8, the calcium nodule emerged, but was not in abundance. This might be because, ZIF-8 was mineralized before co-cultures, and zinc release was not involved in the effects of osteogenesis induction, while zinc in the biomaterials was proven to up-regulate the expressions of osteogenesis-related genes, such as Alp, Runx2, Col I, Opn, and Ocn. This effect accelerated extracellular matrix mineralization by increasing Col secretion synthesis and calcium deposition ^[45]. Our assays did not demonstrate whether mineralized ZIF-8 could directly induce rBMSCs osteogenesis. We postulate that for MEW printed ZIF-8/PCL scaffolds, internal ZIF-8 in PCL would be exposed during PCL degradation, leading to synergistic promotion of osteogenic differentiation by zinc and biomimetic mineralization. The specific process should be further verified. In vivo, MEW printed ZIF-8/PCL scaffolds significantly improved bone defect regeneration, manifested by new bone ingrowth both around the defect edge and between the scaffold fiber, and its volume was significantly higher than that in the control group. This MEW printed ZIF-8/PCL scaffold has limitations for bone regeneration. The diameter of MEW fibers was in the range of several to dozens of micrometers, which is much larger than the nanoscale extracellular matrix (ECM). Besides, the slow PCL degradation may also prevent new bone formation at later stages. Additional studies are required to further investigate these possibilities.

ZIF-8 was disintegrated caused by the attack of Ca²⁺ ions and the competitive binding of ligands and form a large number of hydroxyapatite-like crystal nuclei are precipitated, which effectively promotes the osteogenic differentiation and proliferation of BMSCs without toxicity. Better yet, ZIF-8 could continue its role as a biomimetic mineralization site on the PCL surface. In this study, MEW printed ZIF-8/PCL scaffold promoted osteogenic differentiation in vitro and in vivo owed to the induced mineralization capacity of ZIF-8 indicating the potential use of this scaffold for bone tissue engineering. Importantly, modification of PCL materials using the induced mineralization properties of ZIF-8 is not limited to PCL materials but is also expected to improve the biological activities of other biomaterials.

2-HmIM	2-methylimidazole
Alp	alkaline phosphatase
AV	Apatite volume
AFM	atomic force microscopy
BMD	bone mineral density
BV	bone volume
BV/TV	bone volume/tissue volume
CAP	calcium phosphate
CCK-8	Cell Counting Kit-8
DLS	dynamic light scattering
FBS	fetal bovine serum
FT-IR	Fourier Transform Infrared
LSCM	laser scanning confocal microscopy
MEW	melt electrowrittten
MOF	metal-organic framework
Ocn	osteocalcin
Opn	osteopontin
PBS	phosphate buffered saline
PCL	polycaprolactone
AV/TV	percentage of apatite volume, relative to total volume
PLA	polylactic acid
rBMSCs	rat bone mesenchymal stem cells
Runx2	Runt-related transcription factor 2
SBF	simulated body fluid
SEM	Scanning electron microscopy
(3D	Three-dimensional
TEM	Transmission electron microscopy
XRD	X-ray diffract meter
ZIF-8	Zeolite imidazolate framework-8

Ethics approval and consent to participate

Number [2019]IEC(S1154) was approved by Experimental Animal Ethics Committee of Huazhong

University of Science and Technology

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

The authors declare no conflict of interest.

Funding

Funding for the work was received by the National Nature Science Foundation of China (No.82020108020, 82072198, 81873941 and 81701922).

Authors' contributions

BQ. W and YY. Z were responsible of conceptualization, original draft preparation, experiments and data analysis, while SK. L, MR. Z and HM. F of editing and supervision, ZX. W and JM. S was responsible of project administration and funding acquisition. BQ. W and YY. Z contributed equally to this manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

The authors would like to thank the Analytical & Testing Center of Huazhong University of Science and Technology. They also thank Dr. Zhijun Chen from School of Textile Science and Engineering, Wuhan Textile University for kindly providing us the MEW printing machine.

Ji Z, Freund R, Diercks CS, Hirschle P, Yaghi OM, Wuttke S. From Molecules to Frameworks to Superframework Crystals. Adv Mater. 2021 Oct;33(42):2103808.
Gao L, Chen Q, Gong T, Liu J, Li C. Recent advancement of imidazolate framework (ZIF-8) based nanoformulations for synergistic tumor therapy. Nanoscale. 2019;11(44):21030–45.
Kumar A, Sharma A, Chen Y, Jones MM, Vanyo ST, Li C, et al. Copper@ZIF-8 Core‐Shell Nanowires for Reusable Antimicrobial Face Masks. Adv Funct Mater. 2021 Mar;31(10):2008054.
Simon-Yarza T, Mielcarek A, Couvreur P, Serre C. Nanoparticles of Metal-Organic Frameworks: On the Road to In Vivo Efficacy in Biomedicine. Adv Mater. 2018 Sep;30(37):1707365.
Li G, Zhao S, Zhang Y, Tang Z. Metal-Organic Frameworks Encapsulating Active Nanoparticles as Emerging Composites for Catalysis: Recent Progress and Perspectives. Adv Mater. 2018 Dec;30(51):1800702.
Liang W, Xu H, Carraro F, Maddigan NK, Li Q, Bell SG, et al. Enhanced Activity of Enzymes Encapsulated in Hydrophilic Metal–Organic Frameworks. J Am Chem Soc. 2019 Feb 13;141(6):2348–55.
Mao D, Li W, Zhang F, Yang S, Isak AN, Song Y, et al. Nanocomposite of Peroxidase-Like Cucurbit[6]uril with Enzyme-Encapsulated ZIF-8 and Application for Colorimetric Biosensing. ACS Appl Mater Interfaces. 2021 Aug 25;13(33):39719–29.
Gao X, Xue Y, Zhu Z, Chen J, Liu Y, Cheng X, et al. Nanoscale Zeolitic Imidazolate Framework-8 Activator of Canonical MAPK Signaling for Bone Repair. ACS Appl Mater Interfaces. 2021 Jan 13;13(1):97–111.
Wang X, Miao D, Liang X, Liang J, Zhang C, Yang J, et al. Nanocapsules engineered from polyhedral ZIF-8 templates for bone-targeted hydrophobic drug delivery. Biomater Sci. 2017;5(4):658–62.
Luzuriaga MA, Benjamin CE, Gaertner MW, Lee H, Herbert FC, Mallick S, et al. ZIF-8 degrades in cell media, serum, and some—but not all—common laboratory buffers. Supramolecular Chemistry. 2019 Aug 3;31(8):485–90.
Bauzá A, Mooibroek TJ, Frontera A. Towards design strategies for anion–π interactions in crystal engineering. CrystEngComm. 2016;18(1):10–23.
Wang Y, Yan J, Wen N, Xiong H, Cai S, He Q, et al. Metal-organic frameworks for stimuli-responsive drug delivery. Biomaterials. 2020 Feb;230:119619.
Lotsari A, Rajasekharan AK, Halvarsson M, Andersson M. Transformation of amorphous calcium phosphate to bone-like apatite. Nat Commun. 2018 Dec;9(1):4170.
Dejob L, Toury B, Tadier S, Grémillard L, Gaillard C, Salles V. Electrospinning of in situ synthesized silica-based and calcium phosphate bioceramics for applications in bone tissue engineering: A review. Acta Biomater. 2021 Mar;123:123–53.
Mozafari M, Banijamali S, Baino F, Kargozar S, Hill RG. Calcium carbonate: Adored and ignored in bioactivity assessment. Acta Biomater. 2019 Jun;91:35–47.
Salimi E. Development of bioactive sodium alginate/sulfonated polyether ether ketone/hydroxyapatite nanocomposites: Synthesis and in-vitro studies. Carbohydr Polym. 2021 Sep;267:118236.
Camarero-Espinosa S, Moroni L. Janus 3D printed dynamic scaffolds for nanovibration-driven bone regeneration. Nat Commun. 2021 Dec;12(1):1031.
a) Liu Z, Wan X, Wang ZL, Li L. Electroactive Biomaterials and Systems for Cell Fate Determination and Tissue Regeneration: Design and Applications. Adv Mater. 2021 Aug;33(32):2007429. b) Collins MN, Ren G, Young K, Pina S, Reis RL, Oliveira JM. Scaffold Fabrication Technologies and Structure/Function Properties in Bone Tissue Engineering. Adv. Funct. Mater. 2021, 31, 2010609.
Wang Z, Wang Y, Yan J, Zhang K, Lin F, Xiang L, et al. Pharmaceutical electrospinning and 3D printing scaffold design for bone regeneration. Adv Drug Deliv Rev. 2021 Jul;174:504–34.
Kade JC, Dalton PD. Polymers for Melt Electrowriting. Adv Healthc Mater. 2021 Jan;10(1):2001232.
Abbasi N, Abdal-hay A, Hamlet S, Graham E, Ivanovski S. Effects of Gradient and Offset Architectures on the Mechanical and Biological Properties of 3-D Melt Electrowritten (MEW) Scaffolds. ACS Biomater Sci Eng. 2019 Jul 8;5(7):3448–61.
Abbasi N, Lee RSB, Ivanovski S, Love RM, Hamlet S. In vivo bone regeneration assessment of offset and gradient melt electrowritten (MEW) PCL scaffolds. Biomater Res. 2020 Dec;24(1):17.
Cravillon J, Münzer S, Lohmeier SJ, Feldhoff A, Huber K, Wiebcke M. Rapid Room-Temperature Synthesis and Characterization of Nanocrystals of a Prototypical Zeolitic Imidazolate Framework. Chem Mater. 2009;21:1410–2.
Wang ZX, Chen C, Zhou Q, Wang XS, Zhou G, Liu W, et al. The Treatment Efficacy of Bone Tissue Engineering Strategy for Repairing Segmental Bone Defects Under Osteoporotic Conditions. Tissue Eng Part A. 2015 Sep;21(17–18):2346–55.
Shen Y, Liang L, Zhang S, Huang D, Zhang J, Xu S, et al. Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing. Nanoscale. 2018;10(4):1622–30.
Zhang X, Chen J, Pei X, et al. Enhanced Osseointegration of Porous Titanium Modified with Zeolitic Imidazolate Framework-8. ACS Appl Mater Interfaces. 2017;9(30):25171–83.
Xue Y, Zhu Z, Zhang X, Chen J, Yang X, Gao X, et al. Accelerated Bone Regeneration by MOF Modified Multifunctional Membranes through Enhancement of Osteogenic and Angiogenic Performance. Adv Healthc Mater. 2021 Mar;10(6):2001369.
Kokubo T, Kim HM, Kawashita M. Novel bioactive materials with different mechanical properties. Biomaterials. 2003 Jun;24(13):2161–75.
Kim HD, Amirthalingam S, Kim SL, Lee SS, Rangasamy J, Hwang NS. Biomimetic Materials and Fabrication Approaches for Bone Tissue Engineering. Adv Healthc Mater. 2017 Dec;6(23):1700612.
Gao X, Xue Y, Zhu Z, Chen J, Liu Y, Cheng X, et al. Nanoscale Zeolitic Imidazolate Framework-8 Activator of Canonical MAPK Signaling for Bone Repair. ACS Appl Mater Interfaces. 2021 Jan 13;13(1):97–111.
Su Y, Yang H, Gao J, Qin Y, Zheng Y, Zhu D. Interfacial Zinc Phosphate is the Key to Controlling Biocompatibility of Metallic Zinc Implants. Adv Sci. 2019 Jul;6(14):1900112.
Yao S, Jin B, Liu Z, Shao C, Zhao R, Wang X, et al. Biomineralization: From Material Tactics to Biological Strategy. Adv Mater. 2017 Apr;29(14):1605903.
Jiang J, Li Z, Wang H, Wang Y, Carlson MA, Teusink MJ, et al. Expanded 3D Nanofiber Scaffolds: Cell Penetration, Neovascularization, and Host Response. Adv Healthc Mater. 2016 Dec;5(23):2993–3003.
Yang X, Wang Y, Zhou Y, Chen J, Wan Q. The Application of Polycaprolactone in Three-Dimensional Printing Scaffolds for Bone Tissue Engineering. Polymers. 2021 Aug 17;13(16):2754.
Li L, Li J, Guo J, Zhang H, Zhang X, Yin C, et al. 3D Molecularly Functionalized Cell-Free Biomimetic Scaffolds for Osteochondral Regeneration. Adv Funct Mater. 2019 Feb;29(6):1807356.
Egan. Integrated Design Approaches for 3D Printed Tissue Scaffolds: Review and Outlook. Materials. 2019 Jul 24;12(15):2355.
Bahrami N, Bayat M, Ai A, Ebrahimi-Barough S, Ai J, Ahmadi A, Salehi M, Ghodarzi A, Khanmohammadi M, Karimi R, Mohamadnia A, Rahimi A. Differentiation of Periodontal Ligament Stem Cells into Osteoblasts on Hybrid Alginate/ Polyvinyl Alcohol/ Hydroxyapatite Nanofibrous Scaffolds. Arch Neurosci.In Press; 2018. Jul; e74267.
Kaniuk Ł, Krysiak ZJ, Metwally S, Stachewicz U. Osteoblasts and fibroblasts attachment to poly(3-hydroxybutyric acid-co-3-hydrovaleric acid) (PHBV) film and electrospun scaffolds. Mater Sci Engineering: C. 2020 May;110:110668.
Yang M, Xu W, Chen Z, Chen M, Zhang X, He H, et al. Engineering Hibiscus-Like Riboflavin/ZIF‐8 Microsphere Composites to Enhance Transepithelial Corneal Cross‐Linking. Advanced Materials. 2022 Apr 14;2109865.
Tomasetti L, Breunig M. Preventing Obstructions of Nanosized Drug Delivery Systems by the Extracellular Matrix. Adv Healthc Mater. 2018 Feb;7(3):1700739.
Song Q, Jiao K, Tonggu L, Wang LG, Zhang SL, Yang YD, et al. Contribution of biomimetic collagen-ligand interaction to intrafibrillar mineralization. Sci Adv. 2019 Mar;5(3):eaav9075.
Conte C, Dal Poggetto G, Swartzwelter J, Esposito B, Ungaro D, Laurienzo F P, et al. Surface Exposure of PEG and Amines on Biodegradable Nanoparticles as a Strategy to Tune Their Interaction with Protein-Rich Biological Media. Nanomaterials. 2019 Sep 20;9(10):1354.
Thukkaram M, Coryn R, Asadian M, Esbah Tabaei PS, Rigole P, Rajendhran N, et al. Fabrication of Microporous Coatings on Titanium Implants with Improved Mechanical, Antibacterial, and Cell-Interactive Properties. ACS Appl Mater Interfaces. 2020 Jul 8;12(27):30155–69.
Zhang Y, Jiang W, Yuan S, Zhao Q, Liu Z, Yu W. Impacts of a Nano-Laponite Ceramic on Surface Performance, Apatite Mineralization, Cell Response, and Osseointegration of a Polyimide-Based Biocomposite. IJN. 2020 Nov;Volume 15:9389–405.
Xue Y, Zhu Z, Zhang X, Chen J, Yang X, Gao X, et al. Accelerated Bone Regeneration by MOF Modified Multifunctional Membranes through Enhancement of Osteogenic and Angiogenic Performance. Adv Healthc Mater. 2021 Mar;10(6):2001369.

Download PDF

Version 1

posted

You are reading this latest preprint version

ZIF-8 induced hydroxyapatite-like crystals enabled superior osteogenic ability of MEW printing PCL scaffolds

Status:

Version 1

Abstract

Figures

Background

Methods

Results

3. Fabrication and characterization of PCL, ZIF-8/PCL and MEW printed PCL, ZIF-8/PCL scaffolds

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1