Vitamin D3 Release from MgO Doped 3D Printed TCP Scaffolds for Bone Regeneration

Abstract

Regenerating bone tissue in critical-sized craniofacial bone defects remains challenging and requires the implementation of innovative bone implants with early stage osteogenesis and blood vessel formation. Vitamin D3 is incorporated into MgO-doped 3D-printed scaffolds for defect-specific and patient-specific implants in low load-bearing areas. This novel bone implant also promotes early stage osteogenesis and blood vessel development. Our results show that vitamin D3-loaded MgO-doped 3D-printed scaffolds enhance osteoblast cell proliferation 1.3-fold after being cultured for 7 days. Coculture studies on osteoblasts derived from human mesenchymal stem cells (hMSCs) and osteoclasts derived from monocytes show the upregulation of genes related to osteoblastogenesis and the downregulation of RANK-L, which is essential for osteoclastogenesis. Release of vitamin D3 also inhibits osteoclast differentiation by 1.9-fold after a 21-day culture. After 6 weeks, vitamin D3 release from MgO-doped 3D-printed scaffolds enhances the new bone formation, mineralization, and angiogenic potential. The multifunctional 3D-printed scaffolds can improve early stage osteogenesis and blood vessel formation in craniofacial bone defects.

Graphical Abstract

1. INTRODUCTION

The increasing number of patients suffering from bone related disorders caused by tumors and traumas is becoming a major health issue.¹ Resection of tumors can generate critical-sized bone defects.² The advent of three-dimensional (3D) printing, such as binder jetting, has improved the clinical outcomes of patient-specific bone regeneration by allowing the fabrication of a bone-mimicking microstructure using bone-like materials.^3,4 However, it remains challenging to design defect-specific and patient-specific implants with therapeutic actions of osteogenesis, osteoclastogenesis, and angiogenesis, which are closely associated with bone formation and remodeling.⁵ Here, we have developed a novel bone implant with osteogenic and angiogenic capabilities to enhance early stage bone regeneration.

Vitamins are essential elements that are required for proper metabolic functions.⁶ Studies on vitamins over the last few decades have revealed their potential as regenerative medicines.^7–10 For the musculoskeletal system, vitamin D3 also plays an important role.¹¹ The World Health Organization (WHO) suggests a daily intake of 15–20 μg of vitamin D3 is essential for bone maintenance.¹² However, it has been revealed over the last few decades that 92% of the US population suffers from vitamin-related deficiencies, which can lead to osteomalacia and hypocalcemia.^13–15 Osteomalacia, or bone softening, is often accredited to a vitamin D3 deficiency.¹⁶ Similarly, a deficiency in vitamin D3 can lead to hypocalcemia and rickets.^17,18 Vitamin D3 assists in calcium deposition and mineralization of bone while decreasing bone loss and risk of fracture.^19,20

Bone is a complex structure whose development occurs by simultaneous formation and resorption, termed bone remodeling.^21,22 The cells associated with bone formation and calcium phosphate deposition are known as osteoblast cells, and the cells associated with bone resorption are known as osteoclast cells.²³ Early stage osteogenesis is vital after implantation in large bone defects when it is essential to have enhanced osteoblast proliferation and reduced osteoclast differentiation to promote early stage osteogenesis.^24,25 Vitamin D3 can assist in osteoblast proliferation via upregulating gene expressions such as BGLAP, RUNX2, and ALPL.^26–28 It has been observed that vitamin D3 can inhibit the growth of osteoclast cells and osteoclast differentiation from monocytes via the downregulation of RANK-L.²⁹ Vitamin D3 also has angiogenic potential, which promotes blood vessel formation.³⁰ With the advent of three-dimensional (3D) printing, the fields of medicine and biology have experienced a striking revolution.³¹ 3D-printed scaffolds can imitate the anatomy and chemistry of bone, allowing patient-specific therapy.³² Micropores in 3D-printed scaffolds facilitate transporting nutrients and oxygen that promote new bone growth.³³ The chemical and compositional similarity of tricalcium phosphate (TCP) to bone continues to attract the attention of researchers as a promising bone graft material.³⁴ However, its poor mechanical strength is a concern.³⁵ The MgO doping enhances mechanical properties, including superior compression strength.³⁶ Mg enhances different biological properties and tissue regeneration.^37,38 Magnesium (Mg) as a dopant in tricalcium phosphate (TCP) results in a smaller grain size, which, in turn, facilitates more effective packing during the sintering process. The TCP phase has higher temperature stability when Mg²⁺ ions are present.³⁹ It has been observed that the presence of magnesium oxide (MgO) can enhance the viability of osteoblast cells and promote their proliferation.⁴⁰ Mg²⁺ promotes osteoblasts’ adhesion, proliferation, and differentiation by activating the PI3K/Akt signaling pathway. Mg²⁺ is known to participate in the PI3K/Akt signaling pathway through the ion channel functional protein kinase TRPM7.⁴¹ Mg²⁺ assists in the inhibition of osteoclast differentiation via osteoprotegerin (OPG) secretion while promoting osteoblast proliferation.⁴²

Our primary research question is can we manufacture a functionalized patient-specific and defect-specific implant promoting early stage osteogenesis and angiogenesis. We hypothesize that MgO-doped 3D-printed TCP scaffolds with incorporated vitamin D3 can enhance early stage osteogenic and angiogenic potential. Vitamin D3 release behavior and osteogenic and osteoclastic differentiation in vitro were investigated to evaluate our hypothesis comprehensively. To further evaluate their bone regeneration and blood vessel formation performance in vivo, vitamin D3-loaded MgO-doped TCP scaffolds were implanted into the critical-sized femur defects of rats and analyzed by histological examination. Our results indicate that a novel 3D-printed drug delivery system enhances new bone and blood vessel formation at an early stage.

2. MATERIALS AND METHODS

2.1. Scaffold Preparation.

A binder jet printer (Innovent, ExOne, USA) was used to fabricate the 3D-printed scaffolds using synthesized ß-TCP powder for control TCP scaffolds.⁴³ 1 wt % MgO (Sigma-Aldrich, USA) doped TCP powder was prepared by combining 50 g of TCP powder, and 0.5 g of MgO (99.99%, Sigma-Aldrich, USA) was prepared in 250 mL bottles for MgO doping. The mixture was milled using zirconia milling media with a powder-to-ball ratio of 2:1 for 2 h at 70 rpm.⁴⁴ A hopper was utilized to disperse powder material layers evenly followed by layer compaction. Once the roller had compacted the intermittent powder layer, the inkjet printing head traversed the build bed, depositing liquid droplets of the binder material. This process continued until the entire part had been printed. The 3D-printed samples were cured at a heat of 175 °C for 1.5 h before removing loose powders using air to obtain the green part strength necessary for postprocessing handleability. The unbound powder was removed by air. These green scaffolds were then sintered at 1250 °C for 2 h by using a conventional furnace (Hot Spot 110, Zircar Zirconia, USA). 3D structures were printed applying optimized parameters: layer thickness: 50 μm, binder set time: 2 s, binder drying time: 10 s, and binder saturation: 85%.

2.2. Drug Loading and in Vitro Release.

In vitro vitamin D3 release was measured in a phosphate buffer solution (pH 7.4) and acetate buffer solution (pH 5.0) for 28 days. The phosphate buffer mimics the physiological environment of the human body, and the acetate buffer is similar to the microenvironments of involved body parts postsurgery. Drop casting was used to coat the entire surface of the scaffold with a solution of vitamin D3 (99%, APExBIO, USA) dissolved in ethanol (100%, KOPTEC, USA). Samples loaded with 400 μg of vitamin D3 were kept in glass vials with 4 mL of the buffer; during storage, these vials were shaken at 150 rpm and 37 °C inside a shaker. The buffer media was pulled out and replaced with fresh buffer at each time point. A UV–vis spectroscopy microplate reader (BioTek Synergy 2 SLFPTAD, BioTek, USA) was used to measure the absorbance of the collected solutions at 260 nm to check for the presence of vitamin D3.

2.3. Effective Concentration Study.

To ascertain the optimal vitamin D3 concentration necessary to promote increased osteoblast cell growth, in vitro experiments were conducted by using cultured osteoblast cells. Specifically, human fetal osteoblast cells (hfOB; ATCC, Manassas, VA), derived from human bone tissue and used between passages 4 and 8, were cultured for 7 days. During this time, they were exposed to varying concentrations of vitamin D3, ranging from 1 μg to 35 μg. The viability of the cells was assessed in triplicate using the MTT assay, as previously described. The concentration that resulted in the highest optical density was selected for further consideration.

2.4. In Vitro Osteoblast Cell Culture Study.

Vitamin D3 (99%, APExBIO, USA) was dissolved in ethanol (100%, KOPTEC, USA) at a concentration of 0.3 mg/mL for the in vitro cell culture, and 20 μg of vitamin D3 was loaded on each of the samples. Human fetal osteoblast cells (hFOB, ATCC, USA) were cultured on the samples for 3 and 7 days. A mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/HF12, Sigma-Aldrich, USA) at a ratio of 1:1 was used as the cell culture media. Media supplements consisted of G418 (Sigma-Aldrich, USA), sodium bicarbonate (NaHCO3, Fisher Scientific, USA), fetal bovine serum (10% FBS, ATCC, USA), and penicillin-streptomycin (ATCC, USA). 150 μL of a cell suspension containing 20,000 cells was seeded by unidirectional seeding directly onto each sample. The samples were incubated at 34 °C under a 5% CO₂ atmosphere, with the media changed every 2–3 days.

2.5. In Vitro Osteoclast Cell Culture Study.

THP1 monocytes (ATCC, USA) were seeded onto the samples at a density of 20,000 cells/sample. The osteoclast differentiation media consisted of RPMI-1640 with the addition of 40 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma, USA), 10 ng/mL receptor activator of nuclear factor kappa-B ligand (RANK-L), an antibiotic, nonessential amino acid (NEAA), and FBS. All samples were incubated at 34 °C in a 5% CO₂ atmosphere, and the differentiation media was changed every 2–3 days. The tartrate acid phosphatase (TRAP) assay was conducted to understand osteoclast activity after days 11 and 21. For the resorption pit assay, samples were washed 3 times with a phosphate-buffered saline (PBS) solution after removing the media. Then, samples were ultrasonicated for 10 min with 500 μL of a buffer solution/Triton X-100 mixture with a ratio of 500:1. The samples were washed 3 times with PBS and dehydrated to analyze resorption pit formation through SEM images.

2.6. Viability of Osteoclast Cells.

The viability of osteoclast cells on different scaffolds was evaluated by live–dead staining with 800 mL of a 1:1 mixture of a calcein AM (Biolegend, CA, USA) and propidium iodide (Invitrogen, MA, USA) solution (in PBS). The fluorescence was visualized using a fluorescence microscope (Leica confocal SP5, USA).⁴⁵

2.7. In Vitro Coculture of hMSCs and Monocytes.

The coculture used human bone-marrow-derived mesenchymal stem cells (hMSCs, Lonza, USA) and monocytes (THP-1, ATCC, USA). hMSCs and THP-1 cells were cultured in mesenchymal stem cell growth medium (Lonza, USA) and RPMI-1640 with 0.05 mM 2-mercaptoethanol and 10% FBS. hMSCs and THP-1 cells were seeded on the samples, each at a density of 20,000 cells. An hMSC Osteogenic Differentiation Medium BulletKit (Lonza, USA) was prepared according to manufacturer instructions; additionally, a second osteoclast differentiation medium was prepared, consisting of 10% FBS (ATCC, USA), 10 ng/mL RANK-L (Abcam, USA), and 40 ng/mL phorbol 12-myristate13-acetate (PMA, Abcam, USA) combined in RPMI-1640. A 1:1 mixture of these two mediums was used in the cell cultures. The samples were cultured at 37 °C in a 5% CO₂ humidified atmosphere, changing media every 2–3 days.

2.8. RT-qPCR of Cocultured Samples.

RNA was extracted from the samples on day 11 of the coculture. The extraction was performed using the Aurum TM Total RNA Mini Kit (Bio-Rad, USA), following the manufacturer’s instructions.Fifteen μL of extracted RNA was reverse transcribed to cDNA using an iScript cDNA Synthesis Kit (Bio-Rad, USA), following the manufacturer’s protocol. The study utilized RT-qPCR to evaluate the expression levels of genes, including bone gamma-carboxyglutamic acid-containing protein (BGLAP), also known as osteocalcin; Runt-related transcription factor 2 (Runx2); alkaline phosphatase (ALPL); and receptor activator of nuclear factor-kB ligand (RANK-L). The RT-qPCR was performed using a SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, USA). The 2–ΔΔCt method was employed to analyze the relative gene expression with GAPDH as the control gene.

2.9. The MTT Cell Viability Assay and Cellular Morphology.

The MTT assay evaluated the cell viability for cell cultures. Phosphate buffered saline (PBS) dissolved the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) at a concentration of 5 mg mL⁻¹. 100 μL of an MTT solution was added over each sample, followed by 900 μL of media, and the samples were incubated at 34 °C for 2 h. The solution was removed after the incubation period, and 600 μL of the MTT solubilizer was composed of 0.1 N HCl, 10% Triton X-100, and isopropanol. 100 μL of the solution was transferred to each of 96 well plates to measure the absorbance of the final solution at 570 nm using a microplate reader (BioTek Synergy 2 SLFPTAD, USA).

A Field Emission Scanning Electron Microscope (FESEM, FEI Quanta 200, FEI Inc., USA) was used to examine the cellular morphology. The samples were fixed in 0.1 M phosphate buffer with 2% paraformaldehyde (Electron Microscopy Sciences, USA) and 2% glutaraldehyde (Sigma-Aldrich, USA) overnight at 4 °C. The samples were postfixed with osmium tetroxide (OsO4) at a concentration of 2%. Each sample was dehydrated in an ethanol solution ranging from 30% to 100%, followed by drying with hexamethyldisilane (HMDS, EMS, USA). The samples were gold coated using a sputter coater (Hummer V sputtering system, Hummer, USA) before imaging with FESEM.

2.10. Rat in Vivo Model.

In vivo surgeries were performed with Sprague–Dawley rats (weight range 300–350 g). The surgeries on the animals were conducted following the protocol authorized by the Washington State University Institutional Animal Care and Use Committee (IACUC). A drill in the femur bone created a unicortical defect (approximately 2.5 mm diameter by 4 mm depth). The 3D-printed cylindrical scaffold (diameter = 2.5 mm, length = 4 mm) was implanted in the defect. After 6 weeks, the rats were sacrificed to obtain bone-implant specimens and fixed in 10% neutral buffered formalin for 3 days. The samples for Hematoxylin and Eosin (H&E) staining and anti-vWF staining were first decalcified using ethylenediamine tetra acetate solution and embedded in wax. The samples were then sectioned with 5 μm thickness using microtomes, followed by fixing on slides. The samples were stained with H&E staining to evaluate new bone formation in the defect area, and the blood vessel staining kit (ECM 590, Millipore Sigma, USA) was used to evaluate new blood vessel formation.^36,46 Biological evaluation of new bone formed was conducted by cutting thin sections of resin-fixed bone blocks and preparing them with Sanderson’s Rapid Bone Staining (SRBS) (Sanderson’s RBS + van Gieson (S-CP3), Dorn and Hart, USA).⁴⁶ Quantitative histomorphometric analyses were carried out using ImageJ. The following equation has been used to calculate the % of new bone formation.

$$\begin{array}{l}\text{\% New bone formation}\\ =\frac{\text{total area of scaffold}-\text{total area of new bone induced}}{\text{total area of scaffold}}\times 100\text{\%}\end{array}$$

2.11. Statistical Analyses.

All of the results are based on three biological replicates. Data were analyzed using GraphPad Prism 8 (USA), and comparisons were made using two-way ANOVA and Bonferroni post hoc analysis. Statistical significance was defined as p-values of ≤0.05 (*) and ≤0.001 (**).

3. RESULTS

3.1. Mechanical Characterization of 3D-Printed Scaffolds.

The samples were divided into two groups: TCP scaffolds without a dopant and those with added MgO. The compressive strength of the 3D-printed TCP scaffold was around 8 MPa. In contrast, the MgO-doped 3D-printed TCP scaffold had an average strength of 12 MPa, an increase of 50%. The stress–strain plots are shown in Figure 1B. The final dimensions obtained from the scaffolds are 11 ± 0.6 mm in height and 7 ± 0.3 mm in diameter.

Figure 1.

(A) Optical image of 3D printed in vivo sample and graphical image of 3D printed scaffolds for osteogenesis and angiogenesis. (B) The compressive strength of the 3D printed TCP and MgO doped TCP scaffolds. (C) The vitamin D3 release from control TCP and MgO doped TCP scaffolds in phosphate buffer saline (pH 7.4) and an acetate buffer solution (pH 5). (D) SEM images of the surface morphology of the scaffolds after drug release at pH 5.0 and pH 7.4.

3.2. Release of Vitamin D3 from MgO-Doped 3D-Printed Scaffolds.

Figure 1C depicts the release behavior of vitamin D3 from 3D-printed TCP scaffolds and MgO-doped TCP scaffolds. The samples were stored in buffer solutions with two different pH values. Vitamin D3 was released at a higher rate from samples kept in an acidic buffer. However, when stored at a physiological pH of 7.4, the samples showed a sustained release. The 3D-printed TCP scaffolds in an acidic buffer showed a cumulative release of 38% vitamin D3 at day 28 and a cumulative release of 26% in physiological pH. MgO-doped 3D-printed TCP scaffolds in acidic pH showed a release of 28% at day 28, while those in physiological pH showed a release of 20%.

3.3. In Vitro Osteoblast Cell Material Interaction.

The amount of drug utilized based on the effective concentration study is 20 μg, as shown in Figure S1. The osteoblast study was conducted on days 3 and 7 of the study. The samples loaded with vitamin D3 and doped with MgO showed the highest optical density values using the MTT assay compared to those of other samples. After day 7, the samples doped with MgO showed a 10% increase in the optical density value compared to the control TCP samples by using the MTT assay. This result indicates that MgO can enhance the osteoblast cell proliferation. The release of Vitamin D3 from undoped 3D-printed TCP samples enhanced osteoblast cell proliferation 1.3-fold after day 7. This designed system can enhance early stage osteogenesis via osteoblast cell proliferation (Figure 2A). The FESEM images in Figure 2B validate the results obtained from the MTT values. The samples loaded with vitamin D3 and doped with MgO show more proliferating cells and longer filopodial extensions depicting healthy and mature osteoblast cells.

Figure 2.

Osteoblast cell interaction with 3D printed scaffolds. (A) MTT assay shows that treatment samples do not show cytotoxicity toward osteoblast cells at days 3 and 7. Mg-TCP-VitD3 increased osteoblast cellular viability to 1.3-fold at day 7, compared to the control.(* denotes p ≤0.05, ** denotes p ≤0.001) (B) Osteoblast cellular morphology at days 3 and 7 indicates healthy osteoblast cell attachment and proliferation on Mg-TCP, VitD3, and Mg-TCP-VitD3.

3.4. In Vitro Osteoclast Cell Material Interaction.

The osteoclast study was conducted on days 11 and day 21. The samples were analyzed with TRAP, live/dead, and SEM. The TRAP assay identifies the differentiation of the osteoclast cells, showing that the samples doped with MgO and loaded with vitamin D3 have the slightest differentiation of monocytes into osteoclast cells (Figure 3A). The optical density obtained from the TRAP assay for samples doped with MgO and loaded with vitamin D3 showed a 3.2-fold reduction in osteoclast viability at day 11 and a 1.9-fold reduction at day 21. The optical density obtained from the TRAP assay was 3.1-fold lower for the samples loaded with vitamin D3 and 1.3-fold lower for samples doped with MgO than the control samples on day 11. MgO-doped 3D-printed TCP leads to 1.3-fold lower cellular viability than the control on day 21, while vitamin D3 released from TCP results in 1.6-fold lower cell viability. The difference in resorption pits between the treatments and the control was determined by using SEM images. The 3D-printed TCP shows a more extensive pit formation due to the resorption of TCP by mature osteoclasts (Figure 3B). The treatments show smaller resorption areas, suggesting that treatments inhibit TCP resorption by decreasing the osteoclastic activity. This trend is further confirmed with the FESEM and confocal images of Figures 3C and 3D. Control samples show larger osteoclast cells within the apatite layer on days 11 and 21. The treatment samples show dead cells and cells of smaller size on those days. The confocal images of the live/dead assay in Figure 3D show that the samples doped with MgO have more dead cells (red dots) than the control samples. Undoped and loaded with vitamin D3 samples show more dead cells (red dots) than the control and doped samples. The samples doped and loaded with vitamin D3 show primarily red dots, revealing that the cells were either dead or on the verge of death.

Figure 3.

Osteoclast cell interaction with the 3D printed scaffolds. (A) The TRAP assay indicates that the osteoclast viability of the treatment samples is significantly lower than that of the control on days 11 and 21.(* denotes p ≤ 0.05, ** denotes p ≤0.001) (B) The most extensive pit formation can be found on the control samples than on the treatment samples on days 11 and 21. (C) SEM images showed bigger (white arrow) osteoclast morphology on control samples at all time points. The treatment samples induce cell rupture (red arrow) on day 21. (D) Confocal microscopy images show that the treatment samples predominantly have dead osteoclast cells (red dots), while the control sample primarily presents live cells (green dots).

Micrographs taken from FESEM (Figures 3C and 3D) validate the results from the live/dead assay and TRAP assay. Ruptured membrane morphology is observed in the samples loaded with vitamin D3.

3.5. In Vitro Coculture of hMSCs and Monocytes.

2-Fold gene expression was used to calculate the effects of vitamin D3 on osteoblast and osteoclast differentiation and proliferation (Figure 4A). The values obtained from the samples loaded with vitamin D3 showed a 3.2-fold increase in the gene expression for BGLAP, a 2.6-fold increase for ALPL, and a 4.7-fold increase for RUNX2. The values obtained for BGLAP, ALPL, and RUNX2 were greater than 1, meaning the genes were upregulated. The effect of vitamin D3 release on in vitro osteoclastogenesis was evaluated via RANK-L gene expression. The value obtained was 8-fold less than the control, indicating that the differentiation of monocytes into osteoclasts was inhibited by vitamin D3. The upregulation of genes associated with osteoblastic activities results in the growth and differentiation of osteoblast cells, as shown in Figure 4B. This figure shows that the samples loaded with vitamin D3 have more osteoblast cells with healthy morphology. The samples loaded with vitamin D3 showed unhealthy osteoclast cells. These results indicate that vitamin D3 released from 3D-printed TCP enhances early stage osteogenesis via upregulating BGLAP, RUNX2, and ALPL and downregulating RANK-L gene expression.

Figure 4.

(A) Fold change graph depicts that vitamin D3 upregulates the osteoblast-specific target genes such as BGLAP, ALPL, and RUNX2, whereas it downregulates RANK-L gene-associated osteoclast cells. (* denotes p ≤ 0.05, ** denotes p ≤0.001) (B) SEM image of the coculture study with osteoblasts and osteoclasts on 3D printed scaffolds. All samples show good cellular attachment of osteoblast cells (white arrows). Bigger osteoclast cells (red arrows) can be found in the control samples.

3.6. In Vivo Studies on the Rat Distal Femur Model.

The study was carried out for 6 weeks. The samples were stained using Sandersons’ rapid Bone stain, H&E stain, and Movat pentachrome stain. The deep pinkish H&E stains in Figure 5A mark new bone formation. The red-pinkish mark in the SRBS stain shows the area of bone mineralization in Figure 5B. H&E staining in Figure 5A shows that samples doped with MgO show more areas undergoing new bone formation than undoped TCP. Samples doped with MgO showed an approximately 1.5-fold increase in new bone formation. Samples doped with MgO and loaded with vitamin D3 showed the largest areas undergoing new bone formation. The samples doped with MgO and loaded with vitamin D3 showed close to a 3.1-fold increase in new bone area when stained with H&E. Similar results were obtained from SRBS-stained samples. The samples loaded with vitamin D3 and doped with MgO show the largest areas undergoing new bone formation, which account for an almost 1.3-fold increase in the new bone area. Equivalent results were obtained when the samples were analyzed using Movat pentachrome stain (Figures 6A and 6C). The samples doped with MgO and loaded with vitamin D3 show the largest area undergoing new bone formation, accounting for a greater than 3.0-fold increase in the new bone area. Anti-vWF staining was performed to investigate the growth of blood vessels in and around the scaffolds. Figure 6B shows the formation of blood vessels, as revealed by anti-vWF staining. The area under the blood vessels was calculated using ImageJ and shows a 1.3-fold increase when loaded with vitamin D3 and doped with MgO, compared to the control sample (Figure 6D).

Figure 5.

(A) Optical microscopic images of H&E-stained bone sections. NB (New bone) (B) Stereo images of SRBS-stained sections. NB (New bone) (C) % area of new bone formation after quantification. (** denotes p ≤0.001) (D) The quantification of the new bone area in pixels. Mg-TCP scaffold increases over the control. Mg-TCP-VitD3 scaffolds show more new bone formation.Mg-TCP-VitD3 further enhances compared to the control. (* denotes p ≤0.05, ** denotes p ≤0.001).

Figure 6.

(A) Optical images of the Movat pentachrome stained bones indicate that Mg-TCP and Mg-TCP-VitD3 induce more new bone formation (yellow) than the control sample. (B) Optical cross-sectioned images stained with anti-vWF. In vivo blood vessel staining images show blood vessel growth within the implants. (C) Quantifying the area of new bone formation from Movat pentachrome stain. (* denotes p ≤0.05, ** denots p ≤0.001) (D) Quantification of in blood vessel staining images. Mg-TCP-VitD3 and Mg-TCP samples show more blood vessel formation than the control.. (* denotes p ≤0.05).

4. DISCUSSION

The medicinal efficacy of vitamin D3, in early stage bone regeneration potential, makes it an appropriate option for incorporation into 3D-printed scaffolds as a local drug delivery system for bone tissue engineering. Several studies have shown that vitamin D3 promotes bone health and accelerates early stage bone regeneration.^47,48 Vitamin D3 promotes the growth of osteoblast cells by upregulating osteogenesis-related genes such as BGLAP, ALPL, and RUNX2, which assist in bone formation.^47–49 In line with previous studies, vitamin D3 incorporated 3D-printed scaffolds promote in vitro osteogenesis. An in vitro coculture system was used to investigate the effects of vitamin D3 on the growth and proliferation of osteoblast and osteoclast cells (Figure 4). Following the cell culture study, RT-qPCR was performed in the in vitro coculture system to understand the expression of genes related to osteogenesis and osteoclastogenesis. Vitamin D3 release from 3D-printed TCP scaffolds promotes osteoblast cell growth and differentiation by upregulating genes such as BGLAP, RUNX2, and ALPL. The release of vitamin D3 downregulates the RANK-L expression, which assists osteoclast differentiation from monocytes on day 11 (Figure 4A). FESEM images show that the size of osteoclast cells in the control samples is greater than that in the treatment samples (Figure 4B). These findings indicate that vitamin D3 enhances osteoblast differentiation while inhibiting RANK-L-induced osteoclast maturation.

One of the major advantages of 3D printing ceramics is its ability to manufacture patient-specific and defect-specific implants.⁴⁹ Despite its advantages, the implants’ poor mechanical properties limit them to low-load-bearing applications.⁴³ The MgO dopant was introduced to improve the strength of the 3D-printed TCP scaffolds. Doping TCP with MgO led to a 1.5-fold increase in compressive strength due to the grain boundary strengthening ability during the sintering process.⁵⁰ This result is comparable to the compressive strength of cancellous bone (2–10 MPa). Vitamin D3 was loaded onto the MgO doped and the control TCP scaffolds, and the release of vitamin D3 from these substrates was estimated. The release of vitamin D3 from undoped samples was 38% in acidic pH and 26% in physiological pH over 28 days. In contrast, the release of vitamin D3 from MgO-doped TCP scaffolds was estimated at 28% in acidic pH and 20% in physiological pH. The lower release of vitamin D3 from MgO-doped TCP scaffolds compared to the control TCP scaffolds at both pH 5.0 and pH 7.4 can be attributed to the chelation of vitamin D3 with Mg as the ligand element and to the lower degradation of the scaffolds in the presence of MgO.⁵¹ The higher release of vitamin D3 in acidic pH can be attributed to the higher degradation of the substrate in acidic environments, as shown in Figure 1D. The sustained release of vitamin D3 will assist in osteogenesis for a longer period while promoting other biological properties.

Similarly, MgO is known to stimulate osteoblast cell proliferation. In vitro results from osteoblast cell cultures show that samples loaded with vitamin D3 and doped with MgO have the greatest proliferation, because Mg and vitamin D3 work synergistically to improve osteoblast growth. The growth and proliferation of the osteoblast cells were characterized by an MTT assay and SEM images, as shown in Figures 2A and 2B.

Enhancing osteoblast cell proliferation while lowering osteoclast cell differentiation is desirable to promote early stage osteogenesis.⁵² Monocytes are the precursors that differentiate into osteoclast cells. The quintessential approach to inhibiting the growth of osteoclast cells is not to allow the precursor cells to differentiate. The monocytes differentiate in the presence of RANK-L.⁵³ Results from TRAP, FESEM micrographs, and live/dead assays show that the growth of osteoclast cells is inhibited by the three treatments (Figure 3).

In vitro results show that the 3D printed substrates doped with MgO and loaded with vitamin D3 enhance early stage osteogenic potential. In vivo studies have been conducted to determine whether the same implant system functions within the animal body. Several studies suggest that vitamin D3 assists tissue regeneration by activating multiple pathways. Anti-vWF staining shows that vitamin D3 supports blood vessel formation, as illustrated in Figure 6B. H&E staining, SRBS, and movat pentachrome staining show that new bone formation is enhanced in the presence of vitamin D3 and MgO (Figures 5A and 5B and Figure 6A). Therefore, the release of vitamin D3 from MgO-doped 3D-printed scaffolds enhances early stage osteogenesis both in vitro and in vivo.

5. CONCLUSION

Our work demonstrates that releasing vitamin D3 from MgO-doped 3D-printed scaffolds enhances early stage osteogenesis and angiogenesis while suppressing osteoclastogenesis in vitro and in vivo. In vivo studies on rat distal femur models show that releasing vitamin D3 from MgO-doped 3D-printed scaffolds enhances the new bone formation and mineralization. Anti-vWF staining indicates that the multifunctional 3D-printed scaffolds enhance blood vessel formation inside and around the scaffolds. Results from in vivo studies align with in vitro results that vitamin D3-loaded MgO-doped 3D-printed scaffolds increase in vitro osteoblast proliferation while suppressing osteoclast activity. In this context, vitamin D3 assists in upregulating osteoblast target genes, such as BGLAP, ALPL, and RUNX2. In addition, vitamin D3 inhibits the differentiation of monocytes to mature osteoclasts by downregulating RANK-L expression. Based on these evaluations, vitamin D3-loaded MgO-doped scaffolds can enhance early stage osteogenesis and blood vessel formation, giving a therapeutic alternative to critical-sized bone damage.