Micelle encapsulated curcumin and piperine-laden 3D printed calcium phosphate scaffolds enhance in vitro biological properties

Abstract

Limitations in the current clinical management of critical-sized osseous defects have driven the need for multifunctional bone constructs. The ideal bone scaffold should possess advanced microarchitecture, well-defined pore interconnectivity, and supply biological signals, which actively guide and control tissue regeneration while simultaneously preventing post-implantation complications. Here, a natural medicine-based localized drug delivery from 3D printed scaffold is presented, which offers controlled release of curcumin, piperine from nano-sized polymeric micelles, and burst release of antibacterial carvacrol from the coating endowing the scaffold with their distinct, individual biological properties. This functionalized scaffold exhibits improved osteoblast (hFOB) cell attachment, 4-folds higher hFOB proliferation, and 73% increased hFOB differentiation while simultaneously providing cytotoxicity towards osteosarcoma cells with 61% lesser viability compared to control. In vitro, early tube formation (p < 0.001) indicates that the scaffolds can modulate the endothelial cellular network, critical for faster wound healing. The scaffold also exhibits 94% enhanced antibacterial efficacy (p < 0.001) against gram-positive Staphylococcus aureus, the main causative bacteria for osteomyelitis. Together, the multifunctional scaffolds provide controlled delivery of natural biomolecules from the nano-sized micelle-loaded 3D printed matrix for significant improvement in osteoblast proliferation, endothelial formation, osteosarcoma, and bacterial inhibition, guiding better bone regeneration for post-traumatic defect repair.

Keywords: 3D printing; Bone tissue engineering; Curcumin; Micelle; Multifunctional bone Scaffold.

Fig. 1.

Synthesis of drug encapsulated nano-sized micelle. (A) Pluronic F127 polymer self-assemble in aqueous solution to form spherical micelles with a hydrophobic propylene oxide (PPO) centre surrounded by hydrophilic polyethylene oxide (PEO) shell. During micelle formation, the hydrophobic drugs, curcumin and piperine get entrapped within the PPO core, while hydrated outer shell ensures the solubility of the drug in an aqueous environment, enhancing the bioavailability of the drug. (B) Transmission Electron Microscopy (TEM) showing the micelle structure: Micelle formation is optimized by tailoring the mixing time to achieve critical micelle concentration. 30 min and 60 min mixing time resulted uniform and homogeneous formation of micelle with a size of 47 ± 5 nm, whereas 10 min resulted in a non-distinct vesicle wall, which is attributed to the inadequate time for micelle formation. 120 min mixing time resulted in ruptured micelle wall due to the disruption in the structure.

Fig. 2.

Morphology and drug-loading in 3D printed scaffolds (a) Schematic of drug loading in scaffold showing curcumin-piperine encapsulated micelle is pipetted on 3D printed TCP scaffold, followed by the incorporation of carvacrol as a coating. (b-e) 3D printed TCP scaffold showing the presence of hierarchical porous architecture with 400 μm designed macropores and 5–20 μm residual micropores, which are crucial for enhanced bone ingrowth. (f-g) EDS mapping of elements showing Ca (blue), O (green) and P (Red) suggesting uniform distribution of the elements. Ca, P and O peaks are obtained showing that there is negligible carbonate salt present due to the absence of carbon peak in EDS. (h) UV-Vis spectroscopy data showing a shift in the absorbance and sharper peak in case of micelle-encapsulated curcumin, whereas broad peak and lesser optical density in free curcumin due to presence of non-polar environment in curcumin encapsulated micelle. (i) FTIR results showing a peak shift in 1510–1512, 1581 and 1286–1282 cm⁻¹ depicting stability of curcumin in micellar environment. (j) XRD peaks showing the presence of β-TCP after sintering at 1250 °C. (k) DLS of micelle encapsulated curcumin showing the average micelle diameter being 101 nm.

Fig. 3.

In vitro release kinetics from micelle-loaded 3D printed scaffold and schematic representation of drug release mechanism from 3D printed scaffold. (a) In vitro release kinetics of micelle loaded drugs in physiological pH (pH 7.4) showing a controlled release of curcumin, piperine, and carvacrol with about 79.7%, 89.6%, and 84.7% drug release, respectively in 60 days. (b) In vitro release kinetics of micelle loaded drugs in pH 5.0 showing a significantly higher cumulative release percentage of about 75.6%, 97.0%, 92.1% at pH 5.0 for curcumin, piperine, and carvacrol, respectively within 6 days. The burst release of drugs at pH 5.0 indicates rapid delivery of chemopreventive biomolecules, curcumin and piperine, and antibacterial drug, carvacrol in acidic tumor microenvironment and right after the surgery, whereas controlled release at pH 7.4 indicates sustained drug delivery to provide localized prevention of osteosarcoma cell growth, anti-bacterial protection for secondary infection and subsequent bone regeneration at the defect site for prolonged period. (c-e) The release of drugs (curcumin, piperine and carvacrol) from 3D printed scaffold at initial 24 h are plotted to show the distinct effect of pH on their release.

Fig. 4.

In vitro osteoblast (hFOB) proliferation by 3D printed TCP scaffold (a) MTT cell viability assay showing micelle loaded 3D printed scaffold with statistically significant difference in hFOB proliferation at day 3,7, 11, compared to the control. At day 11, micelle loaded scaffolds show a 4-fold increase in hFOB cell viability compared to the control scaffold. (b) ALP osteoblast differentiation assay showing enhanced hFOB cellular differentiation in the presence of micelle at day 11, compared to control [* * denotes p value< 0.001]. (c) Morphological characterization by FESEM showing firm hFOB attachment, presence of filopodial processes and abundant proliferation in all the samples, suggesting no cytotoxicity of the drug towards the healthy bone cell. (d) FESEM micrograph showing osteoblast cell attachment differentiating from hMSCs after 7-day time point. Samples loaded with micelle encapsulated curcumin showed healthier morphology. (e) ALP staining images showing that there are more number of healthy osteoblast cells in the drug loaded samples as compared to control sample. (f) 2-fold gene expression obtained from RT-qPCR showing upregulation of genes related to osteogenesis in presence of micelle encapsulated curcumin.

Fig. 5.

In vitro cytotoxicity study against human osteosarcoma cells (MG-63). (a) MTT cytotoxicity assay showing no inhibitory effect on osteosarcoma cells in presence of micelle at day 3, however 2- and 3.5-folds lower osteosarcoma cell viability at day 7 and day 11, respectively [* * denotes p value< 0.001]. (b) % cytotoxicity denoting lower than 70% cellular viability at day 7 and 11 suggesting chemopreventive potential of the scaffold loaded with micelle. (c) FESEM images at day 3, 7 and 11 days of cell culture showing the presence of osteosarcoma cell with typical fibroblast-like morphology in control samples, whereas very few osteosarcoma cells in micelle-loaded 3D printed TCP.

Fig. 6.

In vitro tube formation assay by Human Endothelial Vein Endothelial Cells (HUVEC) (A) Schematic diagram of the endothelial cell assay on Matrigel (B) Light microscopy images showing tube formation by HUVEC after 3, 6, 12 and 24 h on Matrigel with control, curcumin and micelle loaded 3D printed TCP scaffolds in transwell inserts. Curcumin and curcumin encapsulated micelles significantly stimulated tube formation at as early as 3 h, compared to the control 3D printed TCP scaffolds, which showed few tubes and minimal network among the branches suggesting enhanced and accelerated early tubular network formation ability by curcumin encapsulated micelle loaded 3D printed TCP scaffold. Maximum tube formation is reached at 6 h for all the compositions, by 12 h, the HUVECs starts to undergo apoptosis and the tubes begin to dissociate, which is a usual phenomenon for HUVEC cell.

Fig. 7.

Quantitative analysis of HUVEC tube formation assay by Angiogenesis Analyzer for ImageJ software (A) A representative image of tube formation showing presence of nodes, meshes, junction, branches and master segment in different colour codes (B-G) Number of nodes, meshes, master segment, branches and total master segment length, total branching length, total segments length and total length are calculated for control (3D printed scaffold), curcumin loaded 3D printed scaffold and curcumin-piperine loaded micelle incorporated 3D printed scaffold at 3 h and 6 h of HUVEC cell culture [* represents p < 0.05, * * represents p < 0.001]. Statistically significant enhancement of nodes, branches and master segments have been observed in presence of curcumin and drug loaded micelle at both 3 and 6 h, indicating their stabilizing effect towards HUVEC formed capillary-like tube structure. (H) FESEM micrographs showing HUVEC cell morphology after a period of 3 days of study. Samples loaded with micelle encapsulated curcumin showed healthier cell morphology. (I) 2-fold gene expression study showing mTOR expression after 3 days. Presence of micelle encapsulated curcumin upregulated the mTOR expression which in turn enhanced cell proliferation.

Fig. 8.

In vitro anti-bacterial efficacy against Staphylococcus aureus (A) Schematic diagram of the antibacterial study. (B-C) Agar plates and related quantification showing the presence of Staphylococcus aureus colonies in control 3DP TCP, after 24 and 48 h of incubation. Lesser number of bacterial colonies are observed in agar plates associated with scaffolds loaded with micellar curcumin (21 ± 3% and around 23 ± 2% efficacy at 24 and 48 h respectively). Further reduction in bacterial colonies are seen with scaffolds loaded with carvacrol with 71 ± 6% and 73 ± 3% antibacterial efficacy after 24 and 38 h. Highest antibacterial efficacy are exhibited with scaffolds loaded with micellar curcumin+carvacrol with 98 ± 1% and 94 ± 4% antibacterial efficacy after 24 and 48 h (** p < 0.001). (D) SEM images depicting higher Staphylococcus aureus colonies in control 3DP TCP, whereas most reduction in the bacterial colony are observed in 3DP TCP loaded with micellar curcumin and carvacrol. (E) Live/dead confocal imaging showing greater live Staphylococcus aureus colonies in control 3DP TCP. Scaffolds loaded with micelle encapsulated curcumin and carvacrol exhibited higher presence dead bacterial colonies in red stain and least number of live bacteria in green. (F) Schematic depicting the mechanism of bactericidal effects of curcumin and carvacrol.