Highly elastic 3D-printed gelatin/HA/placental-extract scaffolds for bone tissue engineering

Abstract

Bioengineering scaffolds have been improved to achieve efficient regeneration of various damaged tissues. In this study, we attempted to fabricate mechanically and biologically activated 3D printed scaffold in which porous gelatin/hydroxyapatite (G/H) as a matrix material provided outstanding mechanical properties with recoverable behavior, and human placental extracts (hPE) embedded in the scaffold were used as bioactive components. Methods: Various cell types (human adipose-derived stem cells; hASCs, pre-osteoblast; MC3T3-E1, human endothelial cell line; EA.hy926, and human dermal fibroblast; hDFs) were used to assess the effect of the hPE on cellular responses. High weight fraction (~ 70 wt%) of hydroxyapatite (HA) in a gelatin solution supplemented with glycerol was used for the G/H scaffold fabrication, and the scaffolds were immersed in hPE for the embedding (G/H/hPE scaffold). The osteogenic abilities of the scaffolds were investigated in cultured cells (hASCs) assaying for ALP activity and expression of osteogenic genes. For the in vivo test, the G/H and G/H/hPE scaffolds were implanted in the rat mastoid obliteration model. Results: The G/H/hPE scaffold presented unique elastic recoverable properties, which are important for efficient usage of implantable scaffolds. The effects of G/H and G/H/hPE scaffold on various in vitro cell-activities including non-toxicity, biocompatibility, and cell proliferation were investigated. The in vitro results indicated that proliferation (G/H = 351.1 ± 13.3%, G/H/hPE = 430.9 ± 8.7% at day 14) and expression of osteogenic markers (ALP: 3.4-fold, Runx2: 3.9-fold, BMP2: 1.7-fold, OPN: 2.4-fold, and OCN: 4.8-fold at day 21) of hASCs grown in the G/H/hPE scaffold were significantly enhanced compared with that in cells grown in the G/H scaffold. In addition, bone formation was also observed in an in vivo model using rat mastoid obliteration. Conclusion:In vitro and in vivo results suggested that the G/H/hPE scaffold is a potential candidate for use in bone tissue engineering.

Keywords: bone; gelatin; placental-extracts; scaffold; tissue engineering.

Figure 1

Schematics of (A) glycerol effects on gelatin/HA/glycerol ink, (B) 3D printing/crosslinking process for the gelatin/HA scaffolds, and (C) the coating of the gelatin/HA (G/H) scaffold with human placental extracts and implantation of the scaffolds into a rat mastoid obliteration model.

Figure 2

(A) Complex viscosity for temperature sweep (15 - 40 °C) of the G/H ink with various glycerol concentrations (0 ~ 30%). (B) Single line test of the G/H with and without glycerol in various printing barrel temperatures and (C) measured diameters of the single struts. (D) Optical images and (E) water evaporation (%) of each ink before and after the condition (37 °C for 3 h). (F) A schematic describing pausing time during printing and optical images showing extruded gelatin/HA ink before and after 10 s printing pause time and (G) extrusion ratio of G/H ink with and without glycerol for various printing pause times (0-20 s).

Figure 3

(A) Optical images showing the printability using the G/H ink with and without glycerol to obtain a cuboid structure (30 × 30 × 3 mm³). (B) Optical and SEM images and (C) pore geometry of the G/H scaffold fabricated using glycerol. (D) HA weight fraction in G/H scaffold before and after printing. (E) FT-IR spectra and (F) XRD results of the G/H scaffold. (G) Optical images of G/H scaffold showing shape recoverable behavior under the condition of 40% strain, 30 mm/min and compressive load-strain and load-time curves for several cycles. (NS = not significant, ^*p < 0.05, ^**p < 0.005, ^***p < 0.001)

Figure 4

(A) DAPI (blue; cell nuclei) and phalloidin (F-actin; green) staining results after 5 days cell-culture; human adipose derived stem cells (hASCs), pre-osteoblast (MC3T3-E1), human endothelial cell line (EA.hy926), and human dermal fibroblast (hDFs) with and without hPE. (B) Cell proliferation, determined using CCK-8, for each cell type. (NS = not significant, ^*p < 0.05, ^**p < 0.005, ^***p < 0.001)

Figure 5

(A) A schematic of hPE-coating process on G/H scaffold (G/H/hPE), and (B) the optical/SEM images of fabricated G/H and G/H/hPE scaffolds. (C) The degradation rate of the G/H and G/H/hPE scaffolds in PBS at 37 °C. (D) Stress-strain curve and (E) the measured compressive modulus of the scaffolds. (F) Photograph series of one-cycle of compressive loading/unloading and (G) compressive load vs. times and compressive load vs. strain graph of G/H/hPE scaffolds. All the tests were performed in wet state.

Figure 6

(A) Live (green)/dead (red) images after 1 day and DAPI (blue)/phalloidin (green) images of hASCs after 7 days in culture with the scaffolds. (B) Cell viability and (C) F-actin area. (D) Cell proliferation, determined using the CCK-8 assay, and (E) alkaline phosphatase (ALP) activities of the cells in the G/H and G/H/hPE scaffolds. (F) DAPI (blue)/phalloidin (green)/osteopontin (OPN; red) fluorescence images of the hASCs cultured on the scaffolds on day 21 and (G) measured OPN positive area. (H) Relative gene expression of ALP, RUNX-2, BMP-2, OPN, and OCN on day 21. (NS = not significant, ^*p < 0.05, ^**p < 0.005, ^***p < 0.001)

Figure 7

(A) Micro-CT images and (B) hematoxylin and eosin (H&E) staining after 12 weeks of the scaffold implantation. 'NB' and 's' indicating new bone formation and scaffold, respectively. (C) Two-photon fluorescence images of alizarin (red)/oxytetracycline (green)/xylenol (yellow) and (D) relative fluorescence expression (%). Immunohistochemistry images of (E) ALP and (F) OCN and immunofluorescence images of (G) DAPI (blue)/CD31 (green). Arrows indicating vascular formation. (NS = not significant, ^*p < 0.05, ^**p < 0.005, ^***p < 0.001)