Nanoscale β-TCP-Laden GelMA/PCL Composite Membrane for Guided Bone Regeneration

Abstract

Major advances in the field of periodontal tissue engineering have favored the fabrication of biodegradable membranes with tunable physical and biological properties for guided bone regeneration (GBR). Herein, we engineered innovative nanoscale beta-tricalcium phosphate (β-TCP)-laden gelatin methacryloyl/polycaprolactone (GelMA/PCL-TCP) photocrosslinkable composite fibrous membranes via electrospinning. Chemo-morphological findings showed that the composite microfibers had a uniform porous network and β-TCP particles successfully integrated within the fibers. Compared with pure PCL and GelMA/PCL, GelMA/PCL-TCP membranes led to increased cell attachment, proliferation, mineralization, and osteogenic gene expression in alveolar bone-derived mesenchymal stem cells (aBMSCs). Moreover, our GelMA/PCL-TCP membrane was able to promote robust bone regeneration in rat calvarial critical-size defects, showing remarkable osteogenesis compared to PCL and GelMA/PCL groups. Altogether, the GelMA/PCL-TCP composite fibrous membrane promoted osteogenic differentiation of aBMSCs in vitro and pronounced bone formation in vivo. Our data confirmed that the electrospun GelMA/PCL-TCP composite has a strong potential as a promising membrane for guided bone regeneration.

Keywords: bone; electrospinning; extracellular matrix; gelatin; regeneration; tissue engineering.

Figure 1.

Morphological and chemical characterizations of manufactured membranes and as-received beta-tricalcium phosphate (TCP). (A) Representative SEM images and histograms showing the fiber diameter frequency and average fiber diameter (AFD) with standard deviation for the manufactured scaffolds before and after crosslinking (n = 3). (B) Bar graph comparing the fiber diameter between groups. Data shown as mean ± SD. (C) Energy-dispersive X-ray spectroscopy (EDS) results confirm the presence of Ca, P, and O with an average Ca/P ratio of 1.7, with representative SEM images for TCP (n = 3).

Figure 2.

Physicochemical characterizations of assembled membranes. (A) Fourier-transform infrared spectroscopy (FTIR) analysis of the scaffolds (n = 3). (B) X-ray diffraction (XRD) analysis of the scaffolds—red asterisk showing characteristic peaks for PCL; green ones for GelMA; clear blue ones for TCP (n = 3). (C) Energy-dispersive X-ray spectroscopy (EDS) analysis of a GelMA/PCL membrane containing 15 wt % of TCP confirms the successful compound incorporation within the fibers. Blue arrows indicate phosphate peaks and green arrows indicate calcium peaks (n = 3) (D) Weight loss of polymers measured by thermogravimetric analysis (TGA); black dashed line marking the 700 °C, a standard temperature used to measure the residual weight (n = 3).

Figure 3.

Swelling, degradation, contact angle, and mechanical characterizations of assembled membranes. (A) The swelling capacity of all membranes at different time points for 24 h (n = 3). Note a higher swelling for GelMA pure and TCP-incorporated membranes. (B) Degradation profile of all membranes at several time points (n = 3). Note an almost complete degradation for pure GelMA membranes after 28 days. (C) Averaged contact angle measurements (n = 3); different lowercase letters denote statistical differences between groups. (D) Individual contact angle data over time points (n = 3). (E) Representative contact angle images between water and the assembled membranes (n = 3). (F) Young’s modulus (MPa, n = 4). (G) Tensile strength (MPa, n = 4). (H) Elongation at break (%, n = 4) under dry (uncrosslinked) and wet (crosslinked) conditions.

Figure 4.

Immunofluorescent staining of F-actin in aBMSCs seeding on membranes, indicating cell attachment after 12 h (A) and 24 h (B)—scale bar: 50 μm (n = 3). (C) Representative SEM images showing cell–membrane interaction after a 7-day seeding on the membranes (n = 3). (D) alarmarBlue Cell Proliferation results of 1, 3, 5, and 7 days indicate that GelMA/PCL-TCP promoted cell proliferation significantly higher than others (n = 4)—different lowercase letters denote statistical differences between groups. (E) Representative images of Micro-CT showing the in vitro bone mineralization nodules formation (n = 3). (F) Quantified bone volume analysis of aBMSCs after 21 days of osteogenic induction (n = 3).

Figure 5.

Osteogenic marker expression after 7 days (A), 14 days (B), and 21 days (C) were evaluated by RT-PCR. Runt-related transcription factor 2 (RUNX2), collagen type 1 (Col1), alkaline phosphatase (ALP), and osteocalcin (OCN) (n = 3). Different lowercase letters denote statistical differences between groups.

Figure 6.

(A) Micro-computed tomography (μCT) of the bone defects with or without (empty) scaffolds after 6 weeks post-implantation (n = 6). Yellow dashed lines highlight the areas assessed for bone quantification. Scale bar: 5 and 1 mm. (B) Quantitative total volume of the defect, no significant differences between groups. (C) Newly regenerated bone volume present inside the defect; note a significantly higher amount of bone formed evoked by the GelMA/PCL-TCP group followed by GelMA/PCL compared to the empty defect. (D) Ratio between bone volume and total volume defect led by different treatments; it is possible to see an increase in BV/TV for the three employed membranes compared to empty defects, with a higher ratio for GelMA/PCL-TCP.

Figure 7.

(A) Hematoxylin and eosin and (B) Masson’s trichrome-stained slices of rat calvaria critical-size defects after 6 weeks. Bone formation was observed in all groups, with a significant amount of mature bone in the GelMA/PCL-TCP scaffold group. The defect area in the empty and PCL groups was filled with a small amount of bone and more fibrous connective tissue. New bone is indicated with (nb), original bone (ob), blood vessel (bv), and membrane remnants over the defect (mb). Three consecutive magnifications 2×, 4×, and 10×, with 500, 250, and 100 μm scale bars, respectively.

Figure 8.

In vivo 6-week rat critical-size defect immunohistochemistry evaluation of bone formation (osteopontin and Runt-related transcription factor 2RUNX2) and angiogenesis (cluster of differentiation 31C—D31 or platelet and endothelial cell adhesion molecule 1—PECAM1 and von Willebrand Factor). For the osteogenic markers, higher immunolabeling is seen for the GelMA/PCL-TCP group, compared to the other three. Even for the empty defect, it is possible to see adequate immunoexpression for angiogenesis-wise markers, similar to the PCL and GelMA/PCL groups; however, undoubtedly, higher immunolabeling can be visualized in the GelMA/PCL-TCP group. Cell nuclei were labeled with DAPI (blue), and antibody binding was visualized using Alexa Fluor 488 (green) secondary antibody. mb: membrane; ob: original bone; nb: new bone. Scale bars: 100 μm and 200 μm for GelMA/PCL-TCP [high magnification]. ImageJ software was utilized to quantify the positively stained area of the four immunomarkers, and they were analyzed by an ordinary one-way ANOVA with Tukey’s multiple comparison test. Bar graphs that exhibited the mean values and their corresponding standard deviations were used to present the results.