Figure 1.
Images show the macroporous structure of CHI (A), CPS-CHI (B), rhBMP2-CHI (C), and rhBMP2-CPS-CHI (D) scaffolds. Bars are 50 µm. rhBMP2-CHI (C), and rhBMP2-CPS-CHI (D) scaffolds. Bars are 50 µm. Inset shows CPS in form of ACP in both CPS-CHI and rhBMP2-CPS-CHI scaffolds. Bar is 30 nm.
Figure 2.
Plots representing the tensile strength versus strain for CHI (—), CPS-CHI (– –), rhBMP2-CHI (···), and rhBMP2-CPS-CHI (–··–) scaffolds.
Table 1.
Rheological properties (Young modulus, tensile strength, and elongation at rupture) and swelling ratio of CHI, CPS-CHI, rhBMP2-CHI, and rhBMP2-CPS-CHI scaffolds.
Figure 3.
A) Graphic shows alkaline phosphatase activity induced in C2C12 cell line. This activity is induced by the active rhBMP-2 which was released from CHI scaffolds after dissolving them in 100 µL of acetic acid 50 mM for 1 hour (black columns) or 200 µL for 2 hours (white columns). B) Total rhBMP-2 detected in culture media in a time course using ELISA assay. C) rhBMP-2 release kinetic using transformed data. All data are adjusted to a first order kinetic.
Table 2.
Release kinetics parameters for rhBMP2-CHI and rhBMP2-CPS-CHI scaffolds using Y = Ymax*(1-exp-kt) after transforming raw data.
Figure 4.
Viability and proliferation assays.
A) Calcein assay: Green fluorescence corresponds to calcein vital staining at day 1 (D1), day 3 (D3) and day 5 (D5). Images show a representative experiment on rhBMP2-CPS-CHI scaffolds; B) Alamar Blue assay: Graph shows a time course quantitative measurement of cell viability for the four scaffolds (CHI, rhBMP2-CHI, CPS-CHI, rhBMP2-CPS-CHI). Relative units are referred to fluorescence units observed at day 1 in order to remark different cell growth between different scaffolds. *P<0.05 comparing with the other scaffolds at the same time point.
Figure 5.
Surgery (A), gross morphology after euthanasia (B, C) and microCT analysis of samples (D).
Surgery images (A) show scaffold implantation process. After euthanasia gross morphology images were obtained at different angles (B,C) (Dotted circle indicates defect location). Defect area is still observed in CPS-CHI implanted tibias (B), while in rhBMP2-CPS-CHI implanted tibias high amount of newly-formed hard tissue, apparently bone, appeared vertically from the defect (C). MicroCT study (D) confirms trabecular bone formation in rhBMP2-CPS-CHI implanted tibias, while it seems no scaffold-resorption in any case. Neither seems a robust new bone formation in the rest of implanted scaffolds compared to empty controls.
Figure 6.
A) Hematoxylin-eosin staining. A1) Empty control, A2) CHI scaffold, A3) rhBMP2-CHI scaffold, A4) CPS-CHI scaffold, A5) rhBMP2-CPS-CHI scaffold. Different lines are limiting newly formed woven bone (WB, ···) with resembles trabecular bone structure (intramedular as well as extramedullar), cortical bone (CB, —) and fibrous tissue (F, – – –). All images were taken at 2×. B) Masson's Trichrome staining. B1) CPS-CHI scaffold. A little amount of bone is formed, and a connective tissue (red arrow) is observed around the matrix, B2-5) rhBMP2-CPS-CHI scaffold. Great amount of new bone is formed, most of it extramedullar trabecular bone (WB). Detailed image (B3) shows a dotted line limiting native cortical bone (CB, —) and newly formed woven bone (WB). Chondral areas (CD) appear in the limits of newly formed tissue (B4, B5) here limited with woven bone indicating new bone formation via osteochondral ossification. Images B1, B2 and B5 were taken at 5×. Images B3 and B4 were taken at 10×. All histological slides were from the closest zone to the center of the defect as possible.
Figure 7.
Image is shown as an example of measurements performed and corresponds to a rhBMP2-CPS-CHI sample. Histogram shows the mean area values obtained in each treatment for newly-formed bone (NS: statistically not significant compared to control, **P<0.01).