Polycaprolactone-coated 3D printed tricalcium phosphate scaffolds for bone tissue engineering: in vitro alendronate release behavior and local delivery effect on in vivo osteogenesis

Abstract

The aim of this work was to evaluate the effect of in vitro alendronate (AD) release behavior through polycaprolactone (PCL) coating on in vivo bone formation using PCL-coated 3D printed interconnected porous tricalcium phosphate (TCP) scaffolds. Higher AD and Ca(2+) ion release was observed at lower pH (5.0) than that at higher pH (7.4). AD and Ca(2+) release, surface morphology, and phase analysis after release indicated a matrix degradation dominated AD release caused by TCP dissolution. PCL coating showed its effectiveness for controlled and sustained AD release. Six different scaffold compositions, namely, (i) TCP (bare TCP), (ii) TCP + AD (AD-coated TCP), (iii) TCP + PCL (PCL-coated TCP), (iv) TCP + PCL + AD, (v) TCP + AD + PCL, and (vi) TCP + AD + PCL + AD were tested in the distal femoral defect of Sprague-Dawley rats for 6 and 10 weeks. An excellent bone formation inside the micro and macro pores of the scaffolds was observed from histomorphology. Histomorphometric analysis revealed maximum new bone formation in TCP + AD + PCL scaffolds after 6 weeks. No adverse effect of PCL on bioactivity of TCP and in vivo bone formation was observed. All scaffolds with AD showed higher bone formation and reduced TRAP (tartrate resistant acid phosphatase) positive cells activity compared to bare TCP and TCP coated with only PCL. Bare TCP scaffolds showed the highest TRAP positive cells activity followed by TCP + PCL scaffolds, whereas TCP + AD scaffolds showed the lowest TRAP activity. A higher TRAP positive cells activity was observed in TCP + AD + PCL compared to TCP + AD scaffolds after 6 weeks. Our results show that in vivo local AD delivery from PCL-coated 3DP TCP scaffolds could further induce increased early bone formation.

Figure 1

(a) Schematic of 3D printing (3DP), (b) sintered 3DP TCP scaffold, and (c, d) surface morphology of the 3D printed bare TCP and PCL-coated 3D printed TCP scaffolds, respectively.

Figure 2

Cumulative AD release at pH 7.4 phosphate buffer (a, b) and at pH 5.0 acetate buffer (c, d) from bare TCP (i.e., no PCL coating: indicated by 0% PCL) scaffolds (a, c), and PCL-coated (1 % PCL in acetone (w/v) solution was used for coating) scaffolds (b, d).

Figure 3

Cumulative Ca²⁺ ion release in the release media from bare TCP (a), PCL-coated TCP scaffolds (b) at pH 5.0, and from bare TCP scaffolds at pH 7.4 (c) as a function of release time (Ca²⁺ release was not observed at all-time points). No detectable Ca²⁺ ion release was observed from PCL-coated scaffolds at pH 7.4 within the ppm (μg/mL) range.

Figure 4

Surface morphology of bare TCP (i.e., no PCL coating: indicated by 0% PCL) and PCL-coated (1 % PCL in acetone (w/v) solution was used for coating) TCP scaffolds before and after AD release at pH 7.4 and 5.0: (a) bare TCP before AD release, (b) PCL coated TCP before AD release, (c) bare TCP after AD release at pH 7.4, (d) PCL-coated TCP scaffold after AD release at pH 7.4, (e) bare TCP after AD release at pH 5.0, and (f) PCL-coated TCP scaffold after AD release at pH 5.0.

Figure 5

XRD patterns of the 3DP TCP scaffolds before and after AD release. The major peak for α-TCP disappeared after AD release at pH 5.0 due to high dissolution of α-TCP as shown in (c). The intensity of the Major peak for α-TCP was decreases at pH 7.4 because of low dissolution of α-TCP at pH 7.4 as shown in (d). Other than this, no phase change was observed.

Figure 6

Photomicrographs of representative histological sections after hematoxylin and eosin (H&E) staining of decalcified tissue sections showing the development of bone formation after 6 and 10 weeks. BM = Bone marrow. Asterisk (*) indicates acellular regions derive from scaffold.

Figure 7

Histomorphometric analysis showing total new percent bone formation comparison between the treatment and control groups (**p < 0.05, *p > 0.05, n = 8 tissue sections of 800 μm width and 800 μm height each).

Figure 8

Histomorphometric analysis showing percent TRAP activity comparison between the treatment and control groups (**p < 0.05, *p > 0.05, n = 8 tissue sections of 800 μm width and 800 μm height each).

Figure 9

Schematic representation of the rate limiting steps for AD (salt or acid form) release. AD is liberated from the TCP surface in the presence of acidic (a) or basic (b) release medium, which is caused by the rate limiting TCP dissolution and/or equilibrium driven shift. In presence of PCL coating, final AD release is caused by the diffusion of AD from PCL coating through the unfavorable interaction between AD and PCL (non-rate limiting step) (c). In absence of PCL coating, AD release is governed by the rate limiting steps.