Laser-direct writing by two-photon polymerization of 3D honeycomb-like structures for bone regeneration

Abstract

A major limitation of existing 3D implantable structures for bone tissue engineering is that most of the cells rapidly attach on the outer edges of the structure, restricting the cells penetration into the inner parts and causing the formation of a necrotic core. Furthermore, these structures generally possess a random spatial arrangement and do not preserve the isotropy on the whole volume. Here, we report on the fabrication and testing of an innovative 3D hierarchical, honeycomb-like structure (HS), with reproducible and isotropic arhitecture, that allows in 'volume' migration of osteoblasts. In particular, we demonstrate the possibility to control the 3D spatial cells growth inside these complex architectures by adjusting the free spaces inside the structures. The structures were made of vertical microtubes arranged in a mulitlayered configuration, fabricated via laser direct writing by two photons polymerization of the IP-L780 photopolymer. In vitro tests performed in MG-63 osteoblast-like cells demonstrated that the cells migration inside the 3D structures is conducted by the separation space between the microtubes layers. Specifically, for layers separation between 2 and 10 μm, the cells gradually penetrated between the microtubes. Furthermore, these structures induced the strongest cells osteogenic differentiation and mineralization, with ALP activity 1.5 times stronger, amount of calcified minerals 1.3 times higher and osteocalcin secretion increased by 2.3 times compared to the other structures. On the opposite, for layers separation less than 2 μm and above 10 μm, the cells were not able to make interconnections and exhibited poor mineralization ability.

Collagen/Chitosan Functionalization of Complex 3D Structures Fabricated by Laser Direct Writing via Two-Photon Polymerization for Enhanced Osteogenesis

“…LDW via TPP has unique advantages among other printing technologies, such as spatial resolution below the diffraction limit, no limitations concerning the 3D architectures that can be obtained and high reproducibility of the 3D structures [7][8][9][10]. For tissue engineering, LDW via TPP provides the means to fabricate 3D structures that sustain the healing of damaged tissue [9,10,[12][13][14]. In this regard, a lot of attention is dedicated to the optimum choice of the structural architecture and of the most suitable polymer.…”

“…Moreover, the 3D printer manufacturers such as Nanoscribe (which is the system we used for structures' fabrication) have developed dedicated photopolymers optimized for being processed with the Nansocribe technology. Among these, IP-L photpolymers have been shown to have high biocompatibility in vitro for bone-forming cells, with promising results for bone regeneration in vivo [13,14].…”

“…It is a biocompatible liquid formulation with optimized sensitivity for fast 3D structuring ref. [13]. We employed IPL-780 for the present work because the producer recommends it for tissue engineering applications and because it has proven good ability to produce structures sized down to 150 nm, with low stress, little shrinkage and good adhesion on glass substrates [13].…”

Fabrication of carbonate apatite honeycomb and its tissue response

“…Furthermore, microporous structure should play an important role in cell growth and differentiation, similar to the niche environment or microenvironment produced by the extracellular matrix . Among many porous structures available, the honeycomb (HC) structure is unique due to its fully interconnected and oriented pores having the same pore size, and shows high mechanical strength in the direction of the pores …”

“…[17][18][19][20][21][22][23][24][25] Furthermore, microporous structure should play an important role in cell growth and differentiation, similar to the niche environment or microenvironment produced by the extracellular matrix. [26][27][28][29][30][31][32][33] Among many porous structures available, the honeycomb (HC) structure is unique due to its fully interconnected and oriented pores having the same pore size, [34][35][36][37][38][39] and shows high mechanical strength in the direction of the pores. 40 Therefore, the feasibility to fabricate CO 3 Ap HC was studied herein, based on the HC extrusion method.…”