Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing

Abstract

While many tissue-engineered constructs aim to treat cartilage defects, most involve chondrocyte or stem cell seeding on scaffolds. The clinical application of cell-based techniques is limited due to the cost of maintaining cellular constructs on the shelf, potential immune response to allogeneic cell lines, and autologous chondrocyte sources requiring biopsy from already diseased or injured, scarce tissue. An acellular scaffold that can induce endogenous influx and homogeneous distribution of native stem cells from bone marrow holds great promise for cartilage regeneration. This study aims to develop such an acellular scaffold using designed, channeled architecture that simultaneously models the native zones of articular cartilage and subchondral bone. Highly porous, hydrophilic chitosan-alginate (Ch-Al) scaffolds were fabricated in three-dimensionally printed (3DP) molds designed to create millimeter scale macro-channels. Different polymer preform casting techniques were employed to produce scaffolds from both negative and positive 3DP molds. Macro-channeled scaffolds improved cell suspension distribution and uptake overly randomly porous scaffolds, with a wicking volumetric flow rate of 445.6 ± 30.3 mm(3) s(-1) for aqueous solutions and 177 ± 16 mm(3) s(-1) for blood. Additionally, directional freezing was applied to Ch-Al scaffolds, resulting in lamellar pores measuring 300 μm and 50 μm on the long and short axes, thus creating micrometer scale micro-channels. After directionally freezing Ch-Al solution cast in 3DP molds, the combined macro- and micro-channeled scaffold architecture enhanced cell suspension uptake beyond either macro- or micro-channels alone, reaching a volumetric flow rate of 1782.1 ± 48 mm(3) s(-1) for aqueous solutions and 440.9 ± 0.5 mm(3) s(-1) for blood. By combining 3DP and directional freezing, we can control the micro- and macro-architecture of Ch-Al to drastically improve cell influx into and distribution within the scaffold, while achieving porous zones that mimic articular cartilage zonal architecture. In future applications, precisely controlled micro- and macro-channels have the potential to assist immediate endogenous bone marrow uptake, stimulate chondrogenesis, and encourage vascularization of bone in an osteochondral scaffold.

Biomimetic synthesis of Mg‐substituted hydroxyapatite nanocomposites and three‐dimensional printing of composite scaffolds for bone regeneration

“…It can not only fabricate 3D bone‐substitute scaffolds suitable to different patient according to their needs quickly and accurately, but precisely control the porous structure and porosity of scaffolds (Hospodiuk, Dey, Sosnoski, & Ozbolat, ; Richards et al, ; Woodruff et al, ; Zheng et al, ). It has been demonstrated that a great amount of materials can be used to prepare porous scaffolds for bone tissue engineering, such as poly(ε‐caprolactone) (Shor, Guceri, Wen, Gandhi, & Sun, ), poly (lactic acid) (Zhang et al, ), gelatin (Gel) (Du et al, ), hydroxyapatite (HA) (Luo, Chen, Shi, & Ma, ), chitosan (CHI) (Reed et al, ) and so on. However, these scaffolds are not perfect due to their limited biocompatibility.…”

Mechanically robust cationic cellulose nanofibril 3D scaffolds with tuneable biomimetic porosity for cell culture

“…The introduction of emulsions, nanoparticles and dilute polymer solutions into the material can further modulate macro-and mesopore formation. 30 Ice crystals grown in a unidirectional fashion leave behind channels of aligned pores, mimicking the vascularity of native tissue 32 and can be applied to a range of scaffold materials, including: hydroxyapatite, 33 silk fibroin, 34 gelatin, 35 dextran, 36 and, more recently, chitosanalginate blends, 37 cellulose-chitosan blends, 38 and cellulose solutions. 39 The resultant scaffolds have the potential to expand the use of these materials from 2D films to 3D porous scaffolds and many authors suggest that the channels can promote vascularisation in vivo, although corroborating cell studies are only reported for some materials.…”

From intricate to integrated: Biofabrication of articulating joints

“…A wide range of smaller osteochondral constructs have been successfully generated using AM alone or in combination with conventional techniques, including casting, freeze‐drying and solvent casting/particle leaching . Even though the generation of a long‐term functional solution in osteochondral tissue engineering remains challenging, in vivo approaches with 3D printed osteochondral plugs have already been reported …”

“…A wide range of smaller osteochondral constructs have been successfully generated using AM alone 34,75,99,100 or in combination with conventional techniques, including casting, freeze-drying and solvent casting/particle leaching. [101][102][103][104] Even though the generation of a long-term functional solution in osteochondral tissue engineering remains challenging, in vivo approaches with 3D printed osteochondral plugs have already been reported. 97,[105][106][107] There is now the opportunity to generate larger personal implants, as biofabrication can provide highly accurate anatomical structures 62,108-110 using different materials, either with 111,112 or without 113,114 the aid of a sacrificial support.…”