Tunable hydrogel composite with two-step processing in combination with innovative hardware upgrade for cell-based three-dimensional bioprinting

Abstract

Three-dimensional (3-D) bioprinting is the layer-by-layer deposition of biological material with the aim of achieving stable 3-D constructs for application in tissue engineering. It is a powerful tool for the spatially directed placement of multiple materials and/or cells within the 3-D sample. Encapsulated cells are protected by the bioink during the printing process. Very few materials are available that fulfill requirements for bioprinting as well as provide adequate properties for cell encapsulation during and after the printing process. A hydrogel composite including alginate and gelatin precursors was tuned with different concentrations of hydroxyapatite (HA) and characterized in terms of rheology, swelling behavior and mechanical properties to assess the versatility of the system. Instantaneous as well as long-term structural integrity of the printed hydrogel was achieved with a two-step mechanism combining the thermosensitive properties of gelatin with chemical crosslinking of alginate. Novel syringe tip heaters were developed for improved temperature control of the bioink to avoid clogging. Human mesenchymal stem cells mixed into the hydrogel precursor survived the printing process and showed high cell viability of 85% living cells after 3 days of subsequent in vitro culture. HA enabled the visualization of the printed structures with micro-computed tomography. The inclusion of HA also favors the use of the bioink for bone tissue engineering applications. By adding factors other than HA, the composite could be used as a bioink for applications in drug delivery, microsphere deposition or soft tissue engineering.

Introduction

Tissue engineering aims to rebuild a tissue by seeding cells onto a three-dimensional (3-D) scaffold and culture the construct in conjunction with biological signals [1]. Although this state-of-the-art approach is being investigated widely, there are still key difficulties to overcome. One issue is the lack of geometrical control during scaffold fabrication when using common methods such as salt-leaching [2], porogen melting [3] or gas foaming [4] and the associated difficulties in up-scaling to clinically relevant sizes. Cell seeding is usually performed by pipetting cells by hand onto the scaffold, which results in inhomogeneous cell distribution and ingrowth into the scaffold [2].

A promising new method to overcome the current limitations is 3-D biofabrication, which is the application of rapid prototyping to tissue engineering, allowing layer-by-layer deposition of biological material [5]. 3-D bioprinting describes the deposition of the biomaterial itself. There are two methods of 3-D bioprinting: pure scaffold printing and 3-D tissue printing. The first method focuses on fabrication of scaffolds by printing pure scaffold material, aiming to control scaffold geometry or pore size. Post-treatment can be performed to enhance scaffold stability, remove cytotoxic crosslinkers or sterilize the scaffold before the addition of living cells. The second method refers to 3-D tissue printing which focuses on the deposition of biological material, where the “bioink” consists of scaffold material and biological components such as cells or growth factors simultaneously. Due to the presence of biological components this printing process requires each single step within the process to be biocompatible, providing gentle processing conditions.

The great power of 3-D tissue printing is the multi-channel assembly of most commercial 3-D bioprinters. This enables the controlled deposition of different materials and/or different cell types adjacent to one another in a predefined 3-D pattern within one process. Fabrication of soft cell-free hydrogel scaffolds has been widely performed with 3-D bioprinting [6], [7]. So far there are only a few studies addressing the challenge of 3-D tissue printing cells into a spatially organized 3-D matrix [8], [9], [10], [11]. In this study, we present a tunable hydrogel composite which provides a physiological environment for cells, mild processing conditions and results in a stable, viable 3-D construct.

A suitable material for 3-D bioprinting of cells embedded in scaffold material must support cell viability and differentiation as well as be printable and maintain construct integrity and mechanical stability after printing. Physical stability requires that a liquid or semi-solid bioink can be chemically or physically crosslinked. Harsh conditions, such as extreme temperatures or pH and high shear stresses, while often necessary for the printing and crosslinking processes, must be avoided. These restrictions often result in a reduced printing resolution compared to the printing of scaffold material without cells.

Biocompatible hydrogels show high potential for 3-D bioprinting due to their ability to induce a phase change from liquid to (semi-)solid by crosslinking. They have been widely used in soft tissue engineering and are lately considered as an emerging material for bone substitutes [12]. Alginate is one of the most used materials for cell-based hydrogel printing due to its favorable characteristics concerning biocompatibility and the capability to support cellular function and differentiation in culture, but also as a carrier for drug delivery [13]. Gelatin, which is also known to enhance cell attachment and promote cell growth [14], [15], was implemented to improve the initial stability of the 3-D printed construct. Hydroxyapatite is known to be an excellent bone substitute material [16]. Enhancing alginate hydrogel with hydroxyapatite (HA) increases cell attachment [17] and leads to osteogenic differentiation of osteogenic progenitor cells in vivo [18].

The hydrogel composite presented in this study is one potential example for the presented two-step processing method. The materials here – alginate and gelatin enhanced with varying amounts of HA – have been specifically selected for bone tissue engineering applications. A possible expansion of the presented processing method for other materials and for other applications will need detailed investigation. Our hypothesis is that combining gelatin and alginate improves instantaneous stability by maintaining initial bonding between single layers due to the gelation of gelatin as well as long-term stability of the whole construct by chemically crosslinking alginate. By adding HA, a versatile material is obtained which additionally leads to radiopacity of the material and thus visibility for X-ray-based micro-computed tomography (μCT), a powerful 3-D imaging technique to assess the printed construct. In this study we assessed the influence of varying amounts of HA on material properties, cell viability after printing and its impact on the printing process. Cells were mixed into the hydrogel precursor before printing and stabilized in their 3-D position once the composite was crosslinked. New hardware consisting of a heatable print head with innovative syringe tip heaters was manufactured to adapt a commercially available 3-D bioprinter for this process.