Mathematically defined tissue engineering scaffold architectures prepared by stereolithography

The technologies employed for the preparation of conventional tissue engineering scaffolds restrict the materials choice and the extent to which the architecture can be designed. Here we show the versatility of stereolithography with respect to materials and freedom of design. Porous scaffolds are designed with computer software and built with either a poly(d,l-lactide)-based resin or a poly(d,l-lactide-co-ε-caprolactone)-based resin. Characterisation of the scaffolds by micro-computed tomography shows excellent reproduction of the designs. The mechanical properties are evaluated in compression, and show good agreement with finite element predictions. The mechanical properties of scaffolds can be controlled by the combination of material and scaffold pore architecture. The presented technology and materials enable an accurate preparation of tissue engineering scaffolds with a large freedom of design, and properties ranging from rigid and strong to highly flexible and elastic.

Photo-cross-linked poly(D,L-lactide)-based networks. Structural characterization by HR-MAS NMR spectroscopy and hydrolytic degradation behavior

To date, biodegradable networks and particularly their kinetic chain lengths have been characterized by analysis of their degradation products in solution. We characterize the network itself by NMR analysis in the solvent-swollen state under magic angle spinning conditions. The networks were prepared by photoinitiated cross-linking of poly(dl-lactide)−dimethacrylate macromers (5 kg/mol) in the presence of an unreactive diluent. Using diffusion filtering and 2D correlation spectroscopy techniques, all network components are identified. By quantification of network-bound photoinitiator fragments, an average kinetic chain length of 9 ± 2 methacrylate units is determined. The PDLLA macromer solution was also used with a dye to prepare computer-designed structures by stereolithography. For these networks structures, the average kinetic chain length is 24 ± 4 methacrylate units. In all cases the calculated molecular weights of the polymethacrylate chains after degradation are maximally 8.8 kg/mol, which is far below the threshold for renal clearance. Upon incubation in phosphate buffered saline at 37 °C, the networks show a similar mass loss profile in time as linear high-molecular-weight PDLLA (HMW PDLLA). The mechanical properties are preserved longer for the PDLLA networks than for HMW PDLLA. The initial tensile strength of 47 ± 2 MPa does not decrease significantly for the first 15 weeks, while HMW PDLLA lost 85 ± 5% of its strength within 5 weeks. The physical properties, kinetic chain length, and degradation profile of these photo-cross-linked PDLLA networks make them most suited materials for orthopedic applications and use in (bone) tissue engineering.

Organ printing

Organ printing techniques offer the potential to produce living 3D tissue constructs to repair or replace damaged or diseased human tissues and organs. Using these techniques, spatial variations along multiple axes with high geometric complexity can be obtained.. The level of control offered by these technologies to develop printed tissues will allow tissue engineers to better study factors that modulate tissue formation and function, and provide a valuable tool to study the effect of anatomy on graft performance. In this chapter we discuss the history behind substrate patterning and cell and organ printing, and the rationale for developing organ printing techniques with respect to limitations of current clinical tissue engineering strategies to effectively repair damaged tissues. We discuss current 2-dimensional and 3-dimesional strategies for assembling cells as well as the necessary support materials such as hydrogels, bioinks and natural and synthetic polymers adopted for organ printing research. Furthermore, given the current state-of-the-art in organ printing technologies, we discuss some of their limitations and provide recommendations for future developments in this rapidly growing field.

A review of rapid prototyping techniques for tissue engineering purposes

Rapid prototyping (RP) is a common name for several techniques, which read in data from computer-aided design (CAD) drawings and manufacture automatically threedimensional objects layer-by-layer according to the virtual design. The utilization of RP in tissue engineering enables the production of three-dimensional scaffolds with complex geometries and very fine structures. Adding micro- and nanometer details into the scaffolds improves the mechanical properties of the scaffold and ensures better cell adhesion to the scaffold surface. Thus, tissue engineering constructs can be customized according to the data acquired from the medical scans to match the each patient’s individual needs. In addition RP enables the control of the scaffold porosity making it possible to fabricate applications with desired structural integrity. Unfortunately, every RP process has its own unique disadvantages in building tissue engineering scaffolds. Hence, the future research should be focused into the development of RP machines designed specifically for fabrication of tissue engineering scaffolds, although RP methods already can serve as a link between tissue and engineering.

Designed biodegradable hydrogel structures prepared by stereolithography using poly(ethylene glycol)/poly(D,L-lactide)-based resins

Designed three-dimensional biodegradable poly(ethylene glycol)/poly(D,L-lactide) hydrogel structures were prepared for the first time by stereolithography at high resolutions. A photopolymerisable aqueous resin comprising PDLLA-PEG-PDLLA-based macromer, visible light photo-initiator, dye and inhibitor in DMSO/water was used to build the structures. Porous and non-porous hydrogels with well-defined architectures and good mechanical properties were prepared. Porous hydrogel structures with a gyroid pore network architecture showed narrow pore size distributions, excellent pore interconnectivity and good mechanical properties. The structures showed good cell seeding characteristics, and human mesenchymal stem cells adhered and proliferated well on these materials.

Fabrication of osteochondral scaffolds with stereolithography

The osteochondral defect is a classical model for a multiple-tissue problem[1]. Tissue engineering of either bone or cartilage imposes different demands on a scaffold concerning porosity, pore size and interconnectivity. Furthermore, local release of tissue-specific growth factors necessitates a tailored architecture. For the fabrication of an osteochondral scaffold with region specific architecture, an advanced technique is required. Stereolithography is a rapid prototyping technique that allows for the creation of such 3D polymer objects with well-defined architecture. Its working principle is the partial irradiation of a resin, causing a liquid-solid transition. By irradiating this resin by a computer-driven light source, a solid 3D object is constructed layer by layer. To make biodegradable polymers applicable in stereolithography, low-molecular weight polymers have to be functionalised with double bonds to enable photo-initiated crosslinking.

Photo-crosslinking of functionalised lactide oligomers for the fabrication of osteochondral tissue engineering scaffolds

In the fabrication of osteochondral tissue engineering scaffolds, the two distinct tissues impose different requirements on the architecture. Stereo-lithography is a rapid prototyping method that can be utilised to make 3D constructs with high spatial control by radical photopolymerization. In this study, biodegradable resins are developed that can be applied in stereo-lithography. Photo-crosslinked poly(lactide) networks with varying physical properties were synthesised, and by photo polymerizing in the presence of leachable particles porous scaffolds could be prepared as well.

Structural analysis of polymeric scaffolds by Micro-CT

The use of porous structures as tissue engineering scaffolds imposes demands on structural parameters such as porosity, pore size and interconnectivity. For the structural analysis of porous scaffolds, micro-computed tomography (μCT) is an ideal tool. μCT is a 3D X-ray imaging method that has several advantages over scanning electron microscopy (SEM) and other conventional characterisation techniques: • visualisation in 3D • quantitative results • non-destructiveness • minimal sample preparation

Designed tissue engineering scaffolds prepared by stereolithography

For the fabrication of tissue engineering scaffolds, the intended tissue formation process imposes requirements on the architecture. The chosen porosity often is a tradeoff between volume and surface area accessible to cells, and mechanical properties of the construct. Interconnectivity of the pores is essential for cell migration through the scaffold and for mass transport. Conventional techniques such as salt leaching often result in heterogeneous structures and do not allow for a precise control of the architecture. Stereolithography is a rapid prototyping method that can be utilised to make 3D constructs with high spatial control by radical photopolymerisation. In this study, a regular structure based on cyclic repetition of cell units were designed through CAD modelling.. One of these structures was built on a stereolithography apparatus (SLA). Furthermore, a polylactide-based resin was developed that can be applied in stereolithography. Polylactide has proven before to be a well-performing polymer in bone tissue engineering. The final objective in this study is to build newly designed PDLLA scaffolds with a precise SLA fabrication technique to study the effect of scaffold architecture on mechanical and biological properties.

Development of a PDLLA-based stereolithography resin for making tissue engineering scaffolds

The use of porous structures as tissue engineering scaffolds imposes high demands on the pore architecture. Stereolithography is a rapid prototyping method based on photo-polymerisation, that can be utilised to make 3D constructs with high spatial control. In this study, biodegradable resins were developed that can find application in stereolithography. Poly(D,L-lactide) (PDLLA) oligomers were synthesised and functionalised with methacrylate end-groups. By mixing the resulting macromers with a diluent, photo-initiator and inhibitor, lowviscosity resins were obtained that were photocrosslinked to yield stiff and strong degradable poly(lactide) networks. Also, porous scaffolds were fabricated on a stereolithography apparatus (SLA) from a nondegradable resin.

Properties of porous structures prepared by stereolithography using a polylactide resin

Rapid prototyping techniques such as stereolithography allow for building designed tissue engineering scaffolds with high accuracy. In this work, a stereolithography resin based on poly(D,L-lactide) was developed. Biodegradable scaffolds with varying porosity were built from this resin. The scaffolds were analysed by μCT-scanning and compression testing. The porous structures showed excellent mechanical properties in the range of trabecular bone.

Properties of photo-cured poly(D,L-lactide) networks

Poly(D,L-lactide) is a degradable polymer with a long history of use in medical applications. It is strong and stiff and degrades over the course of months into lactic acid, a body-own substance. In the field of tissue engineering it is commonly used to fabricate scaffolds. Stereolithography is a high resolution rapid prototyping technique by which designed 3D objects can be built using photo-initiated radical polymerisations. Poly(D,Llactide) (PDLLA) networks can be obtained by photopolymerisation of oligomers functionalised with unsaturated groups. In this work, PDLLA oligomers of varying architectures (arm lengths, numbers of arms) were synthesised and end-functionalised with methacrylate groups. These macromers were photo-crosslinked in solution to yield PDLLA networks of different architectures. The influence of the network architecture on its physical properties was studied.

Micro-CT analysis of tissue engineering scaffold architectures

A novel method was developed for a quantitative assessment of pore interconnectivity using micro-CT data. This method makes use of simulated spherical particles, percolating through the interconnected pore network. For each sphere diameter, the accessible pore volume is calculated. This algorithm was applied to compare pore interconnectivity of two different scaffold architectures; one created by salt-leaching and the other by stereolithography. The algorithm revealed a much higher pore interconnectivity for the latter one.

Mechanical properties of advanced tissue engineering scaffold architectures

In tissue engineering, porous scaffolds are used as a temporal support for tissue regeneration through cell adhesion, proliferation and differentiation. Besides applying a suitable material that is both biocompatible and biodegradable, the architectural design of the porous scaffold can be of essential for successful tissue regeneration. The architecture is of great influence on mechanical properties and transport properties of nutrients and metabolites1.

Poly(D,L-lactide)/hydroxyapatite composite tissue engineering scaffolds prepared by stereolithography

Hydroxyapatite (HAP) is a major component of bone and has osteoconductive and -inductive properties. It has been successfully applied as a substrate in bone tissue engineering, either with or without a biodegradable polymer such as polycaprolactone or polylactide. Recently, we have developed a stereolithography resin based on poly(D,L-lactide) (PDLLA) and a non-reactive diluent, that allows for the preparation of tissue engineering scaffolds with designed architectures. In this work, designed porous composite structures of PDLLA and HAP are prepared by stereolithography.