Dr. Zhengchao Guo

Zengchao Guo (2020) Poly(trimethylene carbonate)-based composites for biomedical applications Enschede DOI: 10.3990/1.9789036550741

Zhengchao Guo was a PhD student in the research group Biomaterials Science and Technology (BST). His supervisor was prof.dr. D.W. Grijpma from the Faculty of Science and Technology (TNW).

The past decades have shown major developments in biomedical engineering technologies, especially in tissue engineering and regenerative medicine. This poses a large demand on the preparation of suitable biomaterials for both research and industry. Among the different classes of biomaterials, polymer-based composites have received special attention in the past years. These composite materials can have several advantages related to presentation of functional bioactivity and improved mechanical properties, next to biocompatibility and biodegradability. The work presented in this PhD thesis introduces two applications of polymer-based composites. One is the use of electrically conductive reduced graphene oxide (rGO) fillers, enhancing the conductivity of scaffolds to be applied in nerve regeneration. Another is the use of sacrificial particle leaching, applied in the manufacturing of porous scaffolds for small-diameter vascular tissue engineering.

Poly(trimethylene carbonate) (PTMC) was chosen as the polymer component, because of its excellent biocompatibility, surface erosion degradation behavior and flexible and elastic properties. Combination of the functional rGO filler with PTMC, was either done by mixing rGO in a PMTC matrix or grafting the polymer onto the rGO and subsequent mixing with PTMC. Porogens such as sodium chloride, sodium fluoride and calcium carbonate were used to fabricate porous scaffolds for vascular tissue engineering.

Next to a description of the material properties of the composites, also several porous scaffold manufacturing methods are described. These include sacrificial particle leaching, electrospinning and additive manufacturing. Moreover, in vitro and in vivo evaluation of prepared scaffolds were performed.

In Chapter 1, a general introduction on the background of the research described in this thesis is presented. The scope and outline of the studies are also introduced. In Chapter 2, the current state of the art on advancements in polymer-based composites, their properties, structure and fabrication techniques is presented. To engineer suitable scaffolds or implants for e.g. tissue engineering and regenerative medicine applications, structure property relationships of composites need to be considered. In this chapter, we focused on and discussed bioactive polymer-based composites, such as bone-forming, electrically conductive, magnetic, bactericidal and oxygen-releasing materials, and non-bioactive polymer-based composites containing reinforcing fillers and porogens. In addition, porous scaffold structures fabricated by particle leaching, electrospinning and additive manufacturing, all subjects of this PhD research, were described. It was concluded that current challenges are related to the limited choice of bioactive and functional fillers, control of the bioactive expression in the polymer matrix and manufacturing techniques for suitable structures. Therefore, studies on the relationship between the material properties of a specific polymer-based composite, its structure, physical and mechanical properties and biological response for a specific biomedical application remain necessary.

Previous studies on PTMC polymer technology developed within the research group, revealed the highly interesting properties of this material, like flexibility, elasticity and degradation without eliciting acidic degradation products in a biological environment. This material is therefore regarded highly suitable for the preparation of tubular scaffolds that can be applied as e.g. engineered nerve guides or blood vessel prostheses. In Chapter 3 a study on the preparation of PTMC/rGO composites with electrical conductivity for peripheral nerve regeneration is described. rGO was obtained through reduction of graphene oxide (GO), which was prepared by a modified Hummers method. A 3-armed PTMC was synthesized by ring-opening polymerization and end-functionalized with methacrylate groups. PTMC-MA/rGO composite films were subsequently prepared by mixing, solvent casting and photo-crosslinking. The PTMC-MA/rGO composite films with 0, 0.5, 1, 2 and 4 wt% rGO showed increasing electro-conductive properties with values up to 6.88*10-2 S/cm. Cell culturing with PC-12 cells showed that after initial cell adhesion, the cells proliferated on the surface of the PTMC-MA/rGO composites, regardless of the rGO loading. These results provide evidence that composite materials of PTMC and rGO have promising properties for application as nerve guide conduits that accelerate nerve regeneration and were the basis for a study described in Chapter 4. In this chapter we describe a study on porous PTMC/rGO composite nerve guides, their preparation and in vivo evaluation. The composite nerve guide conduit with 2 wt% rGO relative to PTMC was prepared by dip-coating, photo-crosslinking and leaching of sodium fluoride porogen particles. The conduit, which had a porosity of 73%, was used to bridge a 15 mm sciatic nerve gap in a pilot study in rabbits. A porous PTMC conduit with the same porosity was used as a control. Macroscopic evaluation 6 weeks after implantation showed the formation of firm tissue in the PTMC/rGO nerve guide, whereas the tissue formed in the PTMC conduit lacked form stability. Histological analysis showed that the number of myelinated axons and the amount of collagen were higher in the PTMC/rGO nerve guide than in the PTMC conduit. The same differences were found in the respective distal nerve stumps.

To improve the compatibility and dispersion behavior of the rGO in the composites, an alternative strategy was chosen for the preparation of the composite material. In Chapter 5 a study on a new composite rGO-graft-PTMC is presented. rGO was synthesized by azido ethanol reaction with GO at a high temperature. The hydroxyl groups on the rGO were used to initiate the ring-opening polymerization of trimethylene carbonate monomer. The obtained rGO-graft-PTMC, comprising of single layer graphene nanosheets, afforded stable rGO-graft-PTMC dispersions in chloroform. The rGO-graft-PTMC composites, with different PTMC molecular weights of 430-7030 g/mol, had electrical conductivities ranging from 0.2-0.016 S/cm. To investigate the biocompatibility of rGO-graft-PTMC, PTMC-based films containing rGO-graft-PTMC were prepared and used in cell culturing experiments. The composite films showed good biocompatibility with PC12 neuronal cells.

Another method than particle leaching to fabricate porous scaffolds is electrospinning. In Chapter 6, we describe a study on the preparation and properties of electrospun PTMC/rGO-graft-PTMC composite fibrous mats. A loading of the rGO-graft-PTMC in the PTMC matrix of up to 6 wt% could be reached. Scanning electron microscopy images showed that the morphology and average diameter of the PTMC/rGO-graft-PTMC composite fibers were affected by the content of rGO-graft-PTMC. Additionally, the incorporation of rGO-graft-PTMC resulted in enhanced thermal stability and hydrophobicity of the PTMC-based fibers. Biological results demonstrated that PC12 cells showed higher cell viability on PTMC/rGO-graft-PTMC fibers of 2.4, 4.0 and 6.0 wt% rGO-graft-PTMC compared to pure PTMC fibers. The results of these studies suggest that PTMC/rGO-graft-PTMC composite structures hold great potential for neural tissue engineering.

In the last chapters and appendix of this thesis, our research on the engineering of porous scaffolds for small-diameter vascular tissue engineering is described. Due to the increasing prevalence of peripheral and cardiac vascular diseases, we focused on vascular grafts with an inner diameter of less than 6 mm for which there is an urgent need. Fabrication of branched micro-vascular channels for biomedical applications is presented in Chapter 7. PTMC/CaCO3 composite films with various amounts of CaCO3 particles were prepared by solvent casting and photo-crosslinking. Particle leaching using a diluted HCl solution afforded micro-porous structures with porosities ranging from 33 to 71% and a pore size around 0.5 mm. A minimum CaCO3 content of 40 vol% was required to get a porous structure with sufficient water permeation at a physiological pressure. The mechanical properties of the micro-porous films were similar to those of native blood vessels. To engineer complex vascular structures, photo-crosslinkable PTMC/CaCO3 composite resins were formulated for stereolithography-based additive manufacturing. We were able to build vascular scaffolds of various shapes and sizes with this PTMC/CaCO3 composite resin. A very small branched vascular structure with a micro-porous wall, having a 482 µm inner diameter, wall thickness of 146 µm, 0.4 µm pore size and 59% porosity could be built, showing that complex structures and scaffolds for biomedical applications can be fabricated using this PTMC/CaCO3 composite resin through additive manufacturing techniques.

In Chapter 8 we describe a method, based on salt leaching, for the manufacturing of porous tubular scaffolds for small-diameter vascular tissue engineering. Using a photo-crosslinkable composite resin composed of methacrylate end-functionalized PTMC macromer, sodium chloride as a sacrificial composite filler and propylene carbonate as a non-reactive diluent in a glass mold, porous structures were prepared after crosslinking, extraction of the diluent and particle leaching. Tubular scaffolds were obtained with an inner diameter of 3 mm, a wall thickness of 1 mm and a length of 4.5 cm. Pore sizes ranged from 0-290 µm and the porosity was around 70%. The pores were homogeneously distributed and interconnected. With increasing PTMC macromer molecular weight from 4 to 22 kg/mol, the E-modulus and maximum tensile strength of the scaffolds in the radial direction increased from 0.56 to 1.12 MPa and 0.12 to 0.55 MPa, respectively. Stress-strain curves of scaffolds made from 13, 17 and 22 kg/mol PTMC macromers, showed a ‘toe’ region characteristic for native arteries, followed by a linear increase until the maximum stress was reached. In view of their mechanical properties, porous tubular scaffolds made from PTMC macromers with a molecular weight of 13–22 kg/mol appeared the preferred scaffolds for vascular tissue engineering.

In the Appendix we present an initial study on the culturing of seeded smooth muscle cells (SMCs) in porous tubular PTMC scaffolds under dynamic conditions in a pulsatile flow bioreactor. Scaffolds with a 3 mm inner diameter, 1 mm wall thickness and an average pore size of 68 µm were used. SMCs cultured in porous PTMC scaffolds under static conditions was used for comparison. Cells were cultured for 14 days and a cell proliferation assay and histological evaluation were performed. A three times higher number of SMCs was found using dynamic culture conditions compared to static culturing. Histological images of the scaffolds cultured under dynamic conditions showed that the SMCs fully filled the porous scaffolds, which was not found after static culturing. Determination of the mechanical properties after dynamic culturing revealed an increase in the maximum stress of the constructs. It was concluded that dynamic culturing of cell-seeded PTMC scaffolds for vascular tissue engineering can be used for future in vivo experiments.

2 Outlook and future perspectives

Polymer-based composites can be regarded as a special class of materials and currently are widely investigated for the preparation of biomedical implants, grafts and other devices. Because polymer composites have advantages, such as low cost of available natural and synthetic polymers, filler types and ease and tunability of manufacturing techniques, it is envisaged that these materials will receive a prominent place in the biomedical field. It can be recognized that the combination of a specific polymer and composite filler will lead to novel material properties. Importantly, fillers not only can be used to change the material properties but can add bioactive properties to the composite. As an example, in the design of an implant the filler can be used to reinforce the polymer material but also be used to deliver bioactive components in a sustained manner. Manufacturing techniques may be used to create a 3D gradient scaffold structure, allowing a spatio-temporal regeneration of the targeted tissue.

As demonstrated in this thesis work, poly(trimethylene carbonate) (PTMC)-based composites with several kinds of functional fillers can be used for biomedical applications such as nerve regeneration conduits and vascular grafts. Several processing techniques were used to prepare those biomedical implants. Functional properties were added by using electrically conductive graphene oxides as fillers. Sacrificial particles were used to create porous tubular scaffolds allowing the diffusion of nutrients and waste products in the tissue regeneration process. While positive results were obtained in these studies, it can be concluded that improvements remain necessary for each application before such biomedical devices can be clinically applied.

2.1 Nerve guide conduits

Due to trauma or disease nerves can be damaged and cause loss of function. The main function of a nerve guide conduit is to provide a temporary guide and protect the regenerating axons from invading cells from the environment, i.e. prevention of scar tissue formation. The wall of the conduit, however, should not be completely closed and allow the sustained delivery of nutrients and removal of cellular waste products. Particle leaching from polymer salt composites is an efficient way to make a porous structure. However, due to the limited access to of submicron leachable particles, porosity is generally not consistently small.

Electrically conductive materials are widely investigated for use in tissue engineering and regenerative medicine, especially in neural tissue engineering due to its bioactive electrical interface which could stimuli cells growing, differentiation and signaling. In addition, based on electrically conductive materials, electrostimulation could be applied to accelerated axonal growth and restoring nerve function. We showed that use of electrically conductive PTMC/rGO composite with a small amount (2wt% relative to PTMC) of highly conductive rGO fillers for the fabrication of nerve guide conduits is a viable strategy to improve peripheral nerve regeneration.

It is envisaged that detailed in vivo experiments should be carried out with these PTMC/rGO composites to show their full potential. A complete nerve function characterization should be carried out to determine the threshold level for undisturbed movement. For future studies, it is recommended to increase the amount of in vivo experiments as well as the implantation time, and to include walking track analysis and electrophysiological measurements. To avoid in vivo implantation studies, the regeneration process may be evaluated using microfluidic techniques.

Although these conduits show promising results in the regeneration of nerves, their use was limited to small defects only. Development of improved conduits will likely focus on composite materials that also will deliver constituents like growth factors that stimulate the regeneration process. This may also stimulate the regeneration of large nerves and large size nerve defects (more than 3 cm).

2.2 Porous vascular networks

Capillary vascular networks are a necessary component of most native tissues and organs. This capillary structure provides the oxygen and nutrient supply for living cells as well as removal of waste products. To engineer tissues and organs for transplantation building of a vascular network plays an important role. Because the capillaries have a micro size inner diameter and wall thickness, these are difficult to fabricate. Selection of a suitable polymeric material, type of salt allows the preparation of scaffolds with sub-micro pore size of the wall and mechanical properties matching those of natural blood vessels. Such artificial vascular structures are needed for both traditional tissue engineering applications but also in studies on disease models. In this respect these materials hold great promise in their combination with microfluidic techniques. Functions, like platelet activation and adhesion dependent blood-graft interfaces may be studied. Such studies will contribute to optimal materials to be applied in blood-vessel tissue development.

2.3 Small diameter vascular grafts

Tissue engineering of small diameter vascular networks can have a significant impact on in vitro generated vascularized tissue and organ constructs for transplantation. 3D printing of vascular grafts recently received a lot of attention due to its efficiency and versatility in design. However, current 3D printing technologies do not allow the fabrication of highly porous scaffolds due to limitations of 3D printing devices and materials. Combining 3D printing, photo-crosslinking and particle leaching may well be a valuable step. Because mechanical stimulation has been shown to affect such processes tissue development using a bioreactor with pulsatile flow may stimulate tissue development. In combination with cell seeding techniques, future in vivo implantation studies may show the potential of these vascular grafts for clinical application.

3 General conclusions

We may conclude that bioactive or other functional fillers will endow biodegradable polymers large possibilities to be used in the repair or regeneration of tissues and other biomedical applications. The current techniques, applying polymer-based composites, in general need fine-tuning to optimize the structure required in the specific application. Polymer-based composite scaffolds already showed not only high cell adhesion, biocompatibility, and biodegradability but also bioactivities in terms of tissue formation, function, stimulation, survival and antibacterial properties during in vitro and in vivo experiments.

However, there are still challenges because of the limited choice of bioactive and functional fillers, control of the bioactive expression in the polymer matrix, and manufacturing techniques of suitable structures. Polymer-based composites should be adapted to the biological microenvironment, thereby accelerating tissue repair and regeneration. Studies on the relationship between the material properties of a specific poly(trimethylene carbonate-based composite, its structure, physical and mechanical properties and biological response for specific biomedical applications remain necessary.