In Vitro Degradation of 3D-Printed Poly(L-lactide-Co-Glycolic Acid) Scaffolds for Tissue Engineering Applications.

The creation of scaffolds for cartilage tissue engineering has faced significant challenges in developing constructs that can provide sufficient biomechanical support and offer suitable degradation characteristics. Ideally, such tissue-engineering techniques necessitate the fabrication of scaffolds that mirror the mechanical characteristics of the articular cartilage while degrading safely without damaging the regenerating tissues. The aim of this study was to create porous, biomechanically comparable 3D-printed scaffolds made from Poly(L-lactide-co-glycolide) 85:15 and to assess their degradation at physiological conditions 37 °C in pH 7.4 phosphate-buffered saline (PBS) for up to 56 days. Furthermore, the effect of scaffold degradation on the cell viability and proliferation of human bone marrow mesenchymal stem cells (HBMSC) was evaluated in vitro. To assess the long-term degradation of the scaffolds, accelerated degradation tests were performed at an elevated temperature of 47 °C for 28 days. The results show that the fabricated scaffolds were porous with an interconnected architecture and had comparable biomechanical properties to native cartilage. The degradative changes indicated stable degradation at physiological conditions with no significant effect on the properties of the scaffold and biocompatibility of the scaffold to HBMSC. Furthermore, the accelerated degradation tests showed consistent degradation of the scaffolds even in the long term without the notable release of acidic byproducts. It is hoped that the fabrication and degradation characteristics of this scaffold will, in the future, translate into a potential medical device for cartilage tissue regeneration.

3D-printed patient-specific pelvis phantom for dosimetry measurements for prostate stereotactic radiotherapy with dominant intraprostatic lesion boost.

AIM: Developing and assessing the feasibility of using a three-dimensional (3D) printed patient-specific anthropomorphic pelvis phantom for dose calculation and verification for stereotactic ablative radiation therapy (SABR) with dose escalation to the dominant intraprostatic lesions. MATERIAL AND METHODS: A 3D-printed pelvis phantom, including bone-mimicking material, was fabricated based on the computed tomography (CT) images of a prostate cancer patient. To compare the extent to which patient and phantom body and bones overlapped, the similarity Dice coefficient was calculated. Modular cylindrical inserts were created to encapsulate radiochromic films and ionization chamber for absolute dosimetry measurements at the location of prostate and at the boost region. Gamma analysis evaluation with 2%/2mm criteria was performed to compare treatment planning system calculations and measured dose when delivering a 10 flattening filter free (FFF) SABR plan and a 10FFF boost SABR plan. RESULTS: Dice coefficients of 0.98 and 0.91 were measured for body and bones, respectively, demonstrating agreement between patient and phantom outlines. For the boost plans the gamma analysis yielded 97.0% of pixels passing 2%/2mm criteria and these results were supported by the chamber average dose difference of 0.47 ± 0.03%. These results were further improved when overriding the bone relative electron density: 97.3% for the 2%/2mm gamma analysis, and 0.05 ± 0.03% for the ionization chamber average dose difference. CONCLUSIONS: The modular patient-specific 3D-printed pelvis phantom has proven to be a highly attractive and versatile tool to validate prostate SABR boost plans using multiple detectors.

Evaluation of the in vitro cytotoxicity and modulation of the inflammatory response by the bioresorbable polymers poly(D,L-lactide-co-glycolide) and poly(L-lactide-co-glycolide).

Bioresorbable polymers composed of poly(D,L-lactide-co-glycolide) (PDLLGA) and poly(L-lactide-co-glycolide) (PLLGA) have become increasingly popular for the preparation of bone substitute constructs. However, there are reports of a delayed inflammatory reaction occurring months or years after implantation. Due to the long polymer degradation times, in vitro tests carried out at physiological temperature, 37°C, tend to assess only the short-term biocompatibility of these materials. The aim of this work is to develop an in vitro protocol that can be used to assess the long-term cytotoxicity of bioresorbable polymers in a time efficient manner. This study used a previously developed and validated accelerated degradation protocol to obtain samples of PDLLGA and PLLGA at increasing levels of degradation. Samples were then applied to standard ISO 10993-5 direct contact cytotoxicity testing and it was found that PDLLGA samples showed increasing levels of cytotoxicity at the later stages of degradation, with PLLGA samples demonstrating significantly less cytotoxic behaviour. Following concern that accumulation of acidic degradation products in a closed multi-well culture environment could overestimate cytotoxicity, we developed and validated a new dynamic flow culture methodology, for testing the cytotoxicity of these degradable materials, by adapting a commercial "organ on a chip" flow culture system, Quasi Vivo®. In addition to cytotoxicity testing, we have carried out profiling of inflammatory cytokines released by cells in response to degraded PDLLGA and PLLGA, and have suggested mechanism by which lactide-based bioresorbable materials could modulate the inflammatory response through the G-protein coupled receptor (GPCR), hydroxycarboxylic acid receptor 1 (HCA1). STATEMENT OF SIGNIFICANCE: Bioresorbable materials naturally disintegrate over time when implanted into the body. They are often used to make screws and clips for repair of broken bones. Unfortunately, some patients can react badly to the material, resulting in painful inflammation. Biomaterials scientists are interested in developing materials that are more compatible with the body. However, it is very difficult to predict the long-term compatibility of bioresorbable materials in the lab. In our study, we have developed a method that will allow us to study the effects of the materials as they continue to break down. This will help us understand why the materials may cause inflammation, and will support research into the development of new and improved materials for bone repair.

Influence of surface condition on the degradation behaviour and biocompatibility of additively manufactured WE43.

The further development of future Magnesium based biodegradable implants must consider not only the freedom of design, but also comprise implant volume reduction, as both aspects are crucial for the development of higher functionalised implants, such as plate systems or scaffold grafts in bone replacement therapy. As conventional manufacturing methods such as turning and milling are often accompanied by limitations concerning implant design and functionality, the process of laser powder bed fusion (LPBF) specifically for Magnesium alloys was recently introduced. In addition, the control of the degradation rate remains a key aspect regarding biodegradable implants. Recent studies focusing on the degradation behaviour of additively manufactured Magnesium scaffolds disclosed additional intricacies when compared to conventionally manufactured Magnesium parts, as a notably larger surface area was exposed to the immersion medium and scaffold struts degraded non-uniformly. Moreover, chemical etching as post processing technique is applied to remove sintered powder particles from the surface, altering surface chemistry. In this study, cylindrical Magnesium specimens were manufactured by LPBF and surfaces were consecutively modified by phosphoric etching and machining. Degradation behaviour and biocompatibility were then investigated, revealing that etched samples exhibited the overall lowest degradation rates, but experienced large pit formation, while the reduction of surface roughness resulted in a delay of degradation.

Development of three-dimensional printing polymer-ceramic scaffolds with enhanced compressive properties and tuneable resorption.

In this study, bone tissue engineered scaffolds fabricated via powder-based 3D printing from hydroxyapatite (HA) and calcium sulphate (CaSO4) powders were investigated. The combination of using a fast resorbing CaSO4 based powder and the relatively slower HA powder represents a promising prospect for tuning the bioresorption of 3D printed (3DP) scaffolds. These properties could then be tailored to coincide with tissue growth rate for different surgical procedures. The manufactured scaffolds were infiltrated with poly(ε­caprolactone) (PCL). The PCL infiltrated the inter-particle spacing within the 3DP structures due to the nature of a loosely-packed powder bed and also covered the surface of ceramic-based scaffolds. Consequently, the average compressive strength, compressive modulus and toughness increased by 314%, 465% and 867%, respectively. The resorption behaviour of the 3DP scaffolds was characterised in vitro using a high-throughput system that mimicked the physiological environment and dynamic flow conditions relevant to the human body. A rapid release of CaSO4 between Day 0 and 28 was commensurate with a reduction in scaffold mass and compressive properties, as well as an increase in medium absorption. In spite of this, HA particles, connected by PCL fibrils, remained within the microstructure after 56 days resorption under dynamic conditions. Consequently, a high level of structural integrity was maintained within the 3DP scaffold. This study presented a porous PCL-HA-CaSO4 3DP structure with the potential to encourage new tissue growth during the initial stages of implantation and also offering sufficient structural and mechanical support during the bone healing phase.

Blueprints for the Next Generation of Bioinspired and Biomimetic Mineralised Composites for Bone Regeneration.

Coccolithophores are unicellular marine phytoplankton, which produce intricate, tightly regulated, exoskeleton calcite structures. The formation of biogenic calcite occurs either intracellularly, forming 'wheel-like' calcite plates, or extracellularly, forming 'tiled-like' plates known as coccoliths. Secreted coccoliths then self-assemble into multiple layers to form the coccosphere, creating a protective wall around the organism. The cell wall hosts a variety of unique species-specific inorganic morphologies that cannot be replicated synthetically. Although biomineralisation has been extensively studied, it is still not fully understood. It is becoming more apparent that biologically controlled mineralisation is still an elusive goal. A key question to address is how nature goes from basic building blocks to the ultrafine, highly organised structures found in coccolithophores. A better understanding of coccolithophore biomineralisation will offer new insight into biomimetic and bioinspired synthesis of advanced, functionalised materials for bone tissue regeneration. The purpose of this review is to spark new interest in biomineralisation and gain new insight into coccolithophores from a material science perspective, drawing on existing knowledge from taxonomists, geologists, palaeontologists and phycologists.

Effects of poly (ε-caprolactone) coating on the properties of three-dimensional printed porous structures.

Powder-based inkjet three-dimensional printing (3DP) to fabricate pre-designed 3D structures has drawn increasing attention. However there are intrinsic limitations associated with 3DP technology due to the weak bonding within the printed structure, which significantly compromises its mechanical integrity. In this study, calcium sulphate ceramic structures demonstrating a porous architecture were manufactured using 3DP technology and subsequently post-processed with a poly (ε-caprolactone) (PCL) coating. PCL concentration, immersion time, and number of coating layers were the principal parameters investigated and improvement in compressive properties was the measure of success. Interparticle spacing within the 3DP structures were successfully filled with PCL material. Consequently the compressive properties, wettability, morphology, and in vitro resorption behaviour of 3DP components were significantly augmented. The average compressive strength, Young׳s modulus, and toughness increased 217%, 250%, and 315%, following PCL coating. Addition of a PCL surface coating provided long-term structural support to the host ceramic material, extending the resorption period from less than 7 days to a minimum of 56 days. This study has demonstrated that application of a PCL coating onto a ceramic 3DP structure was a highly effective approach to addressing some of the limitations of 3DP manufacturing and allows this advanced technology to be potentially used in a wider range of applications.

Surrogate Outcome Measures of In Vitro Osteoclast Resorption of ß Tricalcium Phosphate.

Introduction of porosity to calcium phosphate scaffolds for bone repair has created a new challenge when measuring bioresorption in vitro, rendering traditional outcome measures redundant. The aim of this study is to identify a surrogate endpoint for use with 3D scaffolds. Murine RAW 264.7 cells are cultured on dense discs of ß-tricalcium phosphate in conditions to stimulate osteoclast (OC) formation. Multinucleated OCs are visible from day 6 with increases at days 8 and 10. Resorption pits are first observed at day 6 with much larger pits visible at days 8, 10, and 12. The concentration of calcium ions in the presence of cells is significantly higher than cell-free cultures at days 3 and 9. Using linear regression analysis, Ca ion release could account for 35.9% of any subsequent change in resorption area. The results suggest that Ca ion release is suitable to measure resorption of a beta-tricalcium phosphate ceramic substrate in vitro. This model could replace the more accepted resorption pit assay in circumstances where quantification of pits is not possible, e.g., when characterizing 3D tissue engineered bone scaffolds.

Biocompatibility of calcium phosphate bone cement with optimised mechanical properties: an in vivo study.

This work establishes the in vivo performance of modified calcium phosphate bone cements for vertebroplasty of spinal fractures using a lapine model. A non-modified calcium phosphate bone cement and collagen-calcium phosphate bone cements composites with enhanced mechanical properties, utilising either bovine collagen or collagen from a marine sponge, were compared to a commercial poly(methyl methacrylate) cement. Conical cement samples (8 mm height × 4 mm base diameter) were press-fit into distal femoral condyle defects in New Zealand White rabbits and assessed after 5 and 10 weeks. Bone apposition and tartrate-resistant acid phosphatase activity around cements were assessed. All implants were well tolerated, but bone apposition was higher on calcium phosphate bone cements than on poly(methyl methacrylate) cement. Incorporation of collagen showed no evidence of inflammatory or immune reactions. Presence of positive tartrate-resistant acid phosphatase staining within cracks formed in calcium phosphate bone cements suggested active osteoclasts were present within the implants and were actively remodelling within the cements. Bone growth was also observed within these cracks. These findings confirm the biological advantages of calcium phosphate bone cements over poly(methyl methacrylate) and, coupled with previous work on enhancement of mechanical properties through collagen incorporation, suggest collagen-calcium phosphate bone cement composite may offer an alternative to calcium phosphate bone cements in applications where low setting times and higher mechanical stability are important.

Biocompatibility of calcium phosphate bone cement with optimized mechanical properties.

The broad aim of this work was to investigate and optimize the properties of calcium phosphate bone cements (CPCs) for use in vertebroplasty to achieve effective primary fixation of spinal fractures. The incorporation of collagen, both bovine and from a marine sponge (Chondrosia reniformis), into a CPC was investigated. The biological properties of the CPC and collagen-CPC composites were assessed in vitro through the use of human bone marrow stromal cells. Cytotoxicity, proliferation, and osteoblastic differentiation were evaluated using lactate dehydrogenase, PicoGreen, and alkaline phosphatase activity assays, respectively. The addition of both types of collagen resulted in an increase in cytotoxicity, albeit not to a clinically relevant level. Cellular proliferation after 1, 7, and 14 days was unchanged. The osteogenic potential of the CPC was reduced through the addition of bovine collagen but remained unchanged in the case of the marine collagen. These findings, coupled with previous work showing that incorporation of marine collagen in this way can improve the physical properties of CPCs, suggest that such a composite may offer an alternative to CPCs in applications where low setting times and higher mechanical stability are important.