Advances in biomimetic collagen mineralisation and future approaches to bone tissue engineering.

With an ageing world population and ~20% of adults in Europe being affected by bone diseases, there is an urgent need to develop advanced regenerative approaches and biomaterials capable to facilitate tissue regeneration while providing an adequate microenvironment for cells to thrive. As the main components of bone are collagen and apatite mineral, scientists in the tissue engineering field have attempted in combining these materials by using different biomimetic approaches to favour bone repair. Still, an ideal bone analogue capable of mimicking the distinct properties (i.e., mechanical properties, degradation rate, porosity, etc.) of cancellous bone is to be developed. This review seeks to sum up the current understanding of bone tissue mineralisation and structure while providing a critical outlook on the existing biomimetic strategies of mineralising collagen for bone tissue engineering applications, highlighting where gaps in knowledge exist.

Strategies for Enhancing Polyester-Based Materials for Bone Fixation Applications.

Polyester-based materials are established options, regarding the manufacturing of bone fixation devices and devices in routine clinical use. This paper reviews the approaches researchers have taken to develop these materials to improve their mechanical and biological performances. Polymer blending, copolymerisation, and the use of particulates and fibre bioceramic materials to make composite materials and surface modifications have all been studied. Polymer blending, copolymerisation, and particulate composite approaches have been adopted commercially, with the primary focus on influencing the in vivo degradation rate. There are emerging opportunities in novel polymer blends and nanoscale particulate systems, to tune bulk properties, and, in terms of surface functionalisation, to optimise the initial interaction of devices with the implanted environment, offering the potential to improve the clinical performances of fracture fixation devices.

A Comparison of Osteoblast and Osteoclast In Vitro Co-Culture Models and Their Translation for Preclinical Drug Testing Applications.

As the population of western societies on average ages, the number of people affected by bone remodeling-associated diseases such as osteoporosis continues to increase. The development of new therapeutics is hampered by the high failure rates of drug candidates during clinical testing, which is in part due to the poor predictive character of animal models during preclinical drug testing. Co-culture models of osteoblasts and osteoclasts offer an alternative to animal testing and are considered to have the potential to improve drug development processes in the future. However, a robust, scalable, and reproducible 3D model combining osteoblasts and osteoclasts for preclinical drug testing purposes has not been developed to date. Here we review various types of osteoblast-osteoclast co-culture models and outline the remaining obstacles that must be overcome for their successful translation.

An In Vitro Engineered Osteochondral Model as Tool to Study Osteoarthritis Environment.

Osteoarthritis (OA) is a joint degenerative pathology characterized by mechanical and inflammatory damages affecting synovium, articular cartilage (AC), and subchondral bone (SB). Several in vitro, in vivo, and ex vivo models are developed to study OA, but to date the identification of specific pharmacological targets seems to be hindered by the lack of models with predictive capabilities. This study reports the development of a biomimetic in vitro model of AC and SB interface. Gellan gum methacrylated and chondroitin sulfate/dopamine hydrogels are used for the AC portion, whereas polylactic acid functionalized with gelatin and nanohydroxyapatite for the SB. The physiological behavior of immortalized stem cells (Y201s) and Y201s differentiated in chondrocytes (Y201-Cs), respectively, for the SB and AC, is demonstrated over 21 days of culture in vitro in healthy and pathological conditions, whilst modeling the onset of cytokines-induced OA. The key metrics are: lower glycosaminoglycans production and increased calcification given by a higher Collagen X content, in the AC deep layer; higher expression of pro-angiogenic factor (vegf) and decreased expression of osteogenic markers (coll1, spp1, runx2) in the SB. This novel approach provides a new tool for studying the development and progression of OA.

A cytokine-induced spheroid-based in vitro model for studying osteoarthritis pathogenesis.

Given the lack of in vitro models faithfully reproducing the osteoarthritis (OA) disease on-set, this work aimed at manufacturing a reliable and predictive in vitro cytokine-based Articular Cartilage (AC) model to study OA progression. Cell spheroids of primary human fetal chondrocytes (FCs) and h-TERT mesenchymal stem cells differentiated chondrocytes (Y201-C) were analysed in terms of growth kinetics, cells proliferation and apoptosis over 10 days of culture, in healthy condition or in presence of cytokines (interleukin-1ß, -6 and TNF-α). Then, the spheroids were assembled into chondrospheres using a bottom-up strategy, to obtain an in vitro cytokines-induced OA model. The resulting chondrospheres were evaluated for gene expression and anabolic ECM proteins. Compared to the healthy environment, the simulated OA environment induced chondrocyte hyperproliferation and apoptotic pathway, decreased expression of anabolic ECM proteins, and diminished biosynthetic activity, resembling features of early-stage OA. These characteristics were observed for both Y201-C and HC at high and low concentrations of cytokines. Both HC and Y201-C demonstrated the suitability for the manufacturing of a scaffold-free in vitro OA model to facilitate studies into OA pathogenesis and therapeutic strategies. Our approach provides a faithful reproduction of early-stage osteoarthritis, demonstrating the ability of obtaining different disease severity by tuning the concentration of OA-related cytokines. Given the advantages in easy access and more reproducible performance, Y201-C may represent a more favourable source of chondrocytes for establishing more standardized protocols to obtain OA models.

A computational analysis of a novel therapeutic approach combining an advanced medicinal therapeutic device and a fracture fixation assembly for the treatment of osteoporotic fractures: Effects of physiological loading, interface conditions, and fracture fixation materials.

The occurrence of periprosthetic femoral fractures (PFF) has increased in people with osteoporosis due to decreased bone density, poor bone quality, and stress shielding from prosthetic implants. PFF treatment in the elderly is a genuine concern for orthopaedic surgeons as no effective solution currently exists. Therefore, the goal of this study was to determine whether the design of a novel advanced medicinal therapeutic device (AMTD) manufactured from a polymeric blend in combination with a fracture fixation plate in the femur is capable of withstanding physiological loads without failure during the bone regenerative process. This was achieved by developing a finite element (FE) model of the AMTD together with a fracture fixation assembly, and a femur with an implanted femoral stem. The response of both normal and osteoporotic bone was investigated by implementing their respective material properties in the model. Physiological loading simulating the peak load during standing, walking, and stair climbing was investigated. The results showed that the fixation assembly was the prime load bearing component for this configuration of devices. Within the fixation assembly, the bone screws were found to have the highest stresses in the fixation assembly for all the loading conditions. Whereas the stresses within the AMTD were significantly below the maximum yield strength of the device's polymeric blend material. Furthermore, this study also investigated the performance of different fixation assembly materials and found Ti-6Al-4V to be the optimal material choice from those included in this study.

Embracing additive manufacture: implications for foot and ankle orthosis design.

BACKGROUND: The design of foot and ankle orthoses is currently limited by the methods used to fabricate the devices, particularly in terms of geometric freedom and potential to include innovative new features. Additive manufacturing (AM) technologies, where objects are constructed via a series of sub-millimetre layers of a substrate material, may present the opportunity to overcome these limitations and allow novel devices to be produced that are highly personalised for the individual, both in terms of fit and functionality.Two novel devices, a foot orthosis (FO) designed to include adjustable elements to relieve pressure at the metatarsal heads, and an ankle foot orthosis (AFO) designed to have adjustable stiffness levels in the sagittal plane, were developed and fabricated using AM. The devices were then tested on a healthy participant to determine if the intended biomechanical modes of action were achieved. RESULTS: The adjustable, pressure relieving FO was found to be able to significantly reduce pressure under the targeted metatarsal heads. The AFO was shown to have distinct effects on ankle kinematics which could be varied by adjusting the stiffness level of the device. CONCLUSIONS: The results presented here demonstrate the potential design freedom made available by AM, and suggest that it may allow novel personalised orthotic devices to be produced which are beyond the current state of the art.

A Chondrosphere-Based Scaffold Free Approach to Manufacture an In Vitro Articular Cartilage Model.

In vitro engineering of human articular cartilage (AC) is a regenerative medicine challenge. The main objective of this study was the development of a repeatable scaffold-free in vitro model of chondrocyte spheroid-based treatments of cartilage defects, to allow for systematic study and further optimization of this type of treatment. Human articular chondrocytes (HC) and immortalized mesenchymal cells differentiated in chondrocytes (Y201-Cs) were cultured in round-bottom 96-well plates to produce multicellular spheroids and their growth kinetics, and viability was evaluated over 7 days of culture. Then, the spheroids were assembled and cultured for 21 days on a gelatin-coated poly(lactic-co-glycolic acid) electrospun membrane (10 spheroids/cm2), following a protocol in line with the clinically approved Chondrosphere® (CO.DON AG) technique. Both HC and Y201-C cells formed compact and viable spheroids after 7 days of culture with a reduction of diameter over the 7 days from 1300 ± 150 µm to 600 ± 90 µm and from 1250 ± 60 µm to 800 ± 20 µm for HC and Y201-C, respectively. When the spheroids were transferred onto the support membrane, these adhered on the membrane itself and fused themselves, producing collagen type II (COL2A1) and aggrecan (ACAN), according to gene expression and glycosaminoglycans quantification analyses. We detected higher expression of COL2A1 in HC cells, while the Y201-C constructs were characterized by an increased ACAN expression. The approach we presented allows a standardizable production of spheroids with predictable geometry and the creation of a reproducible scaffold-free in vitro AC-like construct showing high expression of chondrogenic markers, using both HC and Y201-C. In addition, the bankable Y201-C cells provide an effective base model for experimentation with the spheroid approach to further enhance the process. Impact statement This is first work on the development of a repeatable scaffold-free in vitro model based on an optimized protocol in line with a recent clinically approved Chondrosphere® (CO.DON AG) technique. In addition, we demonstrated that a bankable cell type (Y201-C) could produce an engineered cartilage-like construct, giving a repeatable model as a key tool for experimentation of therapeutic treatment ahead of studies with heterogeneous cell populations.

Valuable effect of Manuka Honey in increasing the printability and chondrogenic potential of a naturally derived bioink.

Hydrogel-based bioinks are the main formulations used for Articular Cartilage (AC) regeneration due to their similarity to chondral tissue in terms of morphological and mechanical properties. However, the main challenge is to design and formulate bioinks able to allow reproducible additive manufacturing and fulfil the biological needs for the required tissue. In our work, we investigated an innovative Manuka honey (MH)-loaded photocurable gellan gum methacrylated (GGMA) bioink, encapsulating mesenchymal stem cells differentiated in chondrocytes (MSCs-C), to generate 3D bioprinted construct for AC studies. We demonstrated the beneficial effect of MH incorporation on the bioink printability, leading to the obtainment of a more homogenous filament extrusion and therefore a better printing resolution. Also, GGMA-MH formulation showed higher viscoelastic properties, presenting complex modulus G∗ values of ∼1042 â€‹Pa, compared to ∼730 â€‹Pa of GGMA. Finally, MH-enriched bioink induced a higher expression of chondrogenic markers col2a1 (14-fold), sox9 (3-fold) and acan (4-fold) and AC ECM main element production (proteoglycans and collagen).

Fabrication routes via projection stereolithography for 3D-printing of microfluidic geometries for nucleic acid amplification.

Digital Light Processing (DLP) stereolithography (SLA) as a high-resolution 3D printing process offers a low-cost alternative for prototyping of microfluidic geometries, compared to traditional clean-room and workshop-based methods. Here, we investigate DLP-SLA printing performance for the production of micro-chamber chip geometries suitable for Polymerase Chain Reaction (PCR), a key process in molecular diagnostics to amplify nucleic acid sequences. A DLP-SLA fabrication protocol for printed micro-chamber devices with monolithic micro-channels is developed and evaluated. Printed devices were post-processed with ultraviolet (UV) light and solvent baths to reduce PCR inhibiting residuals and further treated with silane coupling agents to passivate the surface, thereby limiting biomolecular adsorption occurences during the reaction. The printed devices were evaluated on a purpose-built infrared (IR) mediated PCR thermocycler. Amplification of 75 base pair long target sequences from genomic DNA templates on fluorosilane and glass modified chips produced amplicons consistent with the control reactions, unlike the non-silanized chips that produced faint or no amplicon. The results indicated good functionality of the IR thermocycler and good PCR compatibility of the printed and silanized SLA polymer. Based on the proposed methods, various microfluidic designs and ideas can be validated in-house at negligible costs without the requirement of tool manufacturing and workshop or clean-room access. Additionally, the versatile chemistry of 3D printing resins enables customised surface properties adding significant value to the printed prototypes. Considering the low setup and unit cost, design flexibility and flexible resin chemistries, DLP-SLA is anticipated to play a key role in future prototyping of microfluidics, particularly in the fields of research biology and molecular diagnostics. From a system point-of-view, the proposed method of thermocycling shows promise for portability and modular integration of funcitonalitites for diagnostic or research applications that utilize nucleic acid amplification technology.

The interplay between chondrocyte spheroids and mesenchymal stem cells boosts cartilage regeneration within a 3D natural-based hydrogel.

Articular cartilage (AC) lacks the ability to self-repair and cell-based approaches, primarily based on using chondrocytes and mesenchymal stem cells (MSCs), are emerging as effective technology to restore cartilage functionality, because cells synergic functionality may support the maintenance of chondrogenic phenotype and promote extracellular matrix regeneration. This work aims to develop a more physiologically representative co-culture system to investigate the influence of MSCs on the activity of chondrocytes. A thermo-sensitive chitosan-based hydrogel, ionically crosslinked with ß-glycerophosphate, is optimised to obtain sol/gel transition at physiological conditions within 5 minutes, high porosity with pores diameter <30 µm, and in vitro mechanical integrity with compressive and equilibrium Young's moduli of 37 kPa and 17 kPa, respectively. Live/dead staining showed that after 1 and 3 days in culture, the encapsulated MSCs into the hydrogels are viable and characterised by round-like morphology. Furthermore chondrocyte spheroids, seeded on top of gels that contained either MSCs or no cells, show that the encapsulated MSCs stimulate chondrocyte activity within a gel co-culture, both in terms of maintaining the coherence of chondrocyte spheroids, leading to a larger quantity of CD44 (by immunofluorescence) and a higher production of collagen and glycosaminoglycans (by histology) compared with the mono-culture.

Multi-compartment scaffold fabricated via 3D-printing as in vitro co-culture osteogenic model.

The development of in vitro 3D models to get insights into the mechanisms of bone regeneration could accelerate the translation of experimental findings to the clinic, reducing costs and duration of experiments. This work explores the design and manufacturing of multi-compartments structures in poly(ε-caprolactone) (PCL) 3D-printed by Fused Filament Fabrication technique. The construct was designed with interconnected stalls to host stem cells and endothelial cells. Cells were encapsulated within an optimised gellan gum (GG)-based hydrogel matrix, crosslinked using strontium (Sr2+) ions to exploit its bioactivity and finally, assembled within compartments with different sizes. Calcium (Ca2+)-crosslinked gels were also used as control for comparison of Sr2+ osteogenic effect. The results obtained demonstrated that Sr2+ ions were successfully diffused within the hydrogel matrix and increased the hydrogel matrix strength properties under compressive load. The in vitro co-culture of human-TERT mesenchymal stem cells (TERT- hMSCs) and human umbilical vein endothelial cells (HUVECs), encapsulated within Sr2+ ions containing GG-hydrogels and inter-connected by compartmentalised scaffolds under osteogenic conditions, enhanced cell viability and supported osteogenesis, with a significant increase of alkaline phosphatase activity, osteopontin and osteocalcin respect with the Ca2+-crosslinked GG-PCL scaffolds. These outcomes demonstrate that the design and manufacturing of compartmentalised co-culture of TERT-hMSCs and HUVEC populations enables an effective system to study and promote osteogenesis.