Three-dimensional printing (3DP) has emerged as a promising method for creating intricate scaffold designs. This study assessed three 3DP scaffold designs fabricated using biodegradable poly(lactic) acid (PLA) through fused deposition modelling (FDM): mesh, two channels (2C), and four channels (4C). To address the limitations of PLA, such as hydrophobic properties and poor cell attachment, a post-fabrication modification technique employing Polyelectrolyte Multilayers (PEMs) coating was implemented. The scaffolds underwent aminolysis followed by coating with SiCHA nanopowders dispersed in hyaluronic acid and collagen type I, and finally crosslinked the outermost coated layers with EDC/NHS solution to complete the hybrid scaffold production. The study employed rotating wall vessels (RWVs) to investigate how simulating microgravity affects cell proliferation and differentiation. Human mesenchymal stem cells (hMSCs) cultured on these scaffolds using proliferation medium (PM) and osteogenic media (OM), subjected to static (TCP) and dynamic (RWVs) conditions for 21 days, revealed superior performance of 4C hybrid scaffolds, particularly in OM. Compared to commercial hydroxyapatite scaffolds, these hybrid scaffolds demonstrated enhanced cell activity and survival. The pre-vascularisation concept on 4C hybrid scaffolds showed the proliferation of both HUVECs and hMSCs throughout the scaffolds, with a positive expression of osteogenic and angiogenic markers at the early stages.
The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.
Cell seeding via cell-laden hydrogels offers a rapid way of depositing cells onto a substrate or scaffold. When appropriately formulated, hydrogels provide a dense network of fibres for cellular encapsulation and attachment, creating a protective environment that prevents cells to be washed away by media. However, when incorporating hydrogels into a cell seeding strategy the cellular capacity for migration from a hydrogel network and subsequent biofunctionality must be assessed. Here, we compare cell seeding via a bioprinted hydrogel with conventional manual cell seeding in media. To this end, we use a binder jet 3D printed bioceramic scaffold as a model system for bone tissue engineering and the reactive jet impingement (ReJI) bioprinting system to deliver high cell density cell-laden hydrogels onto the surface of the scaffolds. The bioceramic scaffolds were produced in apatite-wollastonite (AW) glass-ceramic, with a total porosity of ~50 %, with pore size predominantly around 50-200 µm. Bone marrow-derived mesenchymal stromal cells were seeded onto the porous AW substrate both in media and via ReJI bioprinting. Cell seeding in media confirmed the osteoinductive nature and the ability of the scaffold to support cell migration within the porous structure. Cell seeding via ReJI bioprinting demonstrated that the cell-laden hydrogel penetrated the porous AW structure upon hydrogel deposition. Furthermore, cells would then migrate out from the hydrogel network and interact with the bioceramic substrate. Overall, levels of cell migration and mineralisation were significant and comparable for both seeding approaches. However, cell seeding via bioprinted hydrogels may serve as an effective strategy for in situ cell seeding into implants, which is desired in clinical tissue engineering procedures, avoiding the time taken for cell attachment from media, and the requirement to maintain a specific orientation until attachment has occurred.
Hydrogels , Tissue Engineering , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Porosity , Cell Movement
The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 µm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.
This work presents a computational model to study the degradation behavior of polyester-based three-dimensional (3D) functionalized scaffolds for bone regeneration. As a case study, we investigated the behavior of a 3D-printed scaffold presenting a functionalized surface with ICOS-Fc, a bioactive protein able to stimulate bone regeneration and healing, inhibiting osteoclast activity. The aim of the model was to optimize the scaffold design to control its degradation and thus the release of grafted protein over time and space. Two different scenarios were considered: (i) a scaffold without macroporosity presenting a functionalized external surface; and (ii) a scaffold presenting an internal functionalized macroporous architecture with open channels to locally deliver the degradation products.
Bone tissue engineering has emerged as a promising strategy to overcome the limitations of current treatments for bone-related disorders, but the trade-off between mechanical properties and bioactivity remains a concern for many polymeric materials. To address this need, novel polymeric blends of poly-L-lactic acid (PLLA), polycaprolactone (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have been explored. Blend filaments comprising PLLA/PCL/PHBV at a ratio of 90/5/5 wt% have been prepared using twin-screw extrusion. The PLLA/PCL/PHBV blends were enriched with nano-hydroxyapatite (nano-HA) and strontium-substituted nano-HA (Sr-nano-HA) to produce composite filaments. Three-dimensional scaffolds were printed by fused deposition modelling from PLLA/PCL/PHBV blend and composite filaments and evaluated mechanically and biologically for their capacity to support bone formation in vitro. The composite scaffolds had a mean porosity of 40%, mean pores of 800 µm, and an average compressive modulus of 32 MPa. Polymer blend and enriched scaffolds supported cell attachment and proliferation. The alkaline phosphatase activity and calcium production were significantly higher in composite scaffolds compared to the blends. These findings demonstrate that thermoplastic polyesters (PLLA and PCL) can be combined with polymers produced via a bacterial route (PHBV) to produce polymer blends with excellent biocompatibility, providing additional options for polymer blend optimization. The enrichment of the blend with nano-HA and Sr-nano-HA powders enhanced the osteogenic potential in vitro.
Bone adhesives offer distinct advantages over the use of screws to attached internal fixation plates (IFPs). As the chemical composition of bone is similar to dentine, it is possible that the types of monomers used to make dentine adhesives could be utilised to affix IFPs to bone. The ability to attach a bio-resorbable IFP to porcine bone was assessed for the monomer 10-methacryloyloxydecyl dihydrogen phosphate (MDP), used either as a homopolymer or a copolymer with urethane dimethacrylate (MDP + U). Additionally, the addition of a priming step (MDP + U + P) was evaluated. The chemical interactions of the monomers with bone were assessed using XRD and imaged using TEM, revealing the formation of nano-layered structures with the MDP primer, something we believe has not been reported on bone. In a 6-week artificial aging study both MDP + U and MDP + U + P demonstrated adequate shear bond strength to affix bio-resorbable IFPs. The cytotoxicity profiles of the adhesive formulations were determined using indirect and direct contact with MC3T3 cells, with indirect conditions suggesting the MDP + U + P is as cytocompatible as the resorbable IFP. The findings of this study suggest our newly developed adhesive has the potential to be used as a bone adhesive to affix bioresorbable IFPs.
PLLA, PCL and PHBV are aliphatic polyesters which have been researched and used in a wide range of medical devices, and all three have advantages and disadvantages for specific applications. Blending of these materials is an attractive way to make a material which overcomes the limitations of the individual polymers. Both PCL and PHBV have been evaluated in polymer blends with PLLA in order to provide enhanced properties for specific applications. This paper explores the use of PCL and PHBV together with PLLA in ternary blends with assessment of the thermal, mechanical and processing properties of the resultant polymer blends, with the aim of producing new biomaterials for orthopaedic applications. DSC characterisation is used to demonstrate that the materials can be effectively blended. Blending PCL and PHBV in concentrations of 5-10% with PLLA produces materials with average modulus improved by up to 25%, average strength improved by up to 50% and average elongation at break improved by 4000%, depending on the concentrations of each polymer used. PHBV impacts most on the modulus and strength of the blends, whilst PCL has a greater impact on creep behaviour and viscosity. Blending PCL and PHBV with PLLA offers an effective approach to the development of new polyester-based biomaterials with combinations of mechanical properties which cannot be provided by any of the materials individually.
Polyesters , Polymers , Biocompatible Materials , Viscosity
The surface of metal implants serves as a powerful signaling cue for cells. Its properties play an essential role in stabilizing the bone-implant interface and facilitating the early osseointegration by encouraging bone deposition on the surface. However, effective strategies to deliver cells to the metal surfaces are yet to be explored. Here, a bioprinting process, called reactive jet impingement (ReJI), is used to deposit high concentrations (4 × 107 cells mL-1 ) of mesenchymal stromal cells (MSCs) within hydrogel matrices directly onto the titanium alloy surfaces that vary in surface roughness and morphology. In this proof-of-concept study, cell-hydrogel-metal systems are fabricated with the aim of enhancing bioactivity through delivering MSCs in hydrogels at the bone-implant interface. These results show that the high cell concentrations encourage quick cell-biomaterial interactions at the hydrogel-metal surface interface, and cell morphology is influenced by the surface type. Cells migrate from the hydrogels and deposit mineralized matrix rich in calcium and phosphorus on the titanium alloy surfaces. The authors demonstrate that ReJI bioprinting is a promising tool to deliver cells in a 3D environment before implantation that can be used when developing a new generation of medical devices for bone tissue engineering.
Bioprinting , Alloys , Bioprinting/methods , Hydrogels/pharmacology , Osseointegration , Titanium/pharmacology
Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof-of-concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures to investigate whether the use of MSCs in ACI procedures would stimulate enhanced ECM production by chondrocytes. Microvalve-based bioprinting uses small-scale solenoid valves (microvalves) to deposit cells suspended in media in a consistent and repeatable manner. In this case, MSCs and chondrocytes have been sequentially printed into an insert-based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate ECM production. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This study, therefore, provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.
Bioprinting , Chondrocytes/cytology , Mesenchymal Stem Cells/cytology , Cell Line , Cell Proliferation , Cell Shape , Cell Survival , Coculture Techniques , Humans , Models, Biological
Drop-on-demand (DoD) inkjet printing has been explored for a range of applications, including those to selectively deposit cellular material, due to the high accuracy and scalability of such systems when compared with alternative bioprinting techniques. Despite this, there remain considerable limitations when handling cell suspensions due to the agglomeration and sedimentation of cells during printing, leading to a deterioration in jetting performance. The objective of this work was to design and assess the effectiveness of a custom agitation system to maintain cellular dispersion within the ink reservoir during printing. The cell printing performance of an inkjet printer was assessed with and without the use of a custom agitation system, with biological characterisation performed to characterise the impact of the agitator on cellular viability and function. Cell printing performance was retained over a 2 h printing period when incorporating an agitated reservoir, with a gradual reduction in performance observed under a non-agitated configuration. Cell assays indicated that the agitation process did not significantly affect the viability, metabolic activity or morphology of the mesenchymal stromal cell (MSC) or chondrocyte cell types. This study therefore provides a new methodology to increase process reliability within DoD printing platforms when jetting cellularised material.
Bioprinting , Chondrocytes , Cell Survival , Printing, Three-Dimensional , Reproducibility of Results
The clinical application of composites seeks to exploit the mechanical and chemical properties of materials which make up the composite, and in researching polymer composites for biomedical applications the aim is usually to enhance the bioactivity of the polymer, while maintaining the mechanical properties. To that end, in this study medical grade Poly(L-lactic) acid (PLLA) has been reinforced with short phosphate-based glass fibers (PGF). The materials were initially mixed by melting PLLA granules with the short fibers, before being extruded to form a homogenous filament, which was pelletized and used as feedstock for compression moulding. As made the composite materials had a bending strength of 51â¯MPa⯱â¯5, and over the course of eight weeks in PBS the average strength of the composite material was in the range 20-50â¯MPa. Human mesenchymal stromal cells were cultured on the surfaces of scaffolds, and the metabolic activity, alkaline phosphatase production and mineralization monitored over a three week period. The short fiber filler made no significant difference to cell proliferation or differentiation, but had a clear and immediate osteoinductive effect, promoting mineralization by cells at the material surface. It is concluded that the PLLA/PGF composite material offers a material with both the mechanical and biological properties for potential application to bone implants and fixation, particularly where an osteoinductive effect would be valuable.
Calcification, Physiologic/drug effects , Glass/chemistry , Phosphates/pharmacology , Polyesters/pharmacology , Biological Assay , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cell Shape/drug effects , Cell Survival/drug effects , Humans , Hydrogen-Ion Concentration , Ions , Osteogenesis/drug effects , Proton Magnetic Resonance Spectroscopy , X-Ray Diffraction
Over the past few decades, there has been growing interest in understanding the molecular mechanisms of cancer pathogenesis and progression, as it is still associated with high morbidity and mortality. Current management of large bone sarcomas typically includes the complex therapeutic approach of limb salvage or sacrifice combined with pre- and postoperative multidrug chemotherapy and/or radiotherapy, and is still associated with high recurrence rates. The development of cellular strategies against specific characteristics of tumour cells appears to be promising, as they can target cancer cells selectively. Recently, Mesenchymal Stromal Cells (MSCs) have been the subject of significant research in orthopaedic clinical practice through their use in regenerative medicine. Further research has been directed at the use of MSCs for more personalized bone sarcoma treatments, taking advantage of their wide range of potential biological functions, which can be augmented by using tissue engineering approaches to promote healing of large defects. In this review, we explore the use of MSCs in bone sarcoma treatment, by analyzing MSCs and tumour cell interactions, transduction of MSCs to target sarcoma, and their clinical applications on humans concerning bone regeneration after bone sarcoma extraction.
[This corrects the article DOI: 10.1186/s42490-019-0015-y.].
BACKGROUND: Mesenchymal stromal cells (MSCs) are widely used in clinical trials for bone repair and regeneration. Despite previous evidence showing a prominent osteogenic potential of 2D cultured CD271 enriched MSCs, the osteogenic potential of CD271 enriched cells cultured on 3D scaffold is unknown. Apatite-wollastonite glass ceramic (A-W) is an osteoconductive biomaterial shown to be compatible with MSCs. This is the first study comparing the attachment, growth kinetics, and osteogenic potential of two MSC populations, namely heterogeneous plastic adherence MSCs (PA-MSCs) and CD271-enriched MSCs (CD271-MSCs), when cultured on A-W 3D scaffold. RESULTS: The paired MSC populations were assessed for their attachment, growth kinetics and ALP activity using confocal and scanning electron microscopy and the quantifications of DNA contents and p-nitrophenyl (pNP) production respectively. While the PA-MSCs and CD271-MSCs had similar expansion and tri-lineage differentiation capacity during standard 2D culture, they showed different proliferation kinetics when seeded on the A-W scaffolds. PA-MSCs displayed a well-spread attachment with more elongated morphology compared to CD271- MSCs, signifying a different level of interaction between the cell populations and the scaffold surface. Following scaffold seeding PA-MSCs fully integrated into the scaffold surface and showed a stronger propensity for osteogenic differentiation as indicated by higher ALP activity than CD271-MSCs. Furthermore, A-W scaffold seeded uncultured non-enriched bone marrow mononuclear cells also demonstrated a higher proliferation rate and greater ALP activity compared to their CD271-enriched counterpart. CONCLUSIONS: Our findings suggest that CD271-positive enrichment of a population is not beneficial for osteogenesis when the cells are seeded on A-W scaffold. Furthermore, unselected heterogeneous MSCs or BMMNCs are more promising for A-W scaffold based bone regeneration. This leads to a conclusion of broader clinical relevance for tissue engineering: on the basis of our observations here the osteogenic potential observed in 2D cell culture should not be considered indicative of likely performance in a 3D scaffold based system, even when one of the cell populations is effectively a subset of the other.
Advances in three-dimensional cell cultures offer new opportunities in biomedical research and drug development. However, there are still challenges to overcome, including the lack of reliability, repeatability and complexity of tissues obtained by these techniques. In this study, we describe a new bioprinting system called reactive jet impingement (ReJI) for the bioprinting of cell-laden hydrogels. Droplets of gel precursor solutions are jetted at one another such that they meet and react in mid-air before the gel droplets fall to the substrate. This technique offers a combination of deposition rate, cell density and cell viability which is not currently matched by any other bioprinting technique. The importance of cell density is demonstrated in the development of bone microtissues derived from immortalised human bone marrow stem cells. The cells were printed with high viability within a collagen-alginate-fibrin gel, and tissue specific gene expression shows significantly higher tissue maturation rates using the ability of the ReJI system to deposit gels with a high cell density.
Bioprinting/methods , Bone Marrow Cells/cytology , Bone and Bones/cytology , Hydrogels/chemistry , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Alginates/chemistry , Bioprinting/instrumentation , Cell Count , Cell Survival , Collagen/chemistry , Humans , Printing, Three-Dimensional , Tissue Engineering/instrumentation
Control of cell migration is fundamental to the performance of materials for cell delivery, as for cells to provide any therapeutic effect, they must migrate out from the delivery material. Here the influence of fibrinogen concentration on the migration of encapsulated human mesenchymal stem cells (hMSCs) from a cell spheroid through fibrin hydrogels is tracked over time. Fibrin was chosen as a model material as it is routinely employed as a haemostatic agent and more recently has been applied as a localised delivery vehicle for potential therapeutic cell populations. The hydrogels consisted of 5 U/mL thrombin and between 5 and 50 mg/mL fibrinogen. Microstructural and viscoelastic properties of different compositions were evaluated using SEM and rheometry. Increasing the fibrinogen concentration resulted in a visibly denser matrix with smaller pores and higher stiffness. hMSCs dispersed within the fibrin gels maintained cell viability post-encapsulation, however, the migration of cells from an encapsulated spheroid revealed that denser fibrin matrices inhibit cell migration. This study provides the first quantitative study on the influence of fibrinogen concentration on 3D hMSC migration within fibrin gels, which can be used to guide material selection for scaffold design in tissue engineering and for the clinical application of fibrin sealants.
A novel apatite-wollastonite/poly(lactic acid) (AW/PLA) composite structure, which matches cortical and cancellous bone properties has been produced and evaluated in vitro and in vivo. The composites structure has been produced using an innovative combination of 3D printed polymer and ceramic macrostructures, thermally bonded to create a hybrid composite structure. In vitro cell assays demonstrated that the AW structure alone, PLA structure alone, and AW/PLA composite were all biocompatible, with the AW structure supporting the proliferation and osteogenic differentiation of rat bone marrow stromal cells. Within a rat calvarial defect model the AW material showed excellent osseointegration with the formation of new bone, and vascularisation of the porous AW structure, both when the AW was implanted alone and when it was part of the AW/PLA composite structure. However, the AW/PLA structure showed the largest amount of the newly formed bone in vivo, an effect which is considered to be a result of the presence of the osteoinductive AW structure stimulating bone growth in the larger pores of the adjacent PLA structure. The layered AW/PLA structure showed no signs of delamination in any of the in vitro or in vivo studies, a result which is attributed to good initial bonding between polymer and ceramic, slow resorption rates of the two materials, and excellent osseointegration. It is concluded that macro-scale composites offer an alternative route to the fabrication of bioactive bone implants which can provide a match to both cortical and cancellous bone properties over millimetre length scales.
Apatites/chemistry , Biocompatible Materials/chemistry , Calcium Compounds/chemistry , Osseointegration/physiology , Polyesters/chemistry , Printing, Three-Dimensional , Silicates/chemistry , Animals , Biocompatible Materials/pharmacology , Cells, Cultured , Male , Osseointegration/drug effects , Porosity , Rats , Rats, Sprague-Dawley
This paper presents an investigation of how different culture media (i.e. basal and osteogenic media) affect the nanomechanical properties and microstructure of the mineralized matrix produced by the human mesenchymal stem cell line Y201, from both an experimental and theoretical approach. A bone nodule (i.e. mineralized matrix) cultured from basal medium shows a more anisotropic microstructure compared to its counterpart cultured from an osteogenic medium. As confirmed by finite element simulations, this anisotropic microstructure explains the bimodal distribution of the corresponding mechanical properties very well. The overall nanomechanical response of the bone nodule from the osteogenic medium is poorer compared to its counterpart from the basal medium. The bone nodules, from both basal and osteogenic media, have shown reverse aging effects in terms of mechanical properties. These are possibly due to the fact that cell proliferation outcompetes the mineralization process.
Cell Culture Techniques/methods , Extracellular Matrix/metabolism , Models, Biological , Nanostructures/chemistry , Cell Culture Techniques/instrumentation , Cell Differentiation , Cell Line , Elastic Modulus , Extracellular Matrix/chemistry , Extracellular Matrix/ultrastructure , Finite Element Analysis , Humans , Mesenchymal Stem Cells/cytology , Osteogenesis , Surface Properties
Three novel glass compositions, identified as NCL2 (SiO2-based), NCL4 (B2O3-based) and NCL7 (SiO2-based), along with apatite-wollastonite (AW) were processed to form sintered dense pellets, and subsequently evaluated for their in vitro bioactive potential, resulting physico-chemical properties and degradation rate. Microstructural analysis showed the carbonated hydroxyapatite (HCA) precipitate morphology following SBF testing to be composition-dependent. AW and the NCL7 formulation exhibited greater HCA precursor formation than the NCL2 and NCL4-derived pellets. Moreover, the NCL4 borate-based samples showed the highest biodegradation rate; with silicate-derived structures displaying the lowest weight loss after SBF immersion. The results of this study suggested that glass composition has significant influence on apatite-forming ability and also degradation rate, indicating the possibility to customise the properties of this class of materials towards the bone repair and regeneration process.
