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Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

University of Sydney

Injectable silk hydrogels are ideal carriers of therapeutic agents due to their biocompatibility and low immunogenicity. Injectable silk hydrogels for bone regeneration have been previously developed but often utilize expensive biologics. In this study, we have developed an injectable silk composite incorporated with a triphasic ceramic called MSM-10 (54 MgSiO, 36 SiSr and 10 MgO (wt%)) capable of simultaneously releasing magnesium, silicon, and strontium ions into its environment. These ions have been previously reported to possess therapeutic effects for bone regeneration. MSM-10 particles were incorporated into the silk hydrogels at various weight percentages [0.1 (SMH-0.1), 0.6 (SMH-0.6), 1 (SMH-1) and 2 (SMH-2)]. The effects of the released ions on the physicochemical and biological properties of the silk hydrogel were comprehensively evaluated. Increased MSM-10 loading was found to hinder the gelation kinetics of the silk hydrogel through the reduction of beta-sheet phase formation, which in turn affected the required sonication time for gelation, compressive strength, force of injection, microstructure and in vitro degradation rate. Primary human osteoblasts seeded on SMH-0.6 demonstrated increased proliferation and early alkaline phosphatase activity, as well as enhanced osteogenic gene expression compared to pure silk hydrogel and SMH-0.1. In vivo results in subcutaneous mouse models showed both decreased fibrous capsule formation and increased number of new blood vessels around the injected SMH-0.1 and SMH-0.6 implants compared to pure silk hydrogels. The results in this study indicate that the ions released from MSM-10 is able to influence the physicochemical and biological properties of silk hydrogels, and SMH-0.6 in particular shows promising properties for bone regeneration.

Modulatory effect of simultaneously released magnesium, strontium, and silicon ions on injectable silk hydrogels for bone regeneration.

The successful regeneration of functional bone tissue in critical-size defects remains a significant clinical challenge. To address this challenge, synthetic bone scaffolds are widely developed, but remarkably few are translated to the clinic due to poor performance in vivo. Here, it is demonstrated how architectural design of 3D printed scaffolds can improve in vivo outcomes. Ceramic scaffolds with different pore sizes and permeabilities, but with similar porosity and interconnectivity, are implanted in rabbit calvaria for 12 weeks, and then the explants are harvested for microcomputed tomography evaluation of the volume and functionality of newly formed bone. The results indicate that scaffold pores should be larger than 390 µm with an upper limit of 590 µm to enhance bone formation. It is also demonstrated that a bimodal pore topology-alternating large and small pores-enhances the volume and functionality of new bone substantially. Moreover, bone formation results indicate that stiffness of new bone is highly influenced by the scaffold's permeability in the direction concerned. This study demonstrates that manipulating pore size and permeability in a 3D printed scaffold architecture provides a useful strategy for enhancing bone regeneration outcomes.

Architectural Design of 3D Printed Scaffolds Controls the Volume and Functionality of Newly Formed Bone.

Here, we present a concise review of current 3D bioprinting technologies applied to induced pluripotent stem cells (iPSC). iPSC have recently received a great deal of attention from the scientific and clinical communities for their unique properties, which include abundant adult cell sources, ability to indefinitely self-renew and differentiate into any tissue of the body. Bioprinting of iPSC and iPSC derived cells combined with natural or synthetic biomaterials to fabricate tissue mimicked constructs, has emerged as a technology that might revolutionize regenerative medicine and patient-specific treatment. This review covers the advantages and disadvantages of bioprinting techniques, influence of bioprinting parameters and printing condition on cell viability, and commonly used iPSC sources, and bioinks. A clear distinction is made for bioprinting techniques used for iPSC at their undifferentiated stage or when used as adult stem cells or terminally differentiated cells. This review presents state of the art data obtained from major searching engines, including Pubmed/MEDLINE, Google Scholar, and Scopus, concerning iPSC generation, undifferentiated iPSC, iPSC bioprinting, bioprinting techniques, cartilage, bone, heart, neural tissue, skin, and hepatic tissue cells derived from iPSC.

iPSC Bioprinting: Where are We at?

In this review, we summarize the challenges of the three-dimensional (3D) printing of porous bioceramics and their translational hurdles to clinical applications. The state-of-the-art of the major 3D printing techniques (powder-based and slurry-based), their limitations and key processing parameters are discussed in detail. The significant roadblocks that prevent implementation of 3D printed bioceramics in tissue engineering strategies, and medical applications are outlined, and the future directions where new research may overcome the limitations are proposed. In recent years, there has been an increasing demand for a nanoscale control in 3D fabrication of bioceramic scaffolds via emerging techniques such as digital light processing, two-photon polymerization, or large area maskless photopolymerization. However, these techniques are still in a developmental stage and not capable of fabrication of large-sized bioceramic scaffolds; thus, there is a lack of sufficient data to evaluate their contribution. This review will also not cover polymer matrix composites reinforced with particulate bioceramics, hydrogels reinforced with particulate bioceramics, polymers coated with bioceramics and non-porous bioceramics.

3D Printing of Bioceramic Scaffolds-Barriers to the Clinical Translation: From Promise to Reality, and Future Perspectives.

Repair or regeneration of load-bearing bones has long been an incentive for the tissue engineering community to develop a plethora of synthetic bone scaffolds. Despite the key role of physical forces and the mechanical environment in bone regeneration, the mechanotransduction concept has rarely been incorporated in structural design of bone tissue scaffolds, particularly those made of bioactive materials such as hydrogels and bioceramics. Herein, we introduce a modular design strategy to fabricate a load bearing device that can support a wide range of hydrogel- and ceramic-based scaffolds against complex in-vivo loading conditions to induce desirable mechanical strains for bone regeneration within the scaffolds. The device is comprised of a fenestrated polymeric shell and ceramic structural pillars arranged in a sophisticated configuration to provide ample internal space for the scaffold, also enabling it to purposely regulate the levels of strains and stresses within the scaffolds. Utilizing this top-down design approach, we demonstrate that the failure load of alginate hydrogels increases 3200-fold in compression, 300-fold in shear and 75-fold in impact, achieving the values that enable them to withstand physiological loads in weight-bearing sites, while allowing generation of osteoinductive strains (i.e., 0.2-0.4%) in the hydrogel. This modular design approach opens a broad range of opportunities to utilize various bioactive but mechanically weak scaffolds for the treatment of load-bearing defects and exploiting mechanobiology strategies to improve bone regeneration.

A modular design strategy to integrate mechanotransduction concepts in scaffold-based bone tissue engineering.

Surface topography is one of the key factors in regulating interactions between materials and cells. While topographies presented to cells  are non-symmetrical and in complex shapes, current fabrication techniques are limited to replicate these complex geometries. In this study, we developed a microcasting technique and successfully produced imprinted hydroxyapatite (HAp) surfaces with nature-inspired (honeycomb, pillars, and isolated islands) topographies. The  biological performance of the developed non-symmetrical topographies was evaluated using adipose-derived stem cells (ADSCs). We demonstrated that ADSCs cultured on all HAp surfaces, except honeycomb patterns, presented well-defined stress fibers and expressed focal adhesion protein (paxillin) molecules. Isolated islands topographies significantly promoted osteogenic differentiation of ADSCs with increased alkaline phosphatase activity and upregulation of key osteogenic markers, compared to the other topographies and the control unmodified (flat) HAp surface. In contrast, honeycomb topographies hampered the ability of the ADSCs to proliferate and differentiate to the osteogenic lineage. This work presents a facile technique to imprint nature-derived topographies on the surface of bioceramics which opens up opportunities for the development of bioresponsive interfaces in tissue engineering and regenerative medicine.

Nature-inspired topographies on hydroxyapatite surfaces regulate stem cells behaviour.

Emerging concepts in bone repair and the premise of soft materials.

β-Tricalcium phosphate (β-TCP) has been extensively used in bone tissue engineering in the form of scaffolds, granules, or as reinforcing phase in organic matrices. Solid-state reaction route at high temperatures (>1000 °C) is the most widely used method for the preparation of β-TCP. The high-temperature synthesis, however, results in the formation of hard agglomerates and fused particles which necessitates postprocessing steps such as milling and sieving operations. This, inadvertently, could lead to introducing unwanted trace elements, promoting particle shape irregularity as well as compromising the biodegradability and bioactivity of β-TCP because of the solid microstructure of particles. In this study, we introduce a one-pot wet-chemical method at low temperatures (between 160 and 170 °C) to synthesize hollow β-TCP (hβ-TCP) submicron particles of an average size of 300 nm with a uniform rhombohedral shape. We assessed the cytocompatibility of the hβ-TCP using primary human osteoblasts (HOB), adipose-derived stem cells (ADSC), and antigen-presenting cells (APCs). We demonstrate the bioactivity of the hβ-TCP when cultured with HOB, ADSC, and APCs at a range of particle concentrations (up to 1000 μg/mL) for up to 7 days. hβ-TCP significantly enhances osteogenic differentiation of ADSC without the addition of osteogenic supplements. These findings offer a new type of β-TCP particles prepared at low temperatures, which present various opportunities for developing β-TCP based biomaterials.

Iman Roohani

School of Biomedical Engineering

University of Sydney

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Iman Roohani

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}School of Biomedical Engineering

University of Sydney

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

School of Biomedical Engineering