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Autograft or metal implants are routinely used in skeletal repair but can fail to provide a long-term clinical resolution, emphasising the need for a functional biomimetic tissue engineering alternative. An attractive sustainable opportunity for tissue regeneration would be the application of human bone waste tissue for the synthesis of a material ink for 3D bioprinting of skeletal tissue.The use of human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate micro-environment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. Extrusion-based deposition was mediated by the blending of human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymer, to engineer a novel material ink (LAB). The inclusion of nanofiller and polymeric material increased the rheological, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularisation following implantation in anex vivochick chorioallantoic membrane (CAM) model. Addition of bone morphogenetic protein-2 (BMP-2) with HBMSCs further enhanced vascularisation together with mineralisation after only 7 days.The current study demonstrates the synergistic combination of nanoclay with biomimetic materials, (alginate and bone-ECM) to support the formation of osteogenic tissue bothin vitroandex vivoand offers a promising novel 3D bioprinting approach to personalised skeletal tissue repair.Graphical AbstractEngineering nanoclay-based bone ECM novel bioink for bone regeneration. Human bone trabecular tissue was demineralised, decellularised and blended with nanoclay (Laponite®) and alginate after digestion. The resulting ink was investigated for printability following rheological and filament fusion investigation. The microstructural arrangement of the blends was examined together with viability and functionality of bioprinted HBMSCs. Finally, the ability of the novel blend to support drug release ex vivo in a CAM model was determined confirming the potential of the bone ECM ink to support bone formation.

Synthetic nanostructured materials incorporating both organic and inorganic components offer a unique, powerful, and versatile class of materials for widespread applications due to the distinct, yet complementary, nature of the intrinsic properties of the different constituents. We report a supramolecular system based on synthetic nanoclay (Laponite,bLap/b) and peptide amphiphiles (PAs,bPAH3/b) rationally designed to coassemble into nanostructured hydrogels with high structural integrity and a spectrum of bioactivities. Spectroscopic and scattering techniques and molecular dynamic simulation approaches were harnessed to confirm thatbPAH3/bnanofibers electrostatically adsorbed and conformed to the surface ofbLap/bnanodisks. Electron and atomic force microscopies also confirmed an increase in diameter and surface area ofbPAH3/bnanofibers after coassembly withbLap/b. Dynamic oscillatory rheology revealed that the coassembledbPAH3-Lap/bhydrogels displayed high stiffness and robust self-healing behavior while gas adsorption analysis confirmed a hierarchical and heterogeneous porosity. Furthermore, this distinctive structure within the three-dimensional (3D) matrix provided spatial confinement for the nucleation and hierarchical organization of high-aspect ratio hydroxyapatite nanorods into well-defined spherical clusters within the 3D matrix. Applicability of the organic-inorganicbPAH3-Lap/bhydrogels was assessediin vitro/iusing human bone marrow-derived stromal cells (hBMSCs) andiex vivo/iusing a chick chorioallantoic membrane (CAM) assay. The results demonstrated that the organic-inorganicbPAH3-Lap/bhydrogels promote human skeletal cell proliferation and, upon mineralization, integrate with the CAM, are infiltrated by blood vessels, stimulate extracellular matrix production, and facilitate extensive mineral deposition relative to the controls.

The past decade has seen a growing appreciation for the role of the innate immune response in mediating repair and biomaterial directed tissue regeneration. The long-held view of the host immune/inflammatory response as an obstacle limiting stem cell regenerative activity, has given way to a fresh appreciation of the pivotal role the macrophage plays in orchestrating the resolution of inflammation and launching the process of remodelling and repair. In the context of bone, work over the past decade has established an essential coordinating role for macrophages in supporting bone repair and sustaining biomaterial driven osteogenesis. In this review evidence for the role of the macrophage in bone regeneration and repair is surveyed before discussing recent biomaterial and drug-delivery based approaches that target macrophage modulation with the goal of accelerating and enhancing bone tissue regeneration.

Synthetic clays are promising biomaterials for delivery of therapeutic molecules in regenerative medicine. However, before their use can be translated into clinical applications, their safety must be assessed in human volunteers. The aim of this study was to test the hypothesis that a synthetic nanoclay (LAPONITE) does not cause irritation to the human skin. To achieve this, a nanoclay gel at two different concentrations (1.5 and 3% w/v) was applied on the forearm of healthy volunteers for 24 h. 1% sodium lauryl sulfate (SLS) and 3% (w/v) polyacrylic acid were used as the positive and negative controls, respectively. The compromise in the skin barrier function was measured by trans-epidermal water loss (TEWL), erythema by spectroscopic measurements, and skin inflammatory biomarkers (IL-1α and IL-1RA) by the enzyme-linked immunosorbent assay. We found that the nanoclay caused no prolonged increase in TEWL, erythema, or induction of inflammatory cytokines. This was in contrast to 1% SLS, a known irritant, which induced significant increases in both skin erythema and TEWL. We conclude that the nanoclay is not an irritant and is thus suitable for therapeutic interventions at the skin surface.

LAPONITE® clay nanoparticles are known to exert osteogenic effects on human bone marrow stromal cells (HBMSCs), most characteristically, an upregulation in alkaline phosphatase activity and increased calcium deposition. The specific properties of LAPONITE® that impart its bioactivity are not known. In this study the role of lithium, a LAPONITE® degradation product, was investigated through the use of lithium salts and lithium modified LAPONITE® formulations. In contrast to intact particles, lithium ions applied at concentrations equivalent to that present in LAPONITE®, failed to induce any significant increase in alkaline phosphatase (ALP) activity. Furthermore, no significant differences were observed in ALP activity with modified clay structures and the positive effect on osteogenic gene expression did not correlate with the lithium content of modified clays. These results suggest that other properties of LAPONITE® nanoparticles, and not their lithium content, are responsible for their bioactivity.

Nanoclays have generated interest in biomaterial design for their ability to enhance the mechanics of polymeric materials and impart biological function. As well as their utility as physical cross-linkers, clays have been explored for sustained localization of biomolecules to promote in vivo tissue regeneration. To date, both biomolecule-clay and polymer-clay nanocomposite strategies have utilised the negatively charged clay particle surface. As such, biomolecule-clay and polymer-clay interactions are set in competition, potentially limiting the functional enhancements achieved. Here, we apply specific bisphosphonate interactions with the positively charged clay particle edge to develop self-assembling hydrogels and functionalized clay nanoparticles with preserved surface exchange capacity. Low concentrations of nanoclay are applied to cross-link hyaluronic acid polymers derivatised with a pendant bisphosphonate to generate hydrogels with enhanced mechanical properties and preserved protein binding able to sustain, for over six weeks in vivo, the localized activity of the clinically licensed growth factor BMP-2., Nanoclays have been used in composites and for drug delivery but have suffered from a trade-off in properties when used for both. Here the authors report on the use of bisphosphonate interactions with nanoclay edges to made drug loaded composites without compromising materials properties or drug loading.

Organ dysfunction is a major cause of morbidity and mortality. Transplantation is typically the only definitive cure, challenged by the lack of sufficient donor organs. Tissue engineering encompasses the development of biomaterial scaffolds to support cell attachment, proliferation, and differentiation, leading to tissue regeneration. For efficient clinical translation, the forming technology utilized must be suitable for mass production. Herein, uniaxial polyhydroxyalkanoate scaffolds manufactured by pressurized gyration, a hybrid scalable spinning technique, are successfully used in bone, nerve, and cardiovascular applications. Chorioallantoic membrane and in vivo studies provided evidence of vascularization, collagen deposition, and cellular invasion for bone tissue engineering. Highly efficient axonal outgrowth was observed in dorsal root ganglion-based 3D ex vivo models. Human induced pluripotent stem cell derived cardiomyocytes exhibited a mature cardiomyocyte phenotype with optimal calcium handling. This study confirms that engineered polyhydroxyalkanoate-based gyrospun fibers provide an exciting and unique toolbox for the development of scalable scaffolds for both hard and soft tissue regeneration.

Biofabrication is revolutionizing substitute tissue manufacturing. Skeletal stem cells (SSCs) can be blended with hydrogel biomaterials and printed to form three-dimensional structures that can closely mimic tissues of interest. Our bioink formulation takes into account the potential for cell printing including a bioink nanocomposite that contains low fraction polymeric content to facilitate cell encapsulation and survival, while preserving hydrogel integrity and mechanical properties following extrusion. Clay inclusion to the nanocomposite strengthens the alginate-methylcellulose network providing a biopaste with unique shear-thinning properties that can be easily prepared under sterile conditions. SSCs can be mixed with the clay-based paste, and the resulting bioink can be printed in 3D structures ready for implantation. In this chapter, we provide the methodology for preparation, encapsulation, and printing of SSCs in a unique clay-based bioink.

Biofabrication is revolutionizing substitute tissue manufacturing. Skeletal stem cells (SSCs) can be blended with hydrogel biomaterials and printed to form three-dimensional structures that can closely mimic tissues of interest. Our bioink formulation takes into account the potential for cell printing including a bioink nanocomposite that contains low fraction polymeric content to facilitate cell encapsulation and survival, while preserving hydrogel integrity and mechanical properties following extrusion. Clay inclusion to the nanocomposite strengthens the alginate-methylcellulose network providing a biopaste with unique shear-thinning properties that can be easily prepared under sterile conditions. SSCs can be mixed with the clay-based paste, and the resulting bioink can be printed in 3D structures ready for implantation. In this chapter, we provide the methodology for preparation, encapsulation, and printing of SSCs in a unique clay-based bioink.

Functionalized scaffolds hold promise for stem cell therapy by controlling stem cell fate and differentiation potential. Here, we have examined the potential of a 2-dimensional (2D) scaffold to stimulate bone regeneration. Solubilized extracellular matrix (ECM) from human bone tissue contains native extracellular cues for human skeletal cells that facilitate osteogenic differentiation. However, human bone ECM displays limited mechanical strength and degradation stability under physiological conditions, necessitating modification of the physical properties of ECM before it can be considered for tissue engineering applications. To increase the mechanical stability of ECM, we explored the potential of synthetic Laponite® (LAP) clay as a counter material to prepare a 2D scaffold using Layer-by-Layer (LbL) self-assembly. The LAP and ECM multilayer nanofilms (ECM/LAP film) were successfully generated through electrostatic and protein-clay interactions. Furthermore, to enhance the mechanical properties of the ECM/LAP film, application of a NaCl solution wash step, instead of deionized water following LAP deposition resulted in the generation of stable, multi-stacked LAP layers which displayed enhanced mechanical properties able to sustain human skeletal progenitor cell growth. The ECM/LAP films were not cytotoxic and, critically, showed enhanced osteogenic differentiation potential as a consequence of the synergistic effects of ECM and LAP. In summary, we demonstrate the fabrication of a novel ECM/LAP nanofilm layer material with potential application in hard tissue engineering.

Gels made from clay could provide an environment able to stimulate stem-cells due to their ability to bind biological molecules. That molecules stick to clay has been known by scientists since the 1960s. Doctors observed that absorption into the blood stream of certain drugs was severely reduced when patients were also receiving clay-based antacid or anti-diarrhoeal treatments. This curious phenomenon was realized to be due to binding of the drugs by clay particles. This interaction is now routinely harnessed in the design of tablets to carefully control the release and action of a drug. Dr Dawson now proposes to use this property of clay to create micro-environments that could stimulate stem cells to regenerate damaged tissues such as bone, cartilage or skin. The rich electrostatic properties of nano (1 millionth of a millimetre) -scale clay particles which mediate these interactions could allow two hurdles facing the development of stem-cell based regenerative therapies to be overcome simultaneously. The first challenge - to deliver and hold stem cells at the right location in the body - is met by the ability of clays to self-organise into gels via the electrostatic interactions of the particles with each other. Cells mixed with a low concentration (less than 4%) of clay particles can be injected into the body and held in the right place by the gel, eliminating, in many situations, the need for surgery. Clay particles can also interact with large structural molecules (polymers) which are frequently used in the development of materials (or 'scaffolds'), designed to host stem cells. These interactions can greatly improve the strength of such structures and could be applied to preserve their stability at the site of injury until regeneration is complete. While several gels and scaffold materials have been designed to deliver and hold stem cells at the site of regeneration, the ability of clay nanoparticles to overcome a second critical hurdle facing stem-cell therapy is what makes them especially exciting. Essential to directing the activity of stem-cells is the carefully controlled provision of key biological signalling molecules. However, the open structures of conventional scaffolds or gels, while essential for the diffusion of nutrients to the cells, means their ability to hold the signalling molecules in the same location as the cells is limited. The ability of clay nano-particles to bind biological molecules presents a unique opportunity to create local environments at a site of injury or disease that can stimulate and control stem-cell driven repair. Dr Dawson's early studies investigated the ability of clay gels to stimulate the growth of new blood vessels by incorporating a key molecular signal that stimulates this process, vascular endothelial growth factor (VEGF). In a manner reminiscent of the observations made in the 60s, Dr Dawson and colleagues observed that adding a drop of clay gel to a solution containing VEGF caused, after a few hours, the disappearance of VEGF from the solution as it became bound to the gel. When placed in an experimental injury model, the gel-bound VEGF stimulated a cluster of new blood vessels to form. These exciting results indicate the potential of clay nanoparticles to create tailor-made micro-environments to foster stem cell regeneration. Dr Dawson is developing this approach as a means of first exploring the biological signals necessary to successfully control stem cell behaviour for regeneration and then, using the same approach, to provide stem cells with these signals to stimulate regeneration in the body. The project will seek to test this approach to regenerate bone lost to cancer or hip replacement failure. If successful the same technology may be applied to harness stem cells for the treatment of a whole host of different scenarios, from burn victims to those suffering with diabetes or Parkinson's.

Free-form printing offers a novel biofabrication approach to generate complex shapes by depositing hydrogel materials within a temporary supportive environment. However, printed hydrogels typically lack the requisite mechanical properties and functionality of the desired tissue, limiting application and, more importantly, safety and efficacy of the implant. The study authors have developed an innovative nanoclay-based bioink to print high shape fidelity functional constructs for potential skeletal application. Laponite® (LAP) nanoclay was combined with gellan gum (GG) to generate a printable hydrogel that was highly stable in vitro, displayed limited swelling ability compared with the silicate-free control and remained stable over time. An agarose fluid gel was found to provide the requisite support for the deposition of the material ink and preservation of the printed structure before crosslinking. Printed C2C12 myoblasts remained viable and displayed extensive proliferation over 21 days in culture. Cell-laden scaffolds demonstrated functionality within 1 day of culture in vitro and that was preserved over 3 weeks. Analysis of absorption and release mechanisms from LAP-GG using model proteins (lysozyme and bovine serum albumin) demonstrated the retention capability of the clay-based materials for compound localisation and absence of burst release. Vascular endothelial growth factor ​was loaded within the agarose fluid gel and absorbed by the material ink via absorption during deposition. The 3D-printed constructs were implanted on the chorioallantoic membrane of a 10-day-old developing chick. Extensive and preferential vasculature infiltration was observed in LAP-GG–loaded vascular endothelial growth factor constructs compared with controls (p, Graphical abstract LAP-GG printing in agarose fluid gel for skeletal tissue engineering. Extrusion of LAP-GG in an agarose bed to generate three-dimensional structures (a). Low polymeric content nanosilicate bioink was used to deliver cells and preserve their viability after extrusion. Agarose fluid gel was harnessed to enable loading of compounds of interests during printing (b) in LAP-GG and GG controls. LAP-GG bioink offers the potential for enhanced compound penetration within the printed strand. LAP-GG extrusion within self-healing agarose can be used to fabricate large functional structures (c) such as 3D sleeve (i and ii) and 3D lattice (iii and iv) structures to fill critical bone defects. Cells were printed in LAP-GG (d) and viability (i and ii) and functionality (iii and iv) were assessed for skeletal tissue formation potential. Integration with chick membrane model and vascular penetration (e) of printed scaffolds implanted within the vascularised membrane of a developing chick. Scale bar: (a) 5 ​mm, (b—i and ii) 100 ​μm, (c—i and ii) 30 ​mm, (c—iii) 30 ​mm, (c—iv) 10 ​mm, (d—i) 100 ​μm, (d—iii and iv) 2 ​mm.Image 1

Recent advances in regenerative medicine have confirmed the potential to manufacture viable and effective tissue engineering 3D constructs comprising living cells for tissue repair and augmentation. Cell printing has shown promising potential in cell patterning in a number of studies enabling stem cells to be precisely deposited as a blueprint for tissue regeneration guidance. Such manufacturing techniques, however, face a number of challenges including; (i) post-printing cell damage, (ii) proliferation impairment and, (iii) poor or excessive final cell density deposition. The use of hydrogels offers one approach to address these issues given the ability to tune these biomaterials and subsequent application as vectors capable of delivering cell populations and as extrusion pastes. While stem cell-laden hydrogel 3D constructs have been widely established in vitro, clinical relevance, evidenced by in vivo long-term efficacy and clinical application, remains to be demonstrated. This review explores the central features of cell printing, cell-hydrogel properties and cell-biomaterial interactions together with the current advances and challenges in stem cell printing. A key focus is the translational hurdles to clinical application and how in vivo research can reshape and inform cell printing applications for an ageing population., Graphical abstract Cell printing - capacity and limitations. Cell type and density are important factors in the printing of living cells. Post-printing consequences including: (i) impaired cell proliferation, (ii) maintenance of phenotype and genotype, (iii) preservation of cell integrity and morphology are issues that need to be considered. Stem cell printing aims to deposit cells in three dimensions within an environment conducive to proliferation and differentiation. Therefore, long-term investigations of cell viability and proliferation in vitro and in vivo are required to elucidate construct maturation and effective tissue regeneration and integration. Importantly, the properties of the hydrogel cell carrier (biocompatibility, bioactivity, physical characteristics) for the select 3D print approach envisaged are crucial for cell encapsulation, protection and support during differentiation and proliferation.Image 1

The retention and sustained activity of therapeutic proteins at delivery sites are goals of regenerative medicine. Vascular endothelial growth factor (VEGF) has significant potential in promoting the growth and regeneration of blood vessels but is intrinsically labile. This is exacerbated by the inflammatory microenvironments at sites requiring regeneration. For VEGF to be efficacious, it may require a carrier that stabilises it, protects it from degradation and retains it at the site of interest. In this study, we tested the hypothesis that injectable nanoclay gels comprising Laponite™ XLG (a synthetic hectorite clay) can stabilise VEGF and retain it in the active form for therapeutic delivery. To achieve this, VEGF was incorporated in Laponite gels and its activity tested at a range of concentrations using in vitro cell culture tubulogenesis assays and in vivo angiogenesis assays. We found that VEGF-Laponite gels enhanced tubulogenesis in a dose-dependent manner in vitro. When administered subcutaneously in vivo, Laponite was retained at the injection site for up to a period of three weeks and promoted a 4-fold increase in blood vessel formation compared with that of alginate or vehicle controls as confirmed by CD31 staining. Notably, as compared to alginate, Laponite gels did not release VEGF, indicating a strong interaction between the growth factor and the nanoclay and suggesting that Laponite enhancement of VEGF efficacy is due to its retention at the implantation site for a prolonged period. Our approach provides a robust method for the delivery of bioactive recombinant VEGF without the necessity for complex hydrogel or protein engineering. STATEMENT OF SIGNIFICANCE: In medicine, it is important to deliver drugs to a particular location in the body. Often, however, the drugs are quickly broken down and carried away in the blood before they can exert their effect. In this study, we used a type of synthetic clay, called Laponite™, to preserve a molecule, named VEGF, that stimulates the growth of blood vessels. Previously, we have been able to bind VEGF to the surface of clays, but the clay is not effective when injected or applied as a gel. Herein, we show that we can mix VEGF with the clay and that it strongly stimulates blood vessel growth. We speculate that this would be a useful material for skin wound healing.

Bone Morphogenic Protein 2 (BMP2) can induce ectopic bone. This ability, which first motivated the widespread application of BMP2 in fracture healing and spinal arthrodesis has, more recently, been indicated as one of several serious adverse effects associated with the supra-physiological doses of BMP2 relied upon for clinical efficacy. Key to harnessing BMPs and other agents safely and effectively will be the ability to localize activity at a target site at substantially reduced doses. Clay (Laponite) nanoparticles can self assemble into gels under physiological conditions and bind growth factors for enhanced and localized efficacy. Here we show the ability to localize and enhance the activity of BMP2 to achieve ectopic bone formation at doses within the sub-microgram per ml range of concentrations sufficient to induce differentiation of responsive cell populations in vitro and at approximately 3000 fold lower than those employed in clinical practice.

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cell-laden constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclay-based bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 d in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 d of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p < 0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p < 0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.

Laponite nanoparticles have attracted attention in the tissue engineering field for their protein interactions, gel-forming properties, and, more recently, osteogenic bioactivity. Despite growing interest in the osteogenic properties of Laponite, the application of Laponite colloidal gels to host the osteogenic differentiation of responsive stem cell populations remains unexplored. Here, the potential to harness the gel-forming properties of Laponite to generate injectable bioactive microenvironments for osteogenesis is demonstrated. A diffusion/dialysis gelation method allows the rapid formation of stable transparent gels from injectable, thixotropic Laponite suspensions in physiological fluids. Upon contact with buffered saline or blood serum, nanoporous gel networks exhibiting, respectively, fivefold and tenfold increases in gel stiffness are formed due to the reorganization of nanoparticle interactions. Laponite diffusion gels are explored as osteogenic microenvironments for skeletal stem cell containing populations. Laponite films support cell adhesion, proliferation, and differentiation of human bone marrow stromal cells in 2D. Laponite gel encapsulation significantly enhances osteogenic protein expression compared with 3D pellet culture controls. In both 2D and 3D conditions, cell associated mineralization is strongly enhanced. This study demonstrates that Laponite diffusion gels offer considerable potential as biologically active and clinically relevant bone tissue engineering scaffolds.

This review explores the application of hydrogels in orthopaedic clinical situations which may benefit from enhanced growth factor delivery and improved osteogenesis of bone graft material. Hydrogels are defined, and in vivo evidence supporting their application in these clinical areas is explored. Our focus is on clinically pertinent properties, such as the chemistry of formation, biocompatibility, efficacy of cell and growth factor delivery, ability to withstand mechanical loading and potential to be delivered via an injection. Naturally derived hydrogels, such as gelatin, hyaluronic acid and fibroin, together with a number of synthetic polyethylene glycol-based gels combined with protease-sensitive domains, have shown excellent biocompatibility. There is significant literature evidence supporting the ability of hydrogels to facilitate growth factor and cell delivery. Burst release of the selected growth factor remains a consistent challenge, which has been overcome in some studies with chemical modifications of the hydrogel. Interestingly, a number of studies detail percutaneous delivery with hydrogels combined with calcium-based minerals to enhance osteogenicity, with mixed results. Few of the studies explored the biomechanical properties of the materials, and none of the studies reviewed demonstrated the ability of a hydrogel/graft material to withstand mechanical loading in a clinically relevant segmental bone defect model.

Clay nanoparticles, composites and hydrogels are emerging as a new class of biomaterial with exciting potential for tissue engineering and regenerative medicine applications. Clay particles have been extensively explored in polymeric nanocomposites for self-assembly and enhanced mechanical properties as well as for their potential as drug delivery modifiers. In recent years, a cluster of studies have explored cellular interactions with clay nanoparticles alone or in combination with polymeric matrices. These pioneering studies have suggested new and unforeseen utility for certain clays as bioactive additives able to enhance cellular functions including adhesion, proliferation and differentiation, most notably for osteogenesis. This review examines the recent literature describing the potential effects of clay-based nanomaterials on cell function and examines the potential role of key clay physicochemical properties in influencing such interactions and their exciting possibilities for regenerative medicine.

Skeletal stem cells confer to bone its innate capacity for regeneration and repair. Bone regeneration strategies seek to harness and enhance this regenerative capacity for the replacement of tissue damaged or lost through congenital defects, trauma, functional/esthetic problems, and a broad range of diseases associated with an increasingly aged population. This review describes the state of the field and current steps to translate and apply skeletal stem cell biology in the clinic and the problems therein. Challenges are described along with key strategies including the isolation and ex vivo expansion of multipotential populations, the targeting/delivery of regenerative populations to sites of repair, and their differentiation toward bone lineages. Finally, preclinical models of bone repair are discussed along with their implications for clinical translation and the opportunities to harness that knowledge for musculoskeletal regeneration. Stem Cells 2014;32:35–44

Bioprinting of living cells is rapidly developing as an advanced biofabrication approach to engineer tissues. Bioinks can be extruded in three-dimensions (3D) to fabricate complex and hierarchical constructs for implantation. However, lack of functionality can often be attributed to poor bioink properties. Indeed, advanced bioinks encapsulating living cells should: (i) present optimal rheological properties and retain 3D structure post-fabrication, (ii) promote cell viability and support cell differentiation, (iii) localise proteins of interest (e.g. vascular endothelial growth factor (VEGF)) to stimulate encapsulated cell activity and tissue ingrowth upon implantation. In this study, we present the results of the inclusion of a synthetic nanoclay, Laponite (LPN) together with a gelatin methacryloyl (GelMA) bioink and the development of a functional cell-instructive bioink. A nanocomposite bioink displaying enhanced shape fidelity retention and interconnected porosity within extrusion-bioprinted fibres was observed. Human bone marrow stromal cell (HBMSC) viability within the nanocomposite showed no significant changes over 21 days of culture in LPN-GelMA (85.60 ± 10.27 %), compared to a significant decrease in GelMA from 7 (95.88 ± 2.90 %) to 21 days (55.54 ± 14.72 %) (p

Mesenchymal stem cells (MSCs) provide an ideal cell source for bone tissue engineering strategies. However, bone marrow stromal cell (BMSC) populations that contain MSCs are highly heterogeneous expressing a wide variety of proliferative and differentiation potentials. Current MSC isolation methods employing magnetic-activated and fluorescent-activated cell sorting can be expensive and time consuming and, in the absence of specific MSC markers, fail to generate homogeneous populations. We have investigated the potential of various colony morphology descriptors to provide correlations with cell growth potential. Density-independent colony forming unit-fibroblastic (CFU-F) capacity is a MSC prerequisite and resultant colonies display an array of shapes and sizes that might be representative of cell function. Parent colonies were initially categorised according to their diameter and cell density and grouped before passage for the subsequent assessment of progeny colonies. Whereas significant morphological differences between distinct parent populations indicated a correlation with immunophenotype, enhanced CFU-F capacity was not observed when individual colonies were isolated according to these morphological parameters. Colony circularity, an alternative morphological measure, displayed a strong correlation with subsequent cell growth potential. The current study indicates the potential of morphological descriptors for predicting cell growth rate and suggests new directions for research into dissection of human BMSC CFU-F populations.

Background: recent approaches have sought to harness the potential of stem cells to regenerate bone that is lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of osteoprogenitors for such applications. However, previous studies indicated that the concentration of osteoprogenitors present in BMA is less than required for robust bone regeneration. We provide further evidence for the importance of BMA enrichment for skeletal tissue engineering strategies using a novel acoustic wave-facilitated filtration strategy to concentrate BMA for osteoprogenitors, clinically applicable for intraoperative orthopedic use.Methods: femoral BMA from 15 patients of an elderly cohort was concentrated for the nucleated cell fraction against erythrocytes and excess plasma volume via size exclusion filtration facilitated by acoustic agitation. The effect of aspirate concentration was assessed by assays for colony formation, flow cytometry, multilineage differentiation and scaffold seeding efficiency.Results: BMA was filtered to achieve a mean 4.2-fold reduction in volume with a corresponding enrichment of viable and functional osteoprogenitors, indicated by flow cytometry and assays for colony formation. Enhanced osteogenic and chondrogenic differentiation was observed using concentrated aspirate and enhanced cell-seeding efficiency onto allogeneic bone graft as an effect of osteoprogenitor concentration relative specifically to the concentration of erythrocytes in the aspirate.Conclusions: these studies provide evidence for the importance of BMA nucleated cell concentration for both cell differentiation and cell seeding efficiency and demonstrate the potential of this approach for intraoperative application to enhance bone healing

New regenerative materials and approaches need to be assessed through reliable and comparable methods for rapid translation to the clinic. There is a considerable need for proven in vitro assays that are able to reduce the burden on animal testing, by allowing assessment of biomaterial utility predictive of the results currently obtained through in vivo studies. The purpose of this multicentre review was to investigate the correlation between existing in vitro results with in vivo outcomes observed for a range of biomaterials. Members from the European consortium BioDesign, comprising 8 universities in a European multicentre study, provided data from 36 in vivo studies and 47 in vitro assays testing 93 different biomaterials. The outcomes of the in vitro and in vivo experiments were scored according to commonly recognised measures of success relevant to each experiment. The correlation of in vitro with in vivo scores for each assay alone and in combination was assessed. A surprisingly poor correlation between in vitro and in vivo assessments of biomaterials was revealed indicating a clear need for further development of relevant in vitro assays. There was no significant overall correlation between in vitro and in vivo outcome. The mean in vitro scores revealed a trend of covariance to in vivo score with 58 %. The inadequacies of the current in vitro assessments highlighted here further stress the need for the development of novel approaches to in vitro biomaterial testing and validated pre-clinical pipelines.

Tissue engineering offers enormous potential for bone regeneration. Despite extensive in vitro and in vivo work, few strategies translate into clinical practice. This paper describes the combination of skeletal stem cells (SSCs) and impaction bone grafting (IBG) for the treatment of patients with bone defects associated with avascular necrosis of the femoral head. SSCs and milled allograft were impacted into necrotic bone in the femoral heads of four patients. Three patients remained asymptomatic at 22-44 month follow-up, but one patient has required total hip replacement (both hips). This has allowed retrieval of the femoral heads, which were analysed structurally and functionally by μCT, histology and mechanical testing. A central channel of impacted bone was found in the femoral heads, which displayed a mature trabecular micro-architecture. The impacted bone was denser than the surrounding trabecular bone, as strong in compression and with histological micro-architecture comparable to that of trabecular bone. Analysis of the retrieved femoral head samples has demonstrated that this tissue-engineering strategy regenerates bone that is both structurally and functionally analogous to normal trabecular bone. SSCs, together with IBG, have proved an effective treatment for avascular necrosis of the femoral head and offer significant potential for the broader spectrum of bone defects.

The application of selected skeletal progenitor cells and appropriate biomimetic microenvironments and nanotopographical surfaces offer the potential for innovative approaches to bone disease treatment and bone regeneration. Skeletal stem cells, commonly referred to as mesenchymal stem cells or human bone marrow stromal stem cells are multipotent progenitor cells with the ability to generate the stromal lineages of bone, cartilage, muscle, tendon, ligament and fat. This review will examine i) the application of innovative nanotopography surfaces that provide cues for human stem cell differentiation in the absence of chemical cues, ii) unique biomimetic microenvironments for skeletal tissue repair as well as iii) data from translational studies from the laboratory through to the clinic demonstrating the potential of skeletal cell based repair using impaction bone grafting as an exemplar. The development of protocols, tools and above all multidisciplinary approaches that integrate biomimetic materials, nanotopography, angiogenic, cell and clinical techniques for skeletal tissue regeneration for de novo tissue formation offers an opportunity to improve the quality of life of many.

Skeletal regeneration and tissue engineering strategies rely critically on the efficient expansion of progenitor cell populations whilst simultaneously preserving multipotentiality and the ability to induce differentiation towards bone and cartilage. Cell population heterogeneity has a significant impact on this process, but is currently poorly quantified, hampering the interpretation of experimental results and the design of optimised expansion protocols. The objective of this study was to characterise individual human bone marrow stromal cell heterogeneity in terms of colony expansion potential. For this purpose, a novel two-stage CFU-F assay was developed in which cells from primary single cell-derived colonies were detached and reseeded again at clonal density as single cells to form new secondary colonies. This clearly demonstrated how secondary colony growth potential varies markedly both between and within primary colonies. Depending on the primary colony, cells either generated small secondary colonies only, or else a wide range of colony sizes. Using computational modelling it was shown how such colony heterogeneity could arise from hierarchical progenitor cell populations and what the limits of such a population structure were in explaining the experimental data. In addition the model demonstrated the significant potential impact of cell mobility on expansion potential and its implications for inducing population heterogeneity. This combined experimental-computational approach will ascertain the impact of cell culture protocols on the expansion potential and functional composition of heterogeneous progenitor populations. Such insights are likely to be of crucial importance for the success of skeletal regeneration strategies.

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen-HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.

In the absence of sufficient cleaning of medical instruments, contamination and infection can result in serious consequences for the health sector and remains a significant unmet challenge. In this paper we describe a novel cleaning system reliant on cavitation action created in a free flowing fluid stream where ultrasonic transmission to a surface, through the stream, is achieved using careful design and control of the device architecture, sound field and the materials employed. Cleaning was achieved with purified water at room temperature, moderate fluid flow rates and without the need for chemical additives or the high power consumption associated with conventional strategies. This study illustrates the potential in harnessing an ultrasonically activated stream to remove biological contamination including brain tissue from surgical stainless steel substrates, S. epidermidis biofilms from glass, and fat/soft tissue matter from bone structures with considerable basic and clinical applications.

This chapter deals with the tissue engineering aspects of one of the mesenchymal tissues—cartilage. It includes a brief description of the different cartilage types and their embryonal origin. Tissue structures including chondrocyte and extracellular matrix components are described in detail. The disease aspect of hyaline cartilage with emphasis on cartilage injuries and the tissue engineering approach to cartilage regeneration with the autologous chondrocyte implantation technique is described in depth. The future aspects of cartilage regeneration techniques with potential cell types other than autologous chondrocytes as well as new promising scaffold techniques are described.

This review explores the application of hydrogels in orthopaedic clinical situations which may benefit from enhanced growth factor delivery and improved osteogenesis of bone graft material. Hydrogels are defined, and in vivo evidence supporting their application in these clinical areas is explored. Our focus is on clinically pertinent properties, such as the chemistry of formation, biocompatibility, efficacy of cell and growth factor delivery, ability to withstand mechanical loading and potential to be delivered via an injection. Naturally derived hydrogels, such as gelatin, hyaluronic acid and fibroin, together with a number of synthetic polyethylene glycol-based gels combined with protease-sensitive domains, have shown excellent biocompatibility. There is significant literature evidence supporting the ability of hydrogels to facilitate growth factor and cell delivery. Burst release of the selected growth factor remains a consistent challenge, which has been overcome in some studies with chemical modifications of the hydrogel. Interestingly, a number of studies detail percutaneous delivery with hydrogels combined with calcium-based minerals to enhance osteogenicity, with mixed results. Few of the studies explored the biomechanical properties of the materials, and none of the studies reviewed demonstrated the ability of a hydrogel/graft material to withstand mechanical loading in a clinically relevant segmental bone defect model.

BACKGROUND & PURPOSE: Skeletal stem cells (SSCs) and impaction bone grafting (IBG) can be combined to produce a mechanically stable living bone composite. This novel strategy has been translated to the treatment of avascular necrosis of the femoral head. Surgical technique, clinical follow-up and retrieval analysis data of this translational case series is presented.METHODS: SSCs and milled allograft were impacted into necrotic bone in five femoral heads of four patients. Cell viability was confirmed by parallel in vitro culture of the cell-graft constructs. Patient follow-up was by serial clinical and radiological examination. Tissue engineered bone was retrieved from two retrieved femoral heads and was analysed by histology, microcomputed tomography (?CT) and mechanical testing.RESULTS: Three patients remain asymptomatic at 22- to 44-month follow-up. One patient (both hips) required total hip replacement due to widespread residual necrosis. Retrieved tissue engineered bone demonstrated a mature trabecular micro-architecture histologically and on ?CT. Bone density and axial compression strength were comparable to trabecular bone.CONCLUSIONS: Clinical follow-up shows this to be an effective new treatment for focal early stage avascular necrosis of the femoral head. Unique retrieval analysis of clinically translated tissue engineered bone has demonstrated regeneration of tissue that is both structurally and functionally analogous to normal trabecular bone.

Seminal recent studies that have shed new light on the remarkable properties of clay interactions suggest unexplored opportunities for biomaterial design and regenerative medicine. Here, recent conceptual and technological developments in the science of clay interactions with biomolecules, polymers, and cells are examined, focusing on the implications for tissue engineering and regenerative strategies. Pioneering studies demonstrating the utility of clay for drug-delivery and scaffold design are reviewed and areas for future research and development highlighted.

The sorptive properties of clay are harnessed to provide niches for tissue regeneration. Synthetic smectite clays self-organize into gels under physiological conditions to allow encapsulation of cells and demonstrate remarkable capacity for the localization of biological molecules without the need for complex chemical modifications.

Bone tissue engineering seeks to develop strategies for the regeneration of diseased or damaged bone. Broadly, the approach seeks to harness the regenerative capacity of local or implanted stem or progenitor cell populations through the application of biodegradable and osteoconductive three-dimensional structures (or scaffolds) together with the temporally and spatially controlled provision of osteoinductive molecules. While natural bone repair provides a unique precedent for stem cell-mediated tissue regeneration, enhancement of bone’s innate regenerative capacity is necessary in order to meet the clinical needs of an aging population. This article describes the concepts underlying bone regeneration and current advances in bone tissue engineering through the enhancement and marrying of biologically informed and clinically relevant strategies.

Advances in our understanding of skeletal stem cells and their role in bone development and repair, offer the potential to open new frontiers in bone regeneration. Tissue engineering seeks to harness the regenerative capacity innate to bone for the replacement of tissue lost or damaged through a broad range of conditions associated with an increasingly aged population. The strategy entails ex vivo expansion of multipotential populations followed by delivery to the site of damage on dynamically durable-biodegradable three-dimensional structures which provide the requisite extracellular microenvironment for stem cell driven tissue development. This review will examine bone stem cell biology, and current advances in skeletal tissue engineering through the enhancement and marrying of biologically informed and clinically relevant strategies.

Skeletal stem cells (SSCs) show great capacity for bone and cartilage repair however, current in vitro cultures are heterogeneous displaying a hierarchy of differentiation potential. SSCs represent the diminutive true multipotent stem cell fraction of bone marrow mononuclear cell (BMMNC) populations. Endeavours to isolate SSCs have generated a multitude of separation methodologies. SSCs were first identified and isolated by their ability to adhere to culture plastic. Once isolated, further separation is achieved via culture in selective or conditioned media (CM). Indeed, preferential SSC growth has been demonstrated through selective in vitro culture conditions. Other approaches have utilised cell morphology (size and shape) as selection criteria. Studies have also targeted SSCs based on their preferential adhesion to specified compounds, individually or in combination, on both macro and microscale platforms. Nevertheless, most of these methods which represent macroscale function with relatively high throughput, yield insufficient purity. Consequently, research has sought to downsize isolation methodologies to the microscale for single cell analysis. The central approach is identification of the requisite cell populations of SSC-specific surface markers that can be targeted for isolation by either positive or negative selection. SELEX and phage display technology provide apt means to sift through substantial numbers of candidate markers. In contrast, single cell analysis is the paramount advantage of microfluidics, a relatively new field for cell biology. Here cells can be separated under continuous or discontinuous flow according to intrinsic phenotypic and physicochemical properties. The combination of macroscale quantity with microscale specificity to generate robust high-throughput (HT) technology for pure SSC sorting, isolation and enrichment offers significant implications therein for skeletal regenerative strategies as a consequence of lab on chip derived methodology.

In the Camargue in southern France, catches of tabanids in a Manitoba trap in 1976–78 gave seasonal, daily and hourly records of numbers. Concurrently, some interactions of the flies with a group of free-ranging horses were observed directly. The tabanids were present from May to October annually, with the main peak of abundance between late May and early July. The number trapped in each hour of the day increased with the ambient temperature in the morning, was stable during the hottest time of the day and declined in the late afternoon. Their alighting on a horse always elicited reactions aimed at dislodging them. When tabanids became very abundant and active, the horses would move as a group from where they were feeding to a traditional open bare area (‘chomadou’) where they would stay without feeding, sometimes for hours, even though large numbers of tabanids were still worrying them. Analyses of data on an index of the abundance of tabanids on or about the horses in a group showed the horses did not reduce the fly load by moving to and resting on their chomadou area. The number of responses by a horse to the tabanids on or about it was shown to be dependent not only on the number of flies but on their activity. Analysis of the responses revealed a negative effect of wind speed on the activity component which was separate from its known negative effect on the number of flies present. A hypothesis is proposed that movement to the chomadou can be explained as the horses going to an area ‘known’ to be usually exposed to the prevailing wind.