A scaffold-free approach to cartilage tissue generation using human embryonic stem cells.

Articular cartilage functions as a shock absorber and facilitates the free movement of joints. Currently, there are no therapeutic drugs that promote the healing of damaged articular cartilage. Limitations associated with the two clinically relevant cell populations, human articular chondrocytes and mesenchymal stem cells, necessitate finding an alternative cell source for cartilage repair. Human embryonic stem cells (hESCs) provide a readily accessible population of self-renewing, pluripotent cells with perceived immunoprivileged properties for cartilage generation. We have developed a robust method to generate 3D, scaffold-free, hyaline cartilage tissue constructs from hESCs that are composed of numerous chondrocytes in lacunae, embedded in an extracellular matrix containing Type II collagen, sulphated glycosaminoglycans and Aggrecan. The elastic (Young's) modulus of the hESC-derived cartilage tissue constructs (0.91 ± 0.08 MPa) was comparable to full-thickness human articular cartilage (0.87 ± 0.09 MPa). Moreover, we have successfully scaled up the size of the scaffold-free, 3D hESC-derived cartilage tissue constructs to between 4.5 mm and 6 mm, thus enhancing their suitability for clinical application.

Live-imaging of Bioengineered Cartilage Tissue using Multimodal Non-linear Molecular Imaging.

Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.

Isolation, Differentiation, and Characterization of Human Bone Marrow Stem Cells In Vitro and In Vivo.

In this chapter, we describe techniques for the isolation and characterisation of skeletal stem cells from human bone marrow. The methods for enrichment of STRO-1+ and STRO-4+ cells using magnetic activated cell sorting are described and we also detail techniques for establishing and characterizing osteogenic, adipogenic, and chondrogenic cultures from these cells. Finally, we present methods for studying the ability of these cells to produce bone in vivo using diffusion chambers which have been implanted subcutaneously into mice.

Quantitative temporal interrogation in 3D of bioengineered human cartilage using multimodal label-free imaging.

The unique properties of skeletal stem cells have attracted significant attention in the development of strategies for skeletal regeneration. However, there remains a crucial unmet need to develop quantitative tools to elucidate skeletal cell development and monitor the formation of regenerated tissues using non-destructive techniques in 3D. Label-free methods such as coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited auto-fluorescence (TPEAF) microscopy are minimally invasive, non-destructive, and present new powerful alternatives to conventional imaging techniques. Here we report a combination of these techniques in a single multimodal system for the temporal assessment of cartilage formation by human skeletal cells. The evaluation of bioengineered cartilage, with a new parameter measuring the amount of collagen per cell, collagen fibre structure and chondrocyte distribution, was performed using the 3D non-destructive platform. Such 3D label-free temporal quantification paves the way for tracking skeletal cell development in real-time and offers a paradigm shift in tissue engineering and regenerative medicine applications.

Augmentation of musculoskeletal regeneration: role for pluripotent stem cells.

The rise in the incidence of musculoskeletal diseases is attributed to an increasing ageing population. The debilitating effects of musculoskeletal diseases, coupled with a lack of effective therapies, contribute to huge financial strains on healthcare systems. The focus of regenerative medicine has shifted to pluripotent stem cells (PSCs), namely, human embryonic stem cells and human-induced PSCs, due to the limited success of adult stem cell-based interventions. PSCs constitute a valuable cell source for musculoskeletal regeneration due to their capacity for unlimited self-renewal, ability to differentiate into all cell lineages of the three germ layers and perceived immunoprivileged characteristics. This review summarizes methods for chondrogenic, osteogenic, myogenic and adipogenic differentiation of PSCs and their potential for therapeutic applications.

Acoustically modulated biomechanical stimulation for human cartilage tissue engineering.

Bioacoustofluidics can be used to trap and levitate cells within a fluid channel, thereby facilitating scaffold-free tissue engineering in a 3D environment. In the present study, we have designed and characterised an acoustofluidic bioreactor platform, which applies acoustic forces to mechanically stimulate aggregates of human articular chondrocytes in long-term levitated culture. By varying the acoustic parameters (amplitude, frequency sweep, and sweep repetition rate), cells were stimulated by oscillatory fluid shear stresses, which were dynamically modulated at different sweep repetition rates (1-50 Hz). Furthermore, in combination with appropriate biochemical cues, the acoustic stimulation was tuned to engineer human cartilage constructs with structural and mechanical properties comparable to those of native human cartilage, as assessed by immunohistology and nano-indentation, respectively. The findings of this study demonstrate the capability of acoustofluidics to provide a tuneable biomechanical force for the culture and development of hyaline-like human cartilage constructs in vitro.

Mesenchymal Stem Cells: Potential Role in the Treatment of Osteochondral Lesions of the Ankle.

Given articular cartilage has a limited repair potential, untreated osteochondral lesions of the ankle can lead to debilitating symptoms and joint deterioration necessitating joint replacement. While a wide range of reparative and restorative surgical techniques have been developed to treat osteochondral lesions of the ankle, there is no consensus in the literature regarding which is the ideal treatment. Tissue engineering strategies, encompassing stem cells, somatic cells, biomaterials, and stimulatory signals (biological and mechanical), have a potentially valuable role in the treatment of osteochondral lesions. Mesenchymal stem cells (MSCs) are an attractive resource for regenerative medicine approaches, given their ability to self-renew and differentiate into multiple stromal cell types, including chondrocytes. Although MSCs have demonstrated significant promise in in vitro and in vivo preclinical studies, their success in treating osteochondral lesions of the ankle is inconsistent, necessitating further clinical trials to validate their application. This review highlights the role of MSCs in cartilage regeneration and how the application of biomaterials and stimulatory signals can enhance chondrogenesis. The current treatments for osteochondral lesions of the ankle using regenerative medicine strategies are reviewed to provide a clinical context. The challenges for cartilage regeneration, along with potential solutions and safety concerns are also discussed.

Generation of a Bag1 homozygous knockout mouse embryonic stem cell line using CRISPR/Cas9.

Bag1 transcribes a multifunctional protein that participates in many important biological processes such as cell apoptosis, proliferation, differentiation and embryo development. Despite numerous published studies, the role of Bag1 in the context of embryonic stem (ES) cells, has not been explored. To investigate the function of Bag1 in ES cells, we generated mutant Bag1-/- ES cells using the CRISPR/Cas9 system. We established that the Bag1 double knockout ES cell line maintained their pluripotency, possessed a normal karyotype and the ability to differentiate into all three germ layers.

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations.

AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1).

BCL-2-associated athanogene-1 (BAG-1) is expressed by osteoblast-lineage cells; early embryonic lethality in Bag-1 null mice, however, has limited the investigation of BAG-1 function in osteoblast development. In the present study, bone morphogenetic protein-2/BMP-2-directed osteogenic differentiation of bone marrow stromal cells (BMSCs) of Bag-1(+/-) (heterozygous) female mice was decreased significantly. Genes crucial for osteogenic differentiation, bone matrix formation and mineralisation were expressed at significantly lower levels in cultures of Bag-1(+/-) BMSCs supplemented with BMP-2, while genes with roles in inhibition of BMP-2-directed osteoblastogenesis were significantly upregulated. 17-ß-estradiol (E2) enhanced responsiveness of BMSCs of wild-type and Bag-1(+/-) mice to BMP-2, and promoted robust BMP-2-stimulated osteogenic differentiation of BMSCs. BAG-1 can modulate cellular responses to E2 by regulating the establishment of functional estrogen receptors (ERs), crucially, via its interaction with heat shock proteins (HSC70/HSP70). Inhibition of BAG-1 binding to HSC70 by the small-molecule chemical inhibitor, Thioflavin-S, and a short peptide derived from the C-terminal BAG domain, which mediates binding with the ATPase domain of HSC70, resulted in significant downregulation of E2/ER-facilitated BMP-2-directed osteogenic differentiation of BMSCs. These studies demonstrate for the first time the significance of BAG-1-mediated protein-protein interactions, specifically, BAG-1-regulated activation of ER by HSC70, in modulation of E2-facilitated BMP-2-directed osteoblast development.

Raman spectroscopy and coherent anti-Stokes Raman scattering imaging: prospective tools for monitoring skeletal cells and skeletal regeneration.

The use of skeletal stem cells (SSCs) for cell-based therapies is currently one of the most promising areas for skeletal disease treatment and skeletal tissue repair. The ability for controlled modification of SSCs could provide significant therapeutic potential in regenerative medicine, with the prospect to permanently repopulate a host with stem cells and their progeny. Currently, SSC differentiation into the stromal lineages of bone, fat and cartilage is assessed using different approaches that typically require cell fixation or lysis, which are invasive or even destructive. Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy present an exciting alternative for studying biological systems in their natural state, without any perturbation. Here we review the applications of Raman spectroscopy and CARS imaging in stem-cell research, and discuss the potential of these two techniques for evaluating SSCs, skeletal tissues and skeletal regeneration as an exemplar.

Nanofibrous poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) scaffolds provide a functional microenvironment for cartilage repair.

Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a 'guiding' structure to support the body's self-healing process.

Extracellular Matrix Deposition in Engineered Micromass Cartilage Pellet Cultures: Measurements and Modelling.

This article explores possible mechanisms governing extracellular matrix deposition in engineered cartilaginous cell pellets. A theoretical investigation is carried out alongside an experimental study measuring proteoglycan and collagen volume fractions within murine chondrogenic (ATDC-5) cell pellets. The simple mathematical model, which adopts a nutrient-dependent proteoglycan production rate, successfully reproduces the periphery-dominated proteoglycan deposition, characteristic of the growth pattern observed experimentally within pellets after 21 days of culture. The results suggest that this inhomogeneous proteoglycan production is due to nutrient deficiencies at the pellet centre. Our model analysis further indicates that a spatially uniform distribution of proteoglycan matrix could be maintained by initiating the culture process with a smaller-sized pellet. Finally, possible extensions are put forward with an aim to improve the model predictions for the early behaviour, where different mechanisms appear to dominate the matrix production within the pellets.

Biofabrication of bone tissue: approaches, challenges and translation for bone regeneration.

The rising incidence of bone disorders has resulted in the need for more effective therapies to meet this demand, exacerbated by an increasing ageing population. Bone tissue engineering is seen as a means of developing alternatives to conventional bone grafts for repairing or reconstructing bone defects by combining biomaterials, cells and signalling factors. However, skeletal tissue engineering has not yet achieved full translation into clinical practice as a consequence of several challenges. The use of additive manufacturing techniques for bone biofabrication is seen as a potential solution, with its inherent capability for reproducibility, accuracy and customisation of scaffolds as well as cell and signalling factor delivery. This review highlights the current research in bone biofabrication, the necessary factors for successful bone biofabrication, in addition to the current limitations affecting biofabrication, some of which are a consequence of the limitations of the additive manufacturing technology itself.

Transient Canonical Wnt Stimulation Enriches Human Bone Marrow Mononuclear Cell Isolates for Osteoprogenitors.

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1(bright) /Glycophorin A(-) phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis.

Bone Tissue Engineering.

Medical advances have led to a welcome increase in life expectancy. However, accompanying longevity introduces new challenges: increases in age-related diseases and associated reductions in quality of life. The loss of skeletal tissue that can accompany trauma, injury, disease or advancing years can result in significant morbidity and significant socio-economic cost and emphasise the need for new, more reliable skeletal regeneration strategies. To address the unmet need for bone augmentation, tissue engineering and regenerative medicine have come to the fore in recent years with new approaches for de novo skeletal tissue formation. Typically, these approaches seek to harness stem cells, innovative scaffolds and biological factors that promise enhanced and more reliable bone formation strategies to improve the quality of life for many. This review provides an overview of recent developments in bone tissue engineering focusing on skeletal stem cells, vascular development, bone formation and the translation from preclinical in vivo models to clinical delivery.

Tracking adipogenic differentiation of skeletal stem cells by label-free chemically selective imaging.

Coherent anti-Stokes Raman scattering (CARS) is a chemically selective label-free imaging technique which is rapidly emerging as a powerful alternative to conventional microscopy in biomedicine. The strength of this imaging approach is the provision of rapid insight into chemical distribution especially of small biomolecules such as lipids. The label-free, non-destructive and non-invasive nature of CARS lends itself for use with stem cells, as labelling or staining will render them otherwise unsuitable for therapy. Isolation, enrichment and characterisation of skeletal stem cells (SSCs) and their progeny is of tremendous significance in regenerative medicine. However, SSCs differentiation into bone, cartilage or fat cell types, is currently assessed using several invasive and, typically, destructive methodologies. Thus, CARS presents an exciting alternative to interrogate the differentiation of SSCs in their natural state. In the current study, we have examined the adipogenic differentiation of SSCs over time using CARS imaging and verified the observed differentiation using molecular analysis of gene expression as well as compared the results to conventional Oil Red O lipid staining. We find that the CARS analysis provides an enhanced resolution and definition of lipid droplets, detectable as early as 24 hours and 72 hours after adipogenic induction. Quantification of the CARS image data sets also showed a change in lipid droplet size distribution during the course of adipogenesis over 14 days. Furthermore, CARS provided a superior and facile approach to monitor changes in SSCs as a result of chemical modulation of adipogenic differentiation. The current studies pave the way for the use of CARS as a powerful chemical imaging tool in therapeutics, regenerative medicine and skeletal stem cell biology.

Chondrogenic potential of human articular chondrocytes and skeletal stem cells: a comparative study.

Regenerative medicine strategies have increasingly focused on skeletal stem cells (SSCs), in response to concerns such as donor site morbidity, dedifferentiation and limited lifespan associated with the use of articular chondrocytes for cartilage repair. The suitability of SSCs for cartilage regeneration, however, remains to be fully determined. This study has examined the chondrogenic potential of human STRO-1-immunoselected SSCs (STRO-1(+) SSCs), in comparison to human articular chondrocytes (HACs), by utilising two bioengineering strategies, namely "scaffold-free" three-dimensional (3-D) pellet culture and culture using commercially available, highly porous, 3-D scaffolds with interconnected pore networks. STRO-1(+) SSCs were isolated by magnetic-activated cell sorting from bone marrow samples of haematologically normal osteoarthritic individuals following routine hip replacement procedures. Chondrocytes were isolated by sequential enzymatic digestion of deep zone articular cartilage pieces dissected from femoral heads of the same individuals. After expansion in monolayer cultures, the harvested cell populations were centrifuged to form high-density 3-D pellets and also seeded in the 3-D scaffold membranes, followed by culture in serum-free chondrogenic media under static conditions for 21 and 28 days, respectively. Chondrogenic differentiation was determined by gene expression, histological and immunohistochemical analyses. Robust cartilage formation and expression of hyaline cartilage-specific markers were observed in both day-21 pellets and day-28 explants generated using HACs. In comparison, STRO-1(+) SSCs demonstrated significantly lower chondrogenic differentiation potential and a tendency for hypertrophic differentiation in day-21 pellets. Culture of STRO-1(+) SSCs in the 3-D scaffolds improved the expression of hyaline cartilage-specific markers in day-28 explants, however, was unable to prevent hypertrophic differentiation of the SSC population. The advantages of application of SSCs in tissue engineering are widely recognised; the results of this study, however, highlight the need for further development of cell culture protocols that may otherwise limit the application of this stem cell population in cartilage bioengineering strategies.

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering.

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects.

Concise review: bridging the gap: bone regeneration using skeletal stem cell-based strategies - where are we now?

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.