Single-cell RNA-sequence analysis of human bone marrow reveals new targets for isolation of skeletal stem cells using spherical nucleic acids.

There is a wealth of data indicating human bone marrow contains skeletal stem cells (SSC) with the capacity for osteogenic, chondrogenic and adipogenic differentiation. However, current methods to isolate SSCs are restricted by the lack of a defined marker, limiting understanding of SSC fate, immunophenotype, function and clinical application. The current study applied single-cell RNA-sequencing to profile human adult bone marrow populations from 11 donors and identified novel targets for SSC enrichment. Spherical nucleic acids were used to detect these mRNA targets in SSCs. This methodology was able to rapidly isolate potential SSCs found at a frequency of <1 in 1,000,000 in human bone marrow, with the capacity for tri-lineage differentiation in vitro and ectopic bone formation in vivo. The current studies detail the development of a platform to advance SSC enrichment from human bone marrow, offering an invaluable resource for further SSC characterisation, with significant therapeutic impact therein.

Evolving applications of the egg: chorioallantoic membrane assay and ex vivo organotypic culture of materials for bone tissue engineering.

The chick chorioallantoic membrane model has been around for over a century, applied in angiogenic, oncology, dental and xenograft research. Despite its often perceived archaic, redolent history, the chorioallantoic membrane assay offers new and exciting opportunities for material and growth factor evaluation in bone tissue engineering. Currently, superior/improved experimental methodology for the chorioallantoic membrane assay are difficult to identify, given an absence of scientific consensus in defining experimental approaches, including timing of inoculation with materials and the analysis of results. In addition, critically, regulatory and welfare issues impact upon experimental designs. Given such disparate points, this review details recent research using the ex vivo chorioallantoic membrane assay and the ex vivo organotypic culture to advance the field of bone tissue engineering, and highlights potential areas of improvement for their application based on recent developments within our group and the tissue engineering field.

Osteoarthritis treatment with a novel nutraceutical acetylated ligstroside aglycone, a chemically modified extra-virgin olive oil polyphenol.

Recent studies have shown that dietary patterns confer protection from certain chronic diseases related to oxidative stress, the immune system and chronic low-grade inflammatory diseases. The aim of this study was to evaluate the anti-inflammatory potential and the capacity to attenuate cartilage degradation using extra-virgin olive oil-derived polyphenols for the treatment of osteoarthritis. Results show that both nutraceuticals ligstroside aglycone and acetylated ligstroside aglycone showed an anti-inflammatory profile. Acetylated ligstroside aglycone significantly reduced the expression of pro-inflammatory genes including NOS2 and MMP13 at both RNA and protein levels; decreased nitric oxide release; and, importantly, reduced proteoglycan loss in human osteoarthritis cartilage explants. Our study demonstrated that a new synthetic acetylated ligstroside aglycone derivative offers enhanced anti-inflammatory profile than the natural nutraceutical compound in osteoarthritis. These results substantiate the role of nutraceuticals in osteoarthritis with implications for therapeutic intervention and our understanding of osteoarthritis pathophysiology.

Regulation of the Bone Vascular Network is Sexually Dimorphic.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High-resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone-derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone-derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Biomimetic oyster shell-replicated topography alters the behaviour of human skeletal stem cells.

The regenerative potential of skeletal stem cells provides an attractive prospect to generate bone tissue needed for musculoskeletal reparation. A central issue remains efficacious, controlled cell differentiation strategies to aid progression of cell therapies to the clinic. The nacre surface from Pinctada maxima shells is known to enhance bone formation. However, to date, there is a paucity of information on the role of the topography of P. maxima surfaces, nacre and prism. To investigate this, nacre and prism topographical features were replicated onto polycaprolactone and skeletal stem cell behaviour on the surfaces studied. Skeletal stem cells on nacre surfaces exhibited an increase in cell area, increase in expression of osteogenic markers ALP (p < 0.05) and OCN (p < 0.01) and increased metabolite intensity (p < 0.05), indicating a role of nacre surface to induce osteogenic differentiation, while on prism surfaces, skeletal stem cells did not show alterations in cell area or osteogenic marker expression and a decrease in metabolite intensity (p < 0.05), demonstrating a distinct role for the prism surface, with the potential to maintain the skeletal stem cell phenotype.

Regenerative medicine in lower limb reconstruction.

Bone is a highly specialized connective tissue and has a rare quality as one of the few tissues that can repair without a scar to regain pre-injury structure and function. Despite the excellent healing capacity of bone, tumor, infection, trauma and surgery can lead to significant bone loss requiring skeletal augmentation. Bone loss in the lower limb poses a complex clinical problem, requiring reconstructive techniques to restore form and function. In the past, amputation may have been the only option; however, there is now an array of reconstructive possibilities and cellular therapies available to salvage a limb. In this review, we will evaluate current applications of bone tissue engineering techniques in limb reconstruction and identify potential strategies for future work.

Application of 3D-printed patient-specific skeletal implants augmented with autologous skeletal stem cells.

Joint replacements have proved a medical success providing symptomatic relief and return to mobility in many patients with arthritis. However, multiple revision surgeries due to joint failure can result in complex revision scenarios with significant bone tissue loss, in an elderly population, which poses a significant clinical challenge. Computer-aided design-computer-assisted manufacturing (CAD-CAM) prototyped bespoke implants are currently being used as an alternative and innovative approach for joint restoration in salvage cases, while the incorporation of autologous skeletal stem cells to optimize regenerative capacity can enhance implant osseointegration. We present a case series of 11 patients with severe disability and significant bone loss due to failed joint replacements. The choice of CAD-CAM prototyped joint implants enhanced with autologous skeletal stem cells resulted in significant patient-reported clinical and radiological improvements.

Polymersome nanoparticles for delivery of Wnt-activating small molecules.

Spatiotemporal control of drug delivery is important for a number of medical applications and may be achieved using polymersome nanoparticles (PMs). Wnt signalling is a molecular pathway activated in various physiological processes, including bone repair, that requires precise control of activation. Here, we hypothesise that PMs can be stably loaded with a small molecule Wnt agonist, 6-bromoindirubin-3'-oxime (BIO), and activate Wnt signalling promoting the osteogenic differentiation in human primary bone marrow stromal cells (BMSCs). We showed that BIO-PMs induced a 40% increase in Wnt signaling activation in reporter cell lines without cytotoxicity induced by free BIO. BMSCs incubated with BIO-PMs showed a significant up-regulation of the Wnt target gene AXIN2 (14 ±â€¯4 fold increase, P < 0.001) and a prolonged activation of the osteogenic gene RUNX2. We conclude that BIO-PMs could represent an innovative approach for the controlled activation of Wnt signaling for promoting bone regeneration after fracture.

Preservation and stability of cell therapy products: recommendations from an expert workshop.

If the field of regenerative medicine is to deliver therapies, rapid expansion and delivery over considerable distances to large numbers of patients is needed. This will demand efficient stabilization and shipment of cell products. However, cryopreservation science is poorly understood by life-scientists in general and in recent decades only limited progress has been made in the technology of preservation and storage of cells. Rapid translation of new developments to a broader range of cell types will be vital, as will assuring a deeper knowledge of the fundamental cell biology relating to successful preservation and recovery of cell cultures. This report presents expert consensus on these and other issues which need to be addressed for more efficient delivery of cell therapies.

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.

The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering.

Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (µCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by µCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.

Prospective isolation of human bone marrow stromal cell subsets: A comparative study between Stro-1-, CD146- and CD105-enriched populations.

Stro-1 has proved an efficacious marker for enrichment of skeletal stem and progenitor cells although isolated populations remain heterogeneous, exhibiting variable colony-forming efficiency and osteogenic differentiation potential. The emerging findings that skeletal stem cells originate from adventitial reticular cells have brought two further markers to the fore including CD146 and CD105 (both primarily endothelial and perivascular). This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1. Fluorescent cell sorting quantification showed that CD146 and CD105 both targeted smaller (2.22% ± 0.59% and 6.94% ± 1.34%, respectively) and potentially different human bone marrow stromal cell fractions compared to Stro-1 (16.29% ± 0.78%). CD146+, but not CD105+, cells exhibited similar alkaline phosphatase-positive colony-forming efficiency in vitro and collagen/proteoglycan deposition in vivo to Stro-1+ cells. Molecular analysis of a number of select osteogenic and potential osteo-predictive genes including ALP, CADM1, CLEC3B, DCN, LOXL4, OPN, POSTN and SATB2 showed Stro-1+ and CD146+ populations possessed similar expression profiles. A discrete human bone marrow stromal cell fraction (2.04% ± 0.41%) exhibited positive immuno-labelling for both Stro-1 and CD146. The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations. However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

Tissue engineering strategies in spinal arthrodesis: the clinical imperative and challenges to clinical translation.

Skeletal disorders requiring the regeneration or de novo production of bone present considerable reconstructive challenges and are one of the main driving forces for the development of skeletal tissue engineering strategies. The skeletal or mesenchymal stem cell is a fundamental requirement for osteogenesis and plays a pivotal role in the design and application of these strategies. Research activity has focused on incorporating the biological role of the mesenchymal stem cell with the developing fields of material science and gene therapy in order to create a construct that is not only capable of inducing host osteoblasts to produce bone, but is also osteogenic in its own right. This review explores the clinical need for reparative approaches in spinal arthrodesis, identifying recent tissue engineering strategies employed to promote spinal fusion, and considers the ongoing challenges to successful clinical translation.