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As a key regulator of bone homeostasis, sclerostin has garnered a lot of interest over the last two decades. Although sclerostin is primarily expressed by osteocytes and is well known for its role in bone formation and remodelling, it is also expressed by a number of other cells and potentially plays a role in other organs. Herein, we aim to bring together recent sclerostin research and discuss the effect of sclerostin on bone, cartilage, muscle, liver, kidney and the cardiovascular and immune systems. Particular focus is placed on its role in diseases, such as osteoporosis and myeloma bone disease, and the novel development of sclerostin as a therapeutic target. Anti-sclerostin antibodies have recently been approved for the treatment of osteoporosis. However, a cardiovascular signal was observed, prompting extensive research into the role of sclerostin in vascular and bone tissue crosstalk. The study of sclerostin expression in chronic kidney disease was followed by the investigation of its role in liver–lipid–bone interactions, and the recent discovery of sclerostin as a myokine prompted new research into sclerostin within the bone–muscle relationship. Potentially, the effects of sclerostin reach beyond that of bone alone. We further summarise recent developments in the use of sclerostin as a potential therapeutic for osteoarthritis, osteosarcoma and sclerosteosis. Overall, these new treatments and discoveries illustrate progress within the field, however, also highlight remaining gaps in our knowledge.

Translational studies in human cholestatic diseases have for years been hindered by various challenges, including the rarity of the disorders, the difficulty in obtaining biliary tissue from across the spectrum of the disease stage, and the difficulty culturing and maintaining primary cholangiocytes. Organoid technology is increasingly being viewed as a technological breakthrough in translational medicine as it allows the culture and biobanking of self-organizing cells from various sources that facilitate the study of pathophysiology and therapeutics, including from individual patients in a personalized approach. This review describes current research using biliary organoids for the study of human cholestatic diseases and the emerging applications of organoids to regenerative medicine directed at the biliary tree. Challenges and possible solutions to the current hurdles in this emerging field, particularly the need for standardization of terminology and clarity on source materials and techniques, are also discussed.

The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation.This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment.The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum.Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing β-estradiol, FGF2, TNFα, TGFβ, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFβ signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature.This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair.

Background Bones have a remarkable capacity to heal upon fracture. Yet, in large defects or compromised conditions healing processes become impaired, resulting in delayed or non-union. Current therapeutic approaches often utilize autologous or allogeneic bone grafts for bone augmentation. However, limited availability of these tissues and lack of predictive biological response result in limitations for clinical demands. Tissue engineering using viable cell-based implants is a strategic approach to address these unmet medical needs. Methods Herein, the in vitro and in vivo cartilage and bone tissue formation potencies of human pluripotent stem cells were investigated. The induced pluripotent stem cells were specified towards the mesodermal lineage and differentiated towards chondrocytes, which subsequently self-assembled into cartilaginous organoids. The tissue formation capacity of these organoids was then challenged in an ectopic and orthotopic bone formation model. Results The derived chondrocytes expressed similar levels of collagen type II as primary human articular chondrocytes and produced stable cartilage when implanted ectopically in vivo. Upon targeted promotion towards hypertrophy and priming with a proinflammatory mediator, the organoids mediated successful bridging of critical size long bone defects in immunocompromised mice. Conclusions These results highlight the promise of induced pluripotent stem cell technology for the creation of functional cartilage tissue intermediates that can be explored for novel bone healing strategies.

ObjectivesInterleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankylosing spondylitis (AS) with several IL-17 blockers currently clinically approved. Despite this, the role of IL-17 in bone pathology is poorly understood. This study aimed to investigate IL-17 signalling in the context of pathological bone formation.MethodsA biomimetic human periosteum-derived cell (hPDC) model of osteogenic differentiation was used in combination with recombinant IL-17 cytokines, T-cell supernatants or serum from patients with AS. IL-17A, IL-17F and bimekizumab monoclonal antibodies were used to block IL-17 cytokine action.ResultsRecombinant IL-17A and IL-17F are pro-osteogenic with respect to hPDC differentiation. T helper 17 or γδ-T cell supernatants also potently stimulated in vitro bone formation, which was blocked deeper by dual inhibition of IL-17A and IL-17F than by neutralisation of IL-17A or IL-17F individually. Osteogenic blockade may be due to an increase in expression of the Wnt antagonist DKK1. Interestingly, osteocommitment was also induced by serum obtained from patients with AS, which was also abrogated by dual neutralisation of IL-17A and IL-17F.ConclusionsThese data show for the first time that IL-17A and IL-17F enhance in vitro osteogenic differentiation and bone formation from hPDCs, inhibition of which may offer an attractive therapeutic strategy to prevent pathological bone formation.

Sclerostin acts as a negative regulator of bone formation by inhibiting wingless-related integration site (Wnt) signaling in osteoblast lineage cells. Pharmacologic inhibition of sclerostin by neutralizing monoclonal antibodies (Scl-Ab) increases bone mass and bone strength while maintaining bone quality by simultaneously increasing bone formation and decreasing bone resorption. Scl-Ab increases bone formation through the activation of bone-lining cells, by increasing bone matrix production by osteoblasts, and through recruitment of osteoprogenitor cells. In addition, Scl-Ab treatment results in changes to the expression of osteoclast mediators, thereby decreasing bone resorption. At the tissue level, Scl-Ab stimulates modeling-based bone formation and increases wall thickness in remodeling sites by decreasing resorption depth and increasing bone formation on preexisting resorption surfaces. In clinical trials, Scl-Ab rapidly increases bone mineral density (BMD) and strength, reduces the risk of skeletal fragility fractures in postmenopausal osteoporotic women, and increases BMD in osteoporotic men.

Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularized xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X-100 (Vac-STx), and sodium dodecyl sulfate (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50 ng/mg of tissue, while retaining 85% of the sulfate glycosaminoglycan content, and as such was selected as the optimal methodology for decellularization. The resultant Vac-OS scaffolds (decellularized extracellular matrix [dcECM]) were also devoid of the immunogenic alpha-Gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry and fluorescence lifetime imaging microscopy. The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells, respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2 macrophage-driven regenerative response both at 2 and 8 weeks postimplantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1 macrophage-mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.

Purpose of Review The development of therapeutics that target anabolic pathways involved in skeletogenesis is of great importance with regard to disease resulting in bone loss, or in cases of impaired bone repair. This review aims to summarize recent developments in this area. Recent Findings A greater understanding of how drugs that modulate signaling pathways involved in skeletogenesis exert their efficacy, and the molecular mechanisms resulting in bone formation has led to novel pharmacological bone repair strategies. Furthermore, crosstalk between pathways and molecules has suggested signaling synergies that may be exploited for enhanced tissue formation. Summary The sequential pharmacological stimulation of the molecular cascades resulting in tissue repair is a promising strategy for the treatment of bone fractures. It is proposed that a therapeutic strategy which mimics the natural cascade of events observed during fracture repair may be achieved through temporal targeting of tissue repair pathways.

Background Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. Methods hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. Results Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. Conclusion Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units. Electronic supplementary material The online version of this article (10.1186/s13287-018-0787-3) contains supplementary material, which is available to authorized users.

Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation towards cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis.

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p

The development of osteoinductive calcium phosphate- (CaP) based biomaterials has, and continues to be, a major focus in the field of bone tissue engineering. However, limited insight into the spatiotemporal activation of signalling pathways has hampered the optimisation of in vivo bone formation and subsequent clinical translation. To gain further knowledge regarding the early molecular events governing bone tissue formation, we combined human periosteum derived progenitor cells with three types of clinically used CaP-scaffolds, to obtain constructs with a distinct range of bone forming capacity in vivo. Protein phosphorylation together with gene expression for key ligands and target genes were investigated 24 hours after cell seeding in vitro, and 3 and 12 days post ectopic implantation in nude mice. A computational modelling approach was used to deduce critical factors for bone formation 8 weeks post implantation. The combined Ca(2+)-mediated activation of BMP-, Wnt- and PKC signalling pathways 3 days post implantation were able to discriminate the bone forming from the non-bone forming constructs. Subsequently, a mathematical model able to predict in vivo bone formation with 96% accuracy was developed. This study illustrates the importance of defining and understanding CaP-activated signalling pathways that are required and sufficient for in vivo bone formation. Furthermore, we demonstrate the reliability of mathematical modelling as a tool to analyse and deduce key factors within an empirical data set and highlight its relevance to the translation of regenerative medicine strategies.

The discovery that two rare autosomal recessive high bone mass conditions were caused by the loss of sclerostin expression prompted studies into its role in bone homeostasis. In this article, we aim to bring together the wealth of information relating to sclerostin in bone though discussion of rare human disorders in which sclerostin is reduced or absent, sclerostin manipulation via genetic approaches and treatment with antibodies that neutralise sclerostin in animal models and in human. Together, these findings demonstrate the importance of sclerostin as a regulator of bone homeostasis and provide valuable insights into its biological mechanism of action. We summarise the current state of knowledge in the field, including the current understanding of the direct effects of sclerostin on the canonical WNT signalling pathway and the actions of sclerostin as an inhibitor of bone formation. We review the effects of sclerostin, and its inhibition, on bone at the cellular and tissue level and discuss new findings that suggest that sclerostin may also regulate adipose tissue. Finally, we highlight areas in which future research is expected to yield additional insights into the biology of sclerostin.

Bone has many functions. It is responsible for protecting the underlying soft organs, it allows locomotion, houses the bone marrow and stores minerals such as calcium and phosphate. Upon damage, bone tissue can efficiently repair itself. However, healing is hampered if the defect exceeds a critical size and/or is in compromised conditions. The isolation or generation of bone-forming progenitors has applicability to skeletal repair and may be used in tissue engineering approaches. Traditionally, bone engineering uses osteochondrogenic stem cells, which are combined with scaffold materials and growth factors. Despite promising preclinical data, limited translation towards the clinic has been observed to date. There may be several reasons for this including the lack of robust cell populations with favourable proliferative and differentiation capacities. However, perhaps the most pertinent reason is the failure to produce an implant that can replicate the developmental programme that is observed during skeletal repair. Pluripotent stem cells (PSCs) can potentially offer a solution for bone tissue engineering by providing unlimited cell sources at various stages of differentiation. In this review, we summarize key embryonic signalling pathways in bone formation coupled with PSC differentiation strategies for the derivation of bone-forming progenitors.This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

Background Pathological bone formation associated with spondyloarthropathies (SpA) is a major cause of structural tissue damage causing permanent disability. A paucity of in vitro models that faithfully replicate human skeletal biology has impeded research into the cellular and molecular triggers for this osteoimmunological phenomenon. Nevertheless, clinical and animal studies have defined IL-17 signalling as a key regulator of SpA disease; however, the role of IL-17 in bone pathology is unclear. IL-17-producing γδ-T cells have a critical function in periosteal bone formation for fracture repair1. The periosteum has also been implicated in pathological bone formation during SpA disease progression2. Objectives To investigate IL-17 signalling in the context of pathological bone formation using a biomimetic human periosteum derived stem cell (hPDSC) model of osteogenic differentiation. Methods hPDSCs were obtained through enzymatic digestion of periosteal biopsies from patients undergoing orthopaedic surgery. Expanded cultures were then treated with recombinant human IL-17A, IL-17F, or both over 96h. The expression of gene markers was then evaluated. Alternatively, hPDSCs were stimulated using a biomimetic protocol in combination with IL-17A and IL-17F, or human T-cell supernatants (SNs) (as a surrogate disease-like inflammatory milieu). Antibodies with strong-affinity to IL-17A, IL-17F, or bimekizumab (a humanised monoclonal IgG1 antibody with strong affinity for both IL-17A and IL-17F) were used to define the role of these cytokines in the SNs. Expression of osteogenic markers and matrix mineralisation was assessed to define in vitro bone formation. Results Under basal conditions IL-17A and IL-17F significantly up-regulated IL-6 expression. Additionally, IL-17A and IL-17F transiently enhanced the expression of the osteogenic transcription factor RUNX-2. When IL-17 cytokines were combined in a biomimetic differentiation protocol, both IL-17A and IL-17F promoted osteogenic differentiation. Importantly, IL-17F enhanced the expression of most osteogenic markers to a greater extent than IL-17A alone following 9 days9 exposure. Conversely, IL-17A treatment resulted in elevated in vitro mineralisation vs IL-17F. Th17 and γδ-T cell SNs potently enhanced hPDSC osteogenic differentiation and mineralisation. Whilst IL-6 expression and in vitro bone formation were blocked by neutralisation of IL-17A or IL-17F, dual neutralisation of IL-17A and IL-17F in the inflammatory milieu exhibited the greatest effect on most of the tested parameters. Conclusions These data show that both IL-17A and IL-17F enhance in vitro osteogenic differentiation and bone formation from hPDSCs. The source of these cytokines has not been established but is likely to involve entheseal resident γδ-T cells. We propose that following their release, IL-17A and IL-17F drive pathological bone formation resulting in enthesophytes at the enthesis/periosteum interface. Current therapeutics display limited efficacy in blocking enthesophyte formation, hence inhibition of both IL-17A and IL-17F offers an attractive therapeutic strategy to prevent this debilitating feature of SpA. References Ono, et al. Nat Commun 2016;7:10928. Lories, et al. Arthritis Res Ther 2009;11:221. Disclosure of Interest M. Shah Grant/research support from: UCB Pharma, Employee of: UCB Pharma, A. Maroof Employee of: UCB Pharma, R. Al-Hosni: None declared, P. Gikas: None declared, N. Gozzard Employee of: UCB Pharma, S. Shaw Employee of: UCB Pharma, S. Roberts Employee of: UCB Pharma

Complications resulting from impaired fracture healing have major clinical implications on fracture management strategies. Novel concepts taken from developmental biology have driven research strategies towards the elaboration of regenerative approaches that can truly harness the complex cellular events involved in tissue formation and repair. Advances in polymer technology and a better understanding of naturally derived scaffolds have given rise to novel biomaterials with an increasing ability to recapitulate native tissue environments. This coupled with advances in the understanding of stem cell biology and technology has opened new avenues for regenerative strategies with true clinical translatability. These advances have provided the impetus to develop alternative approaches to enhance the fracture repair process. We provide an update on these advances, with a focus on the development of novel biomimetic approaches for bone regeneration and their translational potential.

Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/collagen scaffolds (calcium phosphate rich matrix or CPRM), or on decalcified scaffolds (calcium phosphate depleted matrix or CPDM), followed by subcutaneous implantation in nude mice to trigger ectopic bone formation. In this system, osteoblast differentiation occurred in CPRM scaffolds, but not in CPDM scaffolds. Gene expression was assessed by human full-genome microarray at 20 h after seeding, and 2, 8 and 18 days after implantation. In both matrices, implantation of the cell constructs triggered a similar gene expression cascade, however, gene expression dynamics progressed faster in CPRM scaffolds than in CPDM scaffolds. The difference in gene expression dynamics was associated with differential activation of hub genes and molecular signaling pathways related to calcium signaling (CREB), inflammation (TNFα, NFkB, and IL6) and bone development (TGFβ, β-catenin, BMP, EGF, and ERK signaling). Starting from this set of pathways, a growth factor cocktail was developed that robustly enhanced osteogenesis in vitro and in vivo. Taken together, our data demonstrate that through the identification and subsequent stimulation of genes, proteins and signaling pathways associated with calcium phosphate mediated osteoinduction, a focused approach to develop targeted differentiation protocols in adult progenitor cells can be achieved.

Human mesenchymal stromal cells (hMSCs) offer great potential for bone tissue engineering applications, but their in vivo performance remains limited. Preconditioning of these cells with small molecules to improve their differentiation before implantation, or incorporation of growth factors are possible solutions. Insulin-like growth factor-1 (IGF-1) is one of the most abundant growth factors in bone, involved in growth, development, and metabolism, but its effects on hMSCs are still subject of debate. Here we examined the effects of IGF-1 on proliferation and differentiation of hMSCs in vitro and we found that serum abolished the effects of IGF-1. Only in the absence of serum, IGF-1 increased proliferation, alkaline phosphatase expression, and osteogenic gene expression of hMSCs. Furthermore, we examined synergistic effects of bone morphogenetic protein-2 (BMP-2) and IGF-1 and, although IGF-1 enhanced BMP-2-induced mineralization, IGF-1 only slightly affected in vivo bone formation.

Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as 18F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with 18F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Methods: Mouse MSCs and rat MAPCs were used for 18F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with 18F-FDG, and stem cell biodistribution was investigated using small-animal PET. Results: The optimal incubation period for 18F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%–40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by 18F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of 18F-FDG from the cells. Conclusion: MSCs and MAPCs can be successfully labeled with 18F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells’ ultrastructural properties are not influenced by 18F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with 18F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based biomaterials are produced and have been extensively used for dental and orthopaedic applications. This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material property that initiates de novo bone formation), which are attributed to the chemical composition, surface topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarizes the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the research findings on the effects of calcium (Ca(2+)) and phosphate (PO(4)(3-)) ions on the migration, proliferation and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Further, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the material's osteoinductive properties. Examples of different material forms and production techniques are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of cell-material interactions and to gain further insight into the design and optimization of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed. ispartof: Acta Biomaterialia vol:8 issue:11 pages:3876-3887 ispartof: location:England status: published

One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neo-vascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, Sca#8208;1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the pro-angiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Co-implantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential, but also their pro-angiogenic features and thus provide an ideal cell source for bone regeneration therapies. ispartof: Stem Cells vol:30 issue:11 pages:2460-2471 ispartof: location:England status: published

Bone tissue engineering is currently undergoing a paradigm shift regarding the concepts used to develop cell-based therapies for skeletal repair. In place of the "trial and error" approach, researchers aim at developing cellular concepts that mirror developmental and postnatal processes. Herein, we describe a model for in vivo endochondral remodeling of an in vitro derived cartilaginous intermediate and its applicability to bone engineering. In vitro differentiation of the continuous cell line, ATDC5, in pellet culture was enhanced in a medium containing ascorbic acid, insulin-transferrin-selenium, dexamethasone, and transforming growth factor β1, when compared with other tested preparations. This differentiation was characterized by the elevated expression of Collagen type II and X along with glycosaminoglycan (GAG) accumulation and the onset of hypertrophy. On combination with NuOss™, a clinically used bone void filler, and implantation in nude mice, the differentiated pellets further matured into GAG rich cartilaginous intermediates after 4 weeks. This was subsequently partially remodeled into osteocalcin-positive bone tissue after 8 weeks without further external manipulation, indicating the semi-autonomous nature of this implant. Mineralized tissue along with active osteoclast resorption and neo-angiogenesis was apparent throughout the implant. The bone volume was approximately eightfold higher (10.70%±0.99%) when using a cartilaginous intermediate (based on differentiated cell pellets) than when observed with cell-seeded scaffolds (1.19%±0.24% and 1.48%±0.35%), in both a differentiated and an undifferentiated state. This study highlights the potential of endochondral strategies for bone tissue engineering and allows the identification of the key cellular parameters for this process.

Critically ill patients are at increased risk of fractures during rehabilitation, and can experience impaired healing of traumatic and surgical bone fractures. In addition, markers of bone resorption are markedly increased in critically ill patients, while markers of bone formation are decreased. In the current study, we have directly investigated the effect of critical illness on bone metabolism and repair. In a human in vitro model of critical illness, Fluorescence-activated cell sorting (FACS) analysis revealed an increase in circulating CD14+/CD11b+ osteoclast precursors in critically ill patient peripheral blood compared to healthy controls. In addition, the formation of osteoclasts was increased in patient peripheral blood mononuclear cell (PBMC) cultures compared to healthy controls, both in the presence and absence of osteoclastogenic factors receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Culturing PBMCs with 10% critically ill patient serum further increased osteoclast formation and activity in patient PBMCs only, and neutralization studies revealed that immunoglobulin G (IgG) antibody signaling through the immunoreceptor Fc receptor common γ chain III (FcRγIII) played an important role. When analyzing bone formation, no differences in osteogenic differentiation were observed using human periosteal-derived cells (hPDCs) treated with patient serum in vitro, but a decrease in the expression of vascular endothelial growth factor receptor 1 (VEGF-R1) suggested impaired vascularization. This was confirmed using serum-treated hPDCs implanted onto calcium phosphate scaffolds in a murine in vivo model of bone formation, where decreased vascularization and increased osteoclast activity led to a decrease in bone formation in scaffolds with patient serum-treated hPDCs. Together, these findings may help to define novel therapeutic targets to prevent bone loss and optimize fracture healing in critically ill patients.

The Dark Side of Langerhans Cells Several immune cell populations reside in the skin and are thought to provide a protective barrier against infections and to act as sentinels against malignant transformation. However, studies in mice that lack Langerhans cells, a subset of dendritic cells, have suggested that these cells may actually promote tumorigenesis. Using a mouse model of squamous cell carcinoma, Modi et al. (p. 104 ) now reveal how Langerhans cells may promote the transformation of skin epithelial cells. In response to the carcinogen 7,12-dimethylbenz[α]anthracene (DMBA), Langerhans cells increased their expression of the cytochrome P-450 enzyme CYP1B1, which can metabolize DMBA to the mutagenic DMBA- trans -3,4-diol. Thus, besides their functions in regulating the adaptive immune response, Langerhans cells may participate in the metabolism of environmental carcinogens.

Current knowledge suggests that the periosteum, a fibrous tissue which covers the surface of all bones, contains a population of progenitor cells which mediate the repair of bone defects. In an effort to optimise the utilisation of this source of cells for bone engineering, herein we describe the rational selection of calcium phosphate (CaP) containing materials, based on biomaterial properties, and evaluation of their combined bone forming capacity. Five different commercially available orthopaedic 3D matrices composed of CaP particles in an open collagen network (NuOss™, CopiOs™, Bio-Oss(®), Collagraft™ and Vitoss(®)) were evaluated in vitro and in vivo with human periosteal derived cells (hPDCs). It was found that the cell-material combinations behaved quite differently in vivo, despite apparent in vitro similarities in gene expression profiles. Bone formation was highest within the NuOss™/hPDC implant at 13.03%, which also contained the highest incidence of bone marrow formation. The bone formed in this implant was chimeric with approximately 65% originating from implanted cells. Upon analysis of human specific gene expression, although it was found that predominantly osteogenic differentiation was observed within NuOss™/hPDC implants, a lesser induction of chondrogenic genes was also observed. The formation of a cartilage intermediate was confirmed by histology. Additionally the NuOss™/hPDC implant integrated into the mouse environment with apparent active scaffold resorption. This study demonstrates the importance of matching a cell support/biological matrix with a cell type and subsequently has outlined parameters which can be used for the rational selection of biomaterials for bone engineering.

Cell–cell interaction is an integral part of embryoid body (EB) formation controlling 3D aggregation. Manipulation of embryonic stem (ES) cell interactions could provide control over EB formation. Studies have shown a direct relationship between EB formation and ES cell differentiation. We have previously described a cell surface modification and cross-linking method for influencing cell–cell interaction and formation of multicellular constructs. Here we show further characterisation of this engineered aggregation. We demonstrate that engineering accelerates ES cell aggregation, forming larger, denser and more stable EBs than control samples, with no significant decrease in constituent ES cell viability. However, extended culture ≥5 days reveals significant core necrosis creating a layered EB structure. Accelerated aggregation through engineering circumvents this problem as EB formation time is reduced. We conclude that the proposed engineering method influences initial ES cell-ES cell interactions and EB formation. This methodology could be employed to further our understanding of intrinsic EB properties and their effect on ES cell differentiation.

In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft™ carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft™ carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the ‘osteoinductive program’. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies.

We report the ability to move and arrange patterns of live embryonic stem cells using holographic optical tweezers. Single cell suspensions of mouse embryonic stem cells were manipulated with holographic optical tweezers into a variety of patterns including lines, curves and circles. Individual cells were also lifted out of the sample plane highlighting the potential for 3D positional control. Trypan blue dye exclusion and Live/Dead™ staining (CMFDA−1, EthHD−1) showed that the cells were still viable after manipulation with the optical tweezers. The ability to move individual stem cells into specific, pre-defined patterns provides a method to study how arrangement and associated small-scale interactions occur between neighbouring cells.

Background: Current surgical techniques for the repair of the musculoskeletal system can be often limited by the availability, quality and quantity of materials, such as grafts to effect repair. This has led to the exploration and development of novel methods of intervention based on tissue engineering and regenerative medicine. Source of data: This review summarizes the successes and investigations which are happening to date in the field of musculoskeletal tissue engineering. This is based on an extensive literature search and through basic research being performed by the authors. Areas of agreement: Due to the constraints surrounding certain surgical techniques and restrictions on their use, novel procedures are required for the repair and regeneration of damaged tissues. Areas of controversy: The choice of cell type has caused much debate within the tissue-engineering field. However it is widely accepted that currently only autologous primary/adult stem cells are fit for transplantation, until such times that optimized differentiation and selection protocols exist for embryonic stem cells. Growing points: The current results of the clinical cases utilizing tissue engineered constructs for bone and cartilage repair provide insights for improvement of these techniques thus allowing treatments to become increasingly viable. Areas timely for developing research: There is a need to better understand the integration of scaffolds and cell populations into the target tissue. This should provide vital information influencing scaffold manufacturing procedures and cell selection.

The cartilage- and bone-forming properties of the periosteum have long since been recognized. As one of the major sources of skeletal progenitor cells, the periosteum plays a crucial role not only in bone development and growth, but also during bone fracture healing. Aided by the continuous expansion of tools and techniques, we are now starting to acquire more insight into the specific role and regulation of periosteal cells. From a therapeutic point of view, the periosteum has attracted much attention as a cell source for bone tissue engineering purposes. This interest derives not only from the physiological role of the periosteum during bone repair, but is also supported by the unique properties and marked bone-forming potential of expanded periosteum-derived cells. We provide an overview of the current knowledge of periosteal cell biology, focusing on the cellular composition and molecular regulation of this remarkable tissue, as well as the application of periosteum-derived cells in regenerative medicine approaches. This article is part of a Special Issue entitled "Stem Cells and Bone". publisher: Elsevier articletitle: Uncovering the periosteum for skeletal regeneration: The stem cell that lies beneath journaltitle: Bone articlelink: http://dx.doi.org/10.1016/j.bone.2014.08.007 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved. ispartof: BONE vol:70 pages:10-18 ispartof: location:United States status: published

PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase that has previously been implicated in generating inorganic phosphate (P(i)) for matrix mineralization. In this study, we have investigated PHOSPHO1 mRNA expression during embryonic development in the chick. Whole-mount in situ hybridization indicated that PHOSPHO1 expression occurred prior to E6.5 and was initially restricted to the bone collar within the mid-shaft of the diaphysis of long bones but by E11.5 expression was observed over the entire length of the diaphysis. Alcian blue/alizarin red staining revealed that PHOSPHO1 expression seen in the primary regions of ossification preceded the deposition of mineral, suggesting that it is involved in the initial events of mineral formation. We isolated MVs from growth plate chondrocytes and confirmed the presence of high levels of PHOSPHO1 by immunoblotting. Expression of PHOSPHO1, like TNAP activity, was found to be up-regulated in MVs isolated from chondrocytes induced to differentiate by the addition of ascorbic acid. This suggests that both enzymes may be regulated by similar mechanisms. These studies provide for the first time direct evidence that PHOSPHO1 is present in MVs, and its developmental expression pattern is consistent with a role in the early stages of matrix mineralization.

Intraepithelial lymphocytes constitute a group of T cells that express mainly monospecific or oligoclonal T cell receptors (TCRs). Like adaptive TCR alphabeta+ T cells, intraepithelial lymphocytes, a subset enriched in TCR gammadelta+ T cells, are proposed to be positively selected by thymically expressed self agonists, yet no direct evidence for this exists at present. Mouse dendritic epidermal T cells are prototypic intraepithelial lymphocytes, displaying an almost monoclonal TCR gammadelta+ repertoire. Here we describe an FVB substrain of mice in which this repertoire was uniquely depleted, resulting in cutaneous pathology. This phenotype was due to failure of dendritic epidermal T cell progenitors to mature because of a heritable defect in a dominant gene used by the thymic stroma to 'educate' the natural, skin-associated intraepithelial lymphocyte repertoire to be of physiological use.

Upregulation of the inducible gene products MICA (human) and Rae-1 (mouse) may promote tumor surveillance and autoimmunity by engaging the activating receptor NKG2D on natural killer (NK) cells and T cells. Nevertheless, sustained expression of MICA by tumors can also elicit NKG2D downregulation, perhaps indicating 'immunoevasion'. Investigating this paradox, we report here that constitutive Rae-1epsilon transgene expression in normal epithelium elicited local and systemic NKG2D downregulation, generalized but reversible defects in NK cell-mediated cytotoxicity and mild CD8(+) T cell defects. The extent of NKG2D downregulation correlated well with the incidence and progression of cutaneous carcinogenesis, emphasizing the utility of NKG2D as a marker of tumor resistance. Thus, NKG2D engagement is a natural mediator of immunosurveillance, which can be compromised by locally sustained ligand expression but potentially restored by innate immune activation.

Epithelial tissues in which carcinomas develop often contain systemically derived T cell receptor (TCR)alphabeta+ cells and resident intraepithelial lymphocytes that are commonly enriched in TCRgammadelta+ cells. Recent studies have demonstrated that gammadelta cells protect the host against chemically induced cutaneous malignancy, but the role of alphabeta T cells has been enigmatic, with both protective and tumor-enhancing contributions being reported in different systems. This study aims to clarify the contributions of each T cell type to the regulation of squamous cell carcinoma induced in FVB mice by a two-stage regimen of 7,12-dimethylbenz[a]anthracene initiation followed by repetitive application of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. This protocol permits one to monitor the induction of papillomas and the progression of those papillomas to carcinomas. The results show that whereas gammadelta cells are strongly protective, the nonredundant contributions of alphabeta T cells to the host's protection against papillomas are more modest. Furthermore, at both high and low doses of carcinogens, alphabeta T cells can contribute to rather than inhibit the progression of papillomas to carcinomas. As is likely to be the case in humans, this study also shows that the contribution of T cells to tumor immunosurveillance is regulated by modifier genes.

Strategies for bone regeneration are undergoing a paradigm shift, moving away from the replication of end-stage bone tissue and instead aiming to recapture the initial events of fracture repair. Although this is known to resemble endochondral bone formation, chondrogenic cell types with favorable proliferative and hypertrophic differentiation properties are lacking. Recent advances in cellular reprogramming have allowed the creation of alternative cell populations with specific properties through the forced expression of transcription factors. Herein, we investigated the in vitro hypertrophic differentiation and in vivo tissue formation capacity of induced chondrogenic cells (iChon cells) obtained through direct reprogramming. In vitro hypertrophic differentiation was detected in iChon cells that contained a doxycycline-inducible expression system for Klf4, cMyc, and Sox9. Furthermore, endochondral bone formation was detected after implantation in nude mice. The bone tissue was derived entirely from host origin, whereas cartilage tissue contained cells from both host and donor. The results obtained highlight the promise of cellular reprogramming for the creation of functional skeletal cells that can be used for novel bone healing strategies. ispartof: Cellular reprogramming vol:16 issue:1 pages:29-39 ispartof: location:United States status: published