Blocking the expression of both bone sialoprotein (BSP) and osteopontin (OPN) impairs the anabolic action of PTH in mouse calvaria bone.

Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1-84 (iPTH) over the calvaria of BSP knockout (-/-) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and -/- mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from -/- mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5× higher in -/- versus +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in -/- cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP +/+ calvaria, while it blunted iPTH effects in -/- mice, reduced to a modest increase in periosteum thickness. In -/- (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins.

Periostin and transforming growth factor ß-induced protein (TGFßIp) are both expressed by osteoblasts and osteoclasts.

Periostin (Postn) and transforming growth factor ß-induced protein (TGFßIp) are two closely related extracellular matrix (ECM) proteins predominantly distributed in collagen-rich connective tissues submitted to mechanical strain, including bone and more specifically the periosteum. We have investigated the expression of Postn and TGFßIp mRNA by primary osteoblasts isolated from mouse periosteum and calvaria, or by the osteoblast-like MC3T3-E1 cell line, and by osteoclasts from mouse long bones differentiated in vitro. Secretion of Postn was measured with a specific ELISA. Postn and TGFßIp mRNA were concomitantly expressed in all three osteoblast models all along the differentiation process in a time-dependent manner. Both Postn and TGFßIp transcripts appeared early in osteoblast differentiation, and their expression increased 3-10 times in mature osteoblast cells. Expression decreased after differentiation was achieved and when the cultures mineralised. ELISA for secreted Postn showed a similar pattern. When MC3T3-E1 cells were treated with TGF-ß, Postn and TGFßIp mRNA expression and secretion were stimulated, whereas 1.25(OH)(2)D(3) had no detectable effect. Osteoclasts also expressed both Postn and TGFßIp during in vitro differentiation. Expression of both Postn and TGFßIp peaks in the early phases of osteoblast differentiation, and decreases later at the start of mineralisation. A novel finding is that Postn and TGFßIp are expressed by osteoclasts in vitro. Therefore Postn and TGFßIp proteins are potential biomarkers of early osteoblast differentiation and new bone formation.

Tailoring inkjet-printed PEDOT:PSS composition toward green, wearable device fabrication.

Inkjet printing remains one of the most cost-efficient techniques for device prototyping and manufacturing, offering considerable freedom of digital design, non-contact, and additive fabrication. When developing novel wearable devices, a balanced approach is required between functional, user-safe materials and scalable manufacturing processes. Here, we propose a tailor-made ink formulation, based on non-hazardous materials, to develop green electronic devices aimed at interfacing with humans. We demonstrate that developed ink exhibits high-resolution inkjet printability, in line with theoretical prediction, on multiple wearable substrates. The ink's chemical composition ensures the pattern's enhanced electrical properties, mechanical flexibility, and stability in water. The cytocompatibility evaluations show no noxious effects from printed films in contact with human mesenchymal stem cells. Finally, we fabricated a printed wearable touch sensor on a non-woven fabric substrate, capable of tracking human steps. This is a step toward the development of green wearable electronics manufacturing, demonstrating a viable combination of materials and processes for biocompatible devices.

Laser-Based Hybrid Manufacturing of Endosseous Implants: Optimized Titanium Surfaces for Enhancing Osteogenic Differentiation of Human Mesenchymal Stem Cells.

Additive manufacturing (AM) is becoming increasingly important in the orthopedic and dental sectors thanks to two major advantages: the possibility of custom manufacturing and the integration of complex structures. However, at smaller scales, surface conditions of AM products are not mastered. Numerous non-fused powder particles give rise to roughness values (Sa) greater than 10 µm, thus limiting biomedical applications since the surface roughness of, e.g., metal implants plays a major role in the quality and rate of osseointegration. In this study, an innovative hybrid machine combining AM and a femtosecond laser (FS) was used to obtain Ti6Al4V parts with biofunctional surfaces. During the manufacturing process, the FS laser beam "neatly" ablates the surface, leaving in its path nanostructures created by the laser/matter interaction. This step decreases the Sa from 11 to 4 µm and increases the surface wettability. The behavior of human mesenchymal stem cells was evaluated on these new AM+FS surfaces and compared with that on AM surfaces and also on polished surfaces. The number of cells attached 24 h after plating is equivalent on all surfaces, but cell spreading is higher on AM+FS surfaces compared with their AM counterparts. In the longer term (days 7 and 14), fibronectin and collagen synthesis increase on AM+FS surfaces as opposed to AM alone. Alkaline phosphatase activity, osteocalcin production, and mineralization, markers of osteogenic differentiation, are significantly lower on raw AM surfaces, whereas on the AM+FS specimens they display a level equivalent to that on the polished surface. Overall, these results indicate that using an FS laser beam during the fabrication of AM parts optimizes surface morphology to favor osteoblastic differentiation. This new hybrid machine could make it possible to produce AM implants with functional surfaces directly at the end of AM, thereby limiting their post-treatments.

Corrigendum: Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

This corrects the article DOI: 10.1038/ncomms11208.

The role of the SIBLING, Bone Sialoprotein in skeletal biology - Contribution of mouse experimental genetics.

Bone Sialoprotein (BSP) is a member of the "Small Integrin-Binding Ligand N-linked Glycoproteins" (SIBLING) extracellular matrix protein family of mineralized tissues. BSP has been less studied than other SIBLING proteins such as Osteopontin (OPN), which is coexpressed with it in several skeletal cell types. Here we review the contribution of genetically engineered mice (BSP gene knockout and overexpression) to the understanding of the role of BSP in the bone organ. The studies made so far highlight the role of BSP in skeletal mineralization, as well as its importance for proper osteoblast and osteoclast differentiation and activity, most prominently in primary/repair bone. The absence of BSP also affects the local environment of the bone tissue, in particular hematopoiesis and vascularization. Interestingly, lack of BSP induces an overexpression of OPN, and the cognate protein could be responsible for some aspects of the BSP gene knockout skeletal phenotype, while replacing BSP for some of its functions. Such interplay between the partly overlapping functions of SIBLING proteins, as well as the network of cross-regulations in which they are involved should now be the focus of further work.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

In vitro three-dimensional bone tissue models: from cells to controlled and dynamic environment.

Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not reproduce the complex and dynamic three-dimensional (3D) environment experienced by cells in vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology. These in-vitro-engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical, and biomechanical cues that define the in vivo bone environment, while allowing their monitoring. This review focuses on state-of-the-art and the current advances in the development of 3D culture systems for bone biology research. It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, that is, scaffold, cells, and perfusion bioreactor systems. Finally, future challenges for noninvasive imaging technologies are addressed.

The impairment of osteogenesis in bone sialoprotein (BSP) knockout calvaria cell cultures is cell density dependent.

Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.

Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass.

Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing primary mineralization.