Osteoblast Progenitor Cells Research Articles

Doxycycline restores the impaired osteogenic commitment of diabetic-derived bone marrow mesenchymal stromal cells by increasing the canonical WNT signaling

Diabetes mellitus comprehends a group of chronic metabolic disorders, associated with damage and dysfunction of distinct tissues, including bone. At the cellular level, an impaired osteoblastogenesis has been reported, affecting the viability, proliferation and functionality of osteoblasts and precursor populations, hampering the bone metabolic activity, remodeling and healing. Tetracyclines embrace a group of broad-spectrum antibacterial compounds with potential anabolic effects on the bone tissue, through antibacterial-independent mechanisms. Accordingly, this study aims to address the modulatory capability and associated molecular signaling of a low dosage doxycycline – a semi-synthetic tetracycline, in the functional activity of osteoblastic progenitor cells (bone marrow-derived mesenchymal stromal cells), established from a translational diabetic experimental model.Bone marrow-derived mesenchymal stromal cells were isolated from streptozotocin-induced diabetic Wistar rat with proven osteopenia. Cultures were characterized, in the presence of doxycycline (1 μg ml−1) for proliferation, metabolic activity, apoptosis, collagen synthesis and relevant gene expression with the osteogenic and adipogenic program. The activation of the Wnt/β-catenin pathway was further detailed.Doxycycline normalized the viability, proliferation and metabolic activity of the established cultures, further decreasing cell apoptosis, to levels similar to control. The addition of this drug to the culture environment further increased the osteogenic activation, upregulating the expression of osteogenic markers and collagen synthesis, at the same time that a decreased adipogenic priming was attained. These processes were found to me mediated, at least in part, by the restoration of the signaling through the Wnt/β-catenin pathway.

Loss of p53 in mesenchymal stem cells promotes alteration of bone remodeling through negative regulation of osteoprotegerin

p53 plays a pivotal role in controlling the differentiation of mesenchymal stem cells (MSCs) by regulating genes involved in cell cycle and early steps of differentiation process. In the context of osteogenic differentiation of MSCs and bone homeostasis, the osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB (OPG/RANKL/RANK) axis is a critical signaling pathway. The absence or loss of function of p53 has been implicated in aberrant osteogenic differentiation of MSCs that results in higher bone formation versus erosion, leading to an unbalanced bone remodeling. Here, we show by microCT that mice with p53 deletion systemically or specifically in mesenchymal cells possess significantly higher bone density than their respective littermate controls. There is a negative correlation between p53 and OPG both in vivo by analysis of serum from p53+/+, p53+/−, and p53−/− mice and in vitro by p53 knockdown and ChIP assay in MSCs. Notably, high expression of Opg or its combination with low level of p53 are prominent features in clinical cancer lesion of osteosarcoma and prostate cancer respectively, which correlate with poor survival. Intra-bone marrow injection of prostate cancer cells, together with androgen can suppress p53 expression and enhance local Opg expression, leading to an enhancement of bone density. Our results support the notion that MSCs, as osteoblast progenitor cells and one major component of bone microenvironment, represent a cellular source of OPG, whose amount is regulated by the p53 status. It also highlights a key role for the p53-OPG axis in regulating the cancer associated bone remodeling.

Strategic Use of Ultrasonic Frequencies for Targeted Bone Biomodification Following Piezoelectric Bone Surgery in Rats. Part II: Late Phase.

Piezoelectric surgery utilizes ultrasonic vibrations to cut bone more precisely and less traumatically than conventional methods. The regional acceleratory phenomenon following bone injury has a demineralization phase followed by a remineralization phase. Part I of this study on rats assessed the biologic modifications following bone injuries with the piezoelectric knife at 10-Hz and 30-Hz modulation frequencies. Part II focuses on piezoelectric surgery-regulated osteoblast activity and changes occurring in the bone during the regeneration phase. The results indicate that at 30 Hz, the remineralization process starts at day 14 and continues until day 70, with osteoblast progenitor cells observed in the periodontal ligament around acellular new bone as early as day 14. These findings emphasize the potential for regeneration in the late postoperative phase and the possible use of the piezoelectric knife as an adjunct for guided bone regeneration, site development, or site preparation for dental implants.

Mesenchymal stem cells modifications for enhanced bone targeting and bone regeneration.

In pathological bone conditions (e.g., osteoporotic fractures or critical size bone defects), increasing the pool of osteoblast progenitor cells is a promising therapeutic approach to facilitate bone healing. Since mesenchymal stem cells (MSCs) give rise to the osteogenic lineage, a number of clinical trials investigated the potential of MSCs transplantation for bone regeneration. However, the engraftment of transplanted cells is often hindered by insufficient oxygen and nutrients supply and the tendency of MSCs to home to different sites of the body. In this review, we discuss various approaches of MSCs transplantation for bone regeneration including scaffold and hydrogel constructs, genetic modifications and surface engineering of the cell membrane aimed to improve homing and increase cell viability, proliferation and differentiation.

Loss of receptor tyrosine kinase-like orphan receptor 2 impairs the osteogenesis of mBMSCs by inhibiting signal transducer and activator of transcription 3

BackgroundReceptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood. Signal transducer and activator of transcription 3 (Stat3) takes part in maintaining bone homeostasis. The aim of this study is to reveal the role and mechanism of Ror2 in the osteogenic differentiation from mouse bone marrow mesenchymal stem cells (mBMSCs) and to explore the effect of Stat3 on Ror2-mediated osteogenesis.MethodsRor2 CKO mice were generated via the Cre-loxp recombination system using Prrx1-Cre transgenic mice. Quantitative real-time PCR and western blot were performed to assess the expression of Stat3 and osteogenic markers in Ror2-knockdown mBMSCs (mBMSC-sh-Ror2). After being incubated in osteogenic induction medium for 3 weeks, Alizarin Red staining and western blot were used to examine the calcium deposit and osteogenic markers in Stat3 overexpression in mBMSC-sh-Ror2.ResultsLoss of Ror2 in mesenchymal or osteoblast progenitor cells led to a dwarfism phenotype in vivo. The mRNA expression of osteogenic markers (osteocalcin, osteopontin (OPN), and collagen I) in the ulna proximal epiphysis of Ror2 CKO mice was significantly decreased (P < 0.05). The mRNA and protein expression of Stat3 and osteogenic markers (Runx2, osterix, and OPN) decreased in mBMSC-sh-Ror2 cells (P < 0.05). The overexpression of Stat3 in mBMSC-sh-Ror2 cells rescued the calcium deposit and expression of Runx2, osterix, and OPN to a level comparable to normal mBMSCs.ConclusionsRor2 was essential for skeleton development by regulating mBMSCs’ osteogenesis and osteoblast differentiation. Loss of Ror2 may impair the osteogenesis of mBMSCs by inhibiting Stat3.

Piezo1/2 mediate mechanotransduction essential for bone formation through concerted activation of NFAT-YAP1-ß-catenin.

Mechanical forces are fundamental regulators of cell behaviors. However, molecular regulation of mechanotransduction remain poorly understood. Here, we identified the mechanosensitive channels Piezo1 and Piezo2 as key force sensors required for bone development and osteoblast differentiation. Loss of Piezo1, or more severely Piezo1/2, in mesenchymal or osteoblast progenitor cells, led to multiple spontaneous bone fractures in newborn mice due to inhibition of osteoblast differentiation and increased bone resorption. In addition, loss of Piezo1/2 rendered resistant to further bone loss caused by unloading in both bone development and homeostasis. Mechanistically, Piezo1/2 relayed fluid shear stress and extracellular matrix stiffness signals to activate Ca2+ influx to stimulate Calcineurin, which promotes concerted activation of NFATc1, YAP1 and ß-catenin transcription factors by inducing their dephosphorylation as well as NFAT/YAP1/ß-catenin complex formation. Yap1 and ß-catenin activities were reduced in the Piezo1 and Piezo1/2 mutant bones and such defects were partially rescued by enhanced ß-catenin activities.

Glucosylceramide synthase regulates adipo-osteogenic differentiation through synergistic activation of PPARγ with GlcCer.

Dysregulation of the adipo-osteogenic differentiation balance of mesenchymal stem cells (MSCs), which are common progenitor cells of adipocytes and osteoblasts, has been associated with many pathophysiologic diseases, such as obesity, osteopenia, and osteoporosis. Growing evidence suggests that lipid metabolism is crucial for maintaining stem cell homeostasis and cell differentiation; however, the detailed underlying mechanisms are largely unknown. Here, we demonstrate that glucosylceramide (GlcCer) and its synthase, glucosylceramide synthase (GCS), are key determinants of MSC differentiation into adipocytes or osteoblasts. GCS expression was increased during adipogenesis and decreased during osteogenesis. Targeting GCS using RNA interference or a chemical inhibitor enhanced osteogenesis and inhibited adipogenesis by controlling the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ). Treatment with GlcCer sufficiently rescued adipogenesis and inhibited osteogenesis in GCS knockdown MSCs. Mechanistically, GlcCer interacted directly with PPARγ through A/B domain and synergistically enhanced rosiglitazone-induced PPARγ activation without changing PPARγ expression, thereby treatment with exogenous GlcCer increased adipogenesis and inhibited osteogenesis. Animal studies demonstrated that inhibiting GCS reduced adipocyte formation in white adipose tissues under normal chow diet and high-fat diet feeding and accelerated bone repair in a calvarial defect model. Taken together, our findings identify a novel lipid metabolic regulator for the control of MSC differentiation and may have important therapeutic implications.

Suitability and limitations of mesenchymal stem cells to elucidate human bone illness.

Functional impairment of mesenchymal stem cells (MSCs), osteoblast progenitor cells, has been proposed to be a pathological mechanism contributing to bone disorders, such as osteoporosis (the most common bone disease) and other rare inherited skeletal dysplasias. Pathological bone loss can be caused not only by an enhanced bone resorption activity but also by hampered osteogenic differentiation of MSCs. The majority of the current treatment options counteract bone loss, and therefore bone fragility by blocking bone resorption. These so-called antiresorptive treatments, in spite of being effective at reducing fracture risk, cannot be administered for extended periods due to security concerns. Therefore, there is a real need to develop osteoanabolic therapies to promote bone formation. Human MSCs emerge as a suitable tool to study the etiology of bone disorders at the cellular level as well as to be used for cell therapy purposes for bone diseases. This review will focus on the most relevant findings using human MSCs as an in vitro cell model to unravel pathological bone mechanisms and the application and outcomes of human MSCs in cell therapy clinical trials for bone disease.

Abstract 2161: In vitro osteoblast assays for studying the effects of cancer therapeutics on bone biology

Abstract Bone cell activity is an important factor in regulating bone metastases from tumor cells in the skeletal environment. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Bone then releases stimulatory factors that in turn promote the growth and proliferation of cancer cells. This process is called the vicious cycle often leading to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. Osteolytic lesions are common in breast and lung cancer and multiple myeloma. There is a vicious cycle between cancer cells and bone cells also in the context of osteoblastic lesions. In osteoblastic metastases, the vicious cycle observed in osteolytic disease takes place but in addition to this cancer cells produce osteoblast-stimulating factors including bone morphogenetic protein (BMP), epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Osteoblasts also influence osteoclasts by producing RANKL, which stimulates osteoclast differentiation. Many cancer patients with bone metastasis have both osteolytic and osteoblastic lesions. This is common for example in prostate cancer. Our aim was to establish an in vitro cell culture model to study the effects of compounds on osteoblast differentiation and activity. A mouse osteoblast progenitor cell line KS483 was used in the study. BMP-2 was used as a test compound and was added in the beginning of culture and simultaneously with culture medium change every 3-4 days. Ascorbic acid was added into the cell culture medium at day 4. In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8 from cell lysates. Total protein content was measured from the same samples. In the osteoblast activity assay, KS483 mouse osteoprogenitor cells were cultured for 13 days, during which N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to demonstrate effects on organic bone matrix formation. β-glycerophosphate was added to the culture at day 11. The cultures were stopped at day 13 by removing the culture media from the wells and adding hydrochloric acid. Calcium deposition, a marker of inorganic bone formation, was determined at the end of the study. BMP-2 stimulated osteoblast differentiation and activity shown by the increase in ALP, PINP and calcium levels. The results suggest that the KS483 cell line can be used for setting up reliable in vitro models of osteoblast differentiation and activity. These osteoblast assays can be used for studying the effects of compounds on bone formation and cancer related bone events. Citation Format: Jenni H. Mäki-Jouppila, Jussi M. Halleen, Katja Fagerlund. In vitro osteoblast assays for studying the effects of cancer therapeutics on bone biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2161.

Real-Time Imaging of Bacteria/Osteoblast Dynamic Coculture on Bone Implant Material in an in Vitro Postoperative Contamination Model.

Biomedical implants are an important part of evolving modern medicine but have a potential drawback in the form of postoperative pathogenic infection. Accordingly, the "race for surface" combat between invasive bacteria and host cells determines the fate of implants. Hence, proper in vitro systems are required to assess effective strategies to avoid infection. In this study, we developed a real time observation model, mimicking postoperative contamination, designed to follow E. coli proliferation on a titanium surface occupied by human osteoblastic progenitor cells (STRO). This model allowed us to monitor E. coli invasion of human cells on titanium surfaces coated and uncoated with fibronectin. We showed that the surface colonization of bacteria is significantly enhanced on fibronectin coated surfaces irrespective of whether areas were uncovered or covered with human cells. We further revealed that bacterial colonization of the titanium surfaces is enhanced in coculture with STRO cells. Finally, this coculture system provides a comprehensive system to describe in vitro and in situ bacterial and human cells and their localization but also to target biological mechanisms involved in adhesion as well as in interactions with surfaces, thanks to fluorescent labeling. This system is thus an efficient method for studies related to the design and function of new biomaterials.

KIAA1199 is a secreted molecule that enhances osteoblastic stem cell migration and recruitment

Factors mediating mobilization of osteoblastic stem and progenitor cells from their bone marrow niche to be recruited to bone formation sites during bone remodeling are poorly known. We have studied secreted factors present in the bone marrow microenvironment and identified KIAA1199 (also known as CEMIP, cell migration inducing hyaluronan binding protein) in human bone biopsies as highly expressed in osteoprogenitor reversal cells (Rv.C) recruited to the eroded surfaces (ES), which are the future bone formation sites. In vitro, KIAA1199 did not affect the proliferation of human osteoblastic stem cells (also known as human bone marrow skeletal or stromal stem cells, hMSCs); but it enhanced cell migration as determined by scratch assay and trans-well migration assay. KIAA1199 deficient hMSCs (KIAA1199down) exhibited significant changes in cell size, cell length, ratio of cell width to length and cell roundness, together with reduction of polymerization actin (F-actin) and changes in phos-CFL1 (cofflin1), phos-LIMK1 (LIM domain kinase 1) and DSTN (destrin), key factors regulating actin cytoskeletal dynamics and cell motility. Moreover, KIAA1199down hMSC exhibited impaired Wnt signaling in TCF-reporter assay and decreased expression of Wnt target genes and these effects were rescued by KIAA1199 treatment. Finally, KIAA1199 regulated the activation of P38 kinase and its associated changes in Wnt-signaling. Thus, KIAA1199 is a mobilizing factor that interacts with P38 and Wnt signaling, and induces changes in actin cytoskeleton, as a mechanism mediating recruitment of hMSC to bone formation sites.

Composite bilayered scaffolds with bio-functionalized ceramics for cranial bone defects: An in vivo evaluation

Despite having substantial regenerative capabilities, bone regeneration in critical injuries may be insufficient and require an additional intervention. With advancements in material science and production technology it is now possible to generate complex scaffolds with controlled architectures for repairing these injuries. Additionally, these materials can be functionalized with bioactive molecules to enhance osteoinductivity. In the present work, we developed a multifunctional composite bilayered scaffold (BS), integrating a ceramic nanocement (NC) and macroporous composite scaffold (CG) for cranial injuries, mimicking bone architecture. The scaffolds were functionalized with recombinant human bone morphogenetic protein-2 (rhBMP-2) (BMP) (2 μg/scaffold) and zoledronic acid (ZA) (10 μg/scaffold). We hypothesized that the composite scaffolds would support proliferation of osteoblast progenitor cells and provide controlled release of loaded bioactive molecules to induce bone regeneration. Higher amounts of mineralized tissue (MT) deposition was observed with functionalized scaffolds 12 weeks post in vivo implantation in 8.5 mm critical cranial defect in rats. Contrary to our expectations, BS + ZA functionalized scaffolds had highest MT deposition (13.9 mm3), followed by CG + ZA + BMP with 9.2 mm3 and BS + ZA + BMP with 7.6 mm3 of MT deposition, all significantly higher than non-functionalized CG (7.2 mm3) or BS (4.9 mm3) scaffolds and the empty [Teotia et al 2017 ACS Appl. Mater. Interfaces, 9, 6816–6828] groups. The results supported an osteopromotive multifunctional scaffold implantation in critical defects.

Estrogens and selective estrogen receptor modulators differentially antagonize Runx2 in ST2 mesenchymal progenitor cells

Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17ß-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1–1 nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ≥10 nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1–1 nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy.

Growth hormone facilitates 5′-azacytidine-induced myogenic but inhibits 5′-azacytidine-induced adipogenic commitment in C3H10T1/2 mesenchymal stem cells

The C3H10T1/2 cells are considered mesenchymal stem cells (MSCs) because they can be induced to become the progenitor cells for myocytes, adipocytes, osteoblasts, and chondrocytes by the DNA methyltransferase inhibitor 5′-azacytidine. In this study, we determined the effect of growth hormone (GH) on the myogenic and adipogenic lineage commitment in C3H10T1/2 cells. The C3H10T1/2 cells were treated with recombinant bovine GH in the presence or absence of 5′-azacytidine for 4 days. The myogenic commitment in C3H10T1/2 cells was assessed by immunostaining them for MyoD, the marker for myoblasts, and by determining their capacity to differentiate into the multinucleated myotubes. The adipogenic commitment in C3H10T1/2 cells was assessed by determining their ability to differentiate into adipocytes. Myotubes and adipocyteswere identified by immunocytochemistry and Oil Red O staining, respectively. C3H10T1/2 cells treated with 5′-azacytidine and GH for 4 days contained a greater percentage of MyoD-positive cells than those treated with 5′-axacytidine alone (P < 0.05). The former generated more myotubes than the latter upon induced myoblast differentiation (P < 0.05). However, C3H10T1/2 cells treated with GH alone did not form any myotubes. C3H10T1/2 cells treated with 5′-azacytidine formed adipocytes upon adipocyte differentiation induction, whereas C3H10T1/2 cells treated with GH alone did not form any adipocytes. C3H10T1/2 cells treated with both 5′-azacytidine and GH formed fewer adipocytes than those treated with 5′-azacytidine alone (P < 0.05). Both GHR and IGF-I mRNA expression in C3H10T1/2 cells were increased by 5′-azacytidine (P < 0.05), but neither was affected by GH. Overall, this study showed that GH enhanced 5′-azacytidine-induced commitment in C3H10T1/2 cells to myoblasts but inhibited 5′-azacytidine-induced commitment to preadipocytes. These results support the possibility that GH stimulates skeletal muscle growth and inhibits adipose tissue growth in part by stimulating the myogenic commitment and inhibiting the adipogenic commitment, respectively, in mesenchymal stem cells.

Clinical Application of Impact Capacitive – Resistive Electric Transfer 448 kHz on Human Cells

Mesenchymal stem cells (MSCs) can differentiate into more than one type of specialist cells in our body. They are a potential source of progenitor cells for osteoblasts, chondroblasts, adipocytes, skeletal muscles, and cardiomyocytes. They may also differentiate into ecto- and endodermal cell lines, e.g., neural cells, glial cells, hepatocytes and karetinocytes. Mesenchymal cells represent only 0.001–0.01% of all bone marrow cells, are a crucial population of cells participating in the proliferative phase of damage regeneration, and they are present in nearly all body tissues, the largest number of them is in adipose tissue and blood. Properties of MSCs have formed foundations for a new interdisciplinary field, tissue engineering. Its extensive applications include aesthetic medicine, dermatology, orthopaedics, plastic surgery, physioaesthetics, and sports medicine. The aim of this study is to present selected properties of the mesenchymal stem cells exposed to an electric stimulus of frequency of 448 kHz using Capacitive-Resistive Electric Transfer (CRET) technology.

Mouse models to evaluate the role of estrogen receptor α in skeletal maintenance and adaptation

Estrogen signaling and mechanical loading have individual and combined effects on skeletal maintenance and adaptation. Previous work investigating estrogen signaling both in vitro and in vivo using global estrogen receptor α (ERα) gene knockout mouse models has provided information regarding the role of ERα in regulating bone mass and adaptation to mechanical stimulation. However, these models have inherent limitations that confound interpretation of the data. Therefore, recent studies have focused on mice with targeted deletion of ERα from specific bone cells and their precursors. Cell stage, tissue type, and mouse sex all influence the effects of ERα gene deletion. Lack of ERα in osteoblast progenitor and precursor cells generally affects the periosteum of female and male mice. The absence of ERα in differentiated osteoblasts, osteocytes, and osteoclasts in mice generally resulted in reduced cancellous bone mass, with differing reports of the effect by animal sex and greater deficiencies in bone mass typically occurring in cancellous bone in female mice. Limited data exist for the role of bone cell-specific ERα in skeletal adaptation in vivo. Cell-specific ERα gene knockout mice provide an excellent platform for investigating the function of ERα in regulating skeletal phenotype and response to mechanical loading by sex and age.

Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

* Calvarial Bone Regeneration Is Enhanced by Sequential Delivery of FGF-2 and BMP-2 from Layer-by-Layer Coatings with a Biomimetic Calcium Phosphate Barrier Layer.

A drug delivery coating for synthetic bone grafts has been developed to provide sequential delivery of multiple osteoinductive factors to better mimic aspects of the natural regenerative process. The coating is composed of a biomimetic calcium phosphate (bCaP) layer that is applied to a synthetic bone graft and then covered with a poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) film. Bone morphogenetic protein-2 (BMP-2) was applied before the coating process directly on the synthetic bone graft and then, bCaP-PEM was deposited followed by adsorption of fibroblast growth factor-2 (FGF-2) into the PEM layer. Cells access the FGF-2 immediately, while the bCaP-PEM temporally delays the cell access to BMP-2. In vitro studies with cells derived from mouse calvarial bones demonstrated that Sca-1 and CD-166 positive osteoblast progenitor cells proliferated in response to media dosing with FGF-2. Coated scaffolds with BMP-2 and FGF-2 were implanted in mouse calvarial bone defects and harvested at 1 and 3 weeks. After 1 week in vivo, proliferation of cells, including Sca-1+ progenitors, was observed with low dose FGF-2 and BMP-2 compared to BMP-2 alone, indicating that in vivo delivery of FGF-2 activated a similar population of cells as shown by in vitro testing. At 3 weeks, FGF-2 and BMP-2 delivery increased bone formation more than BMP-2 alone, particularly in the center of the defect, confirming that the proliferation of the Sca-1 positive osteoprogenitors by FGF-2 was associated with increased bone healing. Areas of bone mineralization were positive for double fluorochrome labeling of calcium and alkaline phosphatase staining of osteoblasts, along with increased TRAP+ osteoclasts, demonstrating active bone formation distinct from the bone-like collagen/hydroxyapatite scaffold. In conclusion, the addition of a bCaP layer to PEM delayed access to BMP-2 and allowed the FGF-2 stimulated progenitors to populate the scaffold before differentiating in response to BMP-2, leading to improved bone defect healing.

SERPINB2 is a novel TGF\u03b2-responsive lineage fate determinant of human bone marrow stromal cells

TGF-β1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-β1 (10 ng/ml) treatment enhanced OS differentiation as evidenced by increased mineralised matrix production. Conversely, pulsed TGF-β1 administration during the commitment phase increased mature lipid-filled adipocyte numbers. Global gene expression analysis using DNA microarrays in hBMSCs treated with TGF-β1 identified 1587 up- and 1716 down-regulated genes in OS-induced, TGF-β1-treated compared to OS-induced hBMSCs (2.0 fold change (FC), p < 0.05). Gene ontology (GO) analysis revealed enrichment in ‘osteoblast differentiation’ and ‘skeletal system development-associated’ genes and up-regulation of several genes involved in ‘osteoblastic-differentiation related signalling pathways’. In AD-induced, TGF-β1-treated compared to AD-induced hBMSCs, we identified 323 up- and 369 down-regulated genes (2.0 FC, p < 0.05) associated with ‘fat cell differentiation’, ‘fatty acid derivative biosynthesis process’, ‘fatty acid derivative metabolic process’, and ‘inositol lipid-mediated’. Serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2) was down-regulated 3-fold in TGF-β1-treated hBMSCs. siRNA-mediated SERPINB2 inhibition enhanced OS and AD differentiation. Thus, TGF-β signalling is important for hBMSC OS and AD differentiation and SERPINB2 is a TGF-β-responsive gene that plays a negative regulatory role in hBMSC differentiation.

Biofabrication of a co-culture system in an osteoid-like hydrogel matrix

Biofabrication aims to develop functional, biological constructs using automated processes (additive manufacturing, AM) involving different cell types and biomaterials (Groll et al 2016 Biofabrication 13001 1–6). As bone tissue is based on the crosstalk between osteoblasts and osteoclasts at least, evaluating cell–cell and cell–material interactions is of interest to understand bone remodeling. There is increasing interest in the role of osteoclasts not only considering bone resorption, but also their influence on the proliferation, migration and differentiation of osteoblasts. Osteoid-like, non-mineralized matrix is used here for the 3D cultivation of osteoblast and osteoclast progenitor cells to evaluate interactions in an early stage of bone formation. The AM technology bioplotting was used to tailor a 3D environment with defined properties. These results could be helpful to transfer this approach to the fabrication of bone tissue in regenerative medicine approaches. Gelatin is derived from collagen, which is the main phase of osteoid. Oxidized alginate–gelatin crosslinked hydrogel was used to immobilize osteoblastic (ST2) and osteoclastic (RAW) progenitor cells. Cell viability and number, the expression of different proteins like alkaline phosphatase (ALP), osteopontin (OPN) and tartrate resistant acid phosphatase (TRAP) were investigated. Release of vascular endothelial growth factor (VEGF) by the immobilized cells was analyzed. Microscopy techniques were used to evaluate cell morphology during an incubation period of 21 days. The biofabrication process was compatible with the cells. Cells migrated, proliferated and expressed their specific proteins indicating cell differentiation. The co-culture showed increased OPN concentration, which is a major protein of the osteoid involved in the mineralization process. TRAP activity was increased compared to single culture. ST2 single culture showed higher ALP activity compared to the co-culture. VEGF concentration of the co-culture was strongly increased. The results indicate the importance of using co-cultures to fabricate bone tissue by biofabrication. Especially the influence of the osteoblast/osteoclast crosstalk, in an early stage of bone formation, is shown here, using a 3D hydrogel based cell culture model created by biofabrication.