Convergence of Calcium Channel Regulation and Mechanotransduction in Skeletal Regenerative Biomaterial Design.

Cells are known to perceive their microenvironment through extracellular and intracellular mechanical signals. Upon sensing mechanical stimuli, cells can initiate various downstream signaling pathways that are vital to regulating proliferation, growth, and homeostasis. One such physiologic activity modulated by mechanical stimuli is osteogenic differentiation. The process of osteogenic mechanotransduction is regulated by numerous calcium ion channels-including channels coupled to cilia, mechanosensitive and voltage-sensitive channels, and channels associated with the endoplasmic reticulum. Evidence suggests these channels are implicated in osteogenic pathways such as the YAP/TAZ and canonical Wnt pathways. This review aims to describe the involvement of calcium channels in regulating osteogenic differentiation in response to mechanical loading and characterize the fashion in which those channels directly or indirectly mediate this process. The mechanotransduction pathway is a promising target for the development of regenerative materials for clinical applications due to its independence from exogenous growth factor supplementation. As such, also described are examples of osteogenic biomaterial strategies that involve the discussed calcium ion channels, calcium-dependent cellular structures, or calcium ion-regulating cellular features. Understanding the distinct ways calcium channels and signaling regulate these processes may uncover potential targets for advancing biomaterials with regenerative osteogenic capabilities.

ß-Catenin Limits Osteogenesis on Regenerative Materials in a Stiffness-Dependent Manner.

Targeted refinement of regenerative materials requires mechanistic understanding of cell-material interactions. The nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold is shown to promote skull regeneration in vivo without additive exogenous growth factors or progenitor cells, suggesting potential for clinical translation. This work evaluates modulation of MC-GAG stiffness on canonical Wnt (cWnt) signaling. Primary human bone marrow-derived mesenchymal stem cells (hMSCs) are differentiated on two MC-GAG scaffolds (noncrosslinked, NX-MC, 0.3 kPa vs conventionally crosslinked, MC, 3.9 kPa). hMSCs increase expression of activated ß-catenin, the major cWnt intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization on stiffer MC scaffolds. Overall Wnt pathway inhibition reduces activated ß-catenin and osteogenic differentiation, while elevating BMP4 and phosphorylated Smad1/5 (p-Smad1/5) expression on MC, but not NX-MC. Unlike Wnt pathway downregulation, isolated canonical Wnt inhibition with ß-catenin knockdown increases osteogenic differentiation and mineralization specifically on the stiffer MC. ß-catenin knockdown also increases p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material. Thus, while stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis on MC-GAG, activated ß-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials.

The Intersection of Mechanotransduction and Regenerative Osteogenic Materials.

Mechanical signals play a central role in cell fate determination and differentiation in both physiologic and pathologic circumstances. Such signals may be delivered using materials to generate discrete microenvironments for the purposes of tissue regeneration and have garnered increasing attention in recent years. Unlike the addition of progenitor cells or growth factors, delivery of a microenvironment is particularly attractive in that it may reduce the known untoward consequences of the former two strategies, such as excessive proliferation and potential malignant transformation. Additionally, the ability to spatially modulate the fabrication of materials allows for the creation of multiple microenvironments, particularly attractive for regenerating complex tissues. While many regenerative materials have been developed and tested for augmentation of specific cellular responses, the intersection between cell biology and material interactions have been difficult to dissect due to the complexity of both physical and chemical interactions. Specifically, modulating materials to target individual signaling pathways is an avenue of interdisciplinary research that may lead to a more effective method of optimizing regenerative materials. In this work, the aim is to summarize the major mechanotransduction pathways for osteogenic differentiation and to consolidate the known materials and material properties that activate such pathways.

Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds.

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.

Nanoparticulate mineralized collagen glycosaminoglycan materials directly and indirectly inhibit osteoclastogenesis and osteoclast activation.

The ability of the extracellular matrix (ECM) to direct cell fate has generated the potential for developing a materials-only strategy for tissue regeneration. Previously, we described a nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material that efficiently induced osteogenic differentiation of human mesenchymal stem cells (hMSCs) and calvarial bone healing without exogenous growth factors or progenitor cell expansion. In this work, we evaluated the interactions between MC-GAG and primary human osteoclasts (hOCs). In the absence of hMSCs, mineralized Col-GAG materials directly inhibited hOC viability, proliferation, and resorption in contrast to nonmineralized Col-GAG, which demonstrated a modest inhibition of resorptive activity only. Cocultures containing differentiating hMSCs with hOCs demonstrated increased hOC-mediated resorption only on Col-GAG while MC-GAG cocultures continued to inhibit resorption. Unlike Col-GAG, hMSCs on MC-GAG expressed increased amounts of osteoprotegerin (OPG) protein, the major endogenous osteoclast inhibitor. Interestingly, OPG expression was found to be antagonized by small mothers against decapentaplegic1/5 (Smad1/5) phosphorylation, an obligate pathway for osteogenic differentiation of hMSCs on MC-GAG, and potentiated by extracellular signal-regulated kinase (ERK1/2) phosphorylation. Collectively, these results suggested that the MC-GAG material both directly inhibited the osteoclast viability, proliferation, and resorptive activity as well as induced hMSCs to secrete osteoprotegerin, an antiosteoclastogenic factor, via a signalling pathway distinct from osteogenic differentiation.

Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells.

The instructive capabilities of extracellular matrix components in progenitor cell differentiation have recently generated significant interest in the development of bioinspired materials for regenerative applications. Previously, a correlation was described between the osteogenic capabilities of nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC-GAG) and an autogenous activation of small mothers against decapentaplegic ( Smad1/5) in the canonical bone morphogenetic protein receptor (BMPR) pathway with a diminished extracellular signal regulated kinase 1/2 (ERK1/2) activation when compared to nonmineralized collagen glycosaminoglycan scaffolds (Col-GAG). This work utilizes a canonical BMPR inhibitor (dorsomorphin homologue 1, DMH1) and an inhibitor of the mitogen activated protein kinase/ERK kinase (MEK)/(ERK) cascade (PD98059) to characterize the necessity of each pathway for osteogenesis. While DMH1 inhibits runt-related transcription factor 2 (Runx2) and bone sialoprotein II (BSPII) gene expression of primary human mesenchymal stem cells (hMSCs) on MC-GAG, PD98059 inhibits BSPII expression on Col-GAG independent of Runx2 expression. DMH1 inhibits mineralization on both Col-GAG and MC-GAG, however, PD98059 only inhibits mineralization on Col-GAG. DMH1 inhibits both Smad1/5 phosphorylation and Runx2 protein expression, whereas PD98059 inhibits ERK1/2 and c-Jun amino-terminal kinase 1/2 (JNK1/2) phosphorylation without affecting Runx2. Thus, activation of the canonical BMPR signaling is necessary for osteogenic differentiation and mineralization of hMSCs on Col-GAG or MC-GAG. The MEK/ERK cascade, intimately tied to JNK activation, is necessary for Runx2-independent osteogenesis on Col-GAG, while completely dispensable in osteogenesis on MC-GAG.

Metabolic relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus zinc treatment.

BACKGROUND: Association of Alzheimer's Disease (AD) with Type 2 Diabetes (T2D) has been well established. Cyclo(His-Pro) plus zinc (Cyclo-Z) treatment ameliorated diabetes in rats and similar improvements have been seen in human patients. Treatment of amyloid precursor protein (APP) transgenic mice with Cyclo-Z exhibited memory improvements and significantly reduced Aß-40 and Aß-42 protein levels in the brain tissues of the mice. SCOPE OF REVIEW: Metabolic relationship between AD and T2D will be described with particular attention to insulin sensitivity and Aß degradation in brain and plasma tissues. Mechanistic effect of insulin degrading enzyme (IDE) in decreasing blood glucose and brain Aß levels will be elucidated. Cyclo-Z effects on these biochemical parameters will be discussed. MAJOR CONCLUSION: Stimulation of IDE synthesis is effective for the clinical treatment of metabolic diseases including AD and T2D. GENERAL SIGNIFICANCE: Cyclo-Z might be the effective treatment of AD and T2D by stimulating IDE synthesis.

Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.

Nanoparticulate mineralized collagen scaffolds induce in vivo bone regeneration independent of progenitor cell loading or exogenous growth factor stimulation.

Current strategies for skeletal regeneration often require co-delivery of scaffold technologies, growth factors, and cellular material. However, isolation and expansion of stem cells can be time consuming, costly, and requires an additional procedure for harvest. Further, the introduction of supraphysiologic doses of growth factors may result in untoward clinical side effects, warranting pursuit of alternative methods for stimulating osteogenesis. In this work, we describe a nanoparticulate mineralized collagen glycosaminoglycan scaffold that induces healing of critical-sized rabbit cranial defects without addition of expanded stem cells or exogenous growth factors. We demonstrate that the mechanism of osteogenic induction corresponds to an increase in canonical BMP receptor signalling secondary to autogenous production of BMP-2 and -9 early and BMP-4 later during differentiation. Thus, nanoparticulate mineralized collagen glycosaminoglycan scaffolds may provide a novel growth factor-free and ex vivo progenitor cell culture-free implantable method for bone regeneration.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

INTRODUCTION: Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. METHODS: Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. RESULTS: Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. CONCLUSIONS: Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway.

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.

Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling.

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs). METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs. RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels. CONCLUSION: CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Th17 cells in inflammation and autoimmunity.

T helper 17 (Th17), a distinct subset of CD4(+) T cells with IL-17 as their major cytokine, orchestrate the pathogenesis of inflammatory and autoimmune diseases. Dysregulated Th17 cells contribute to inflammatory and autoimmune diseases. Candidate biologics are in development for targeting IL-17, IL-17 receptors or IL-17 pathways. Several drugs that impact the IL-17 pathway are already in clinical trials for the treatment of autoimmune diseases. In this review we provide evidence for the role of Th17 cells in immune-mediated diseases. An understanding of the role of Th17 in these conditions will provide important insights and unravel novel targets for therapeutic intervention.

"Ins" and "Outs" of mesenchymal stem cell osteogenesis in regenerative medicine.

Repair and regeneration of bone requires mesenchymal stem cells that by self-renewal, are able to generate a critical mass of cells with the ability to differentiate into osteoblasts that can produce bone protein matrix (osteoid) and enable its mineralization. The number of human mesenchymal stem cells (hMSCs) diminishes with age and ex vivo replication of hMSCs has limited potential. While propagating hMSCs under hypoxic conditions may maintain their ability to self-renew, the strategy of using human telomerase reverse transcriptase (hTERT) to allow for hMSCs to prolong their replicative lifespan is an attractive means of ensuring a critical mass of cells with the potential to differentiate into various mesodermal structural tissues including bone. However, this strategy must be tempered by the oncogenic potential of TERT-transformed cells, or their ability to enhance already established cancers, the unknown differentiating potential of high population doubling hMSCs and the source of hMSCs (e.g., bone marrow, adipose-derived, muscle-derived, umbilical cord blood, etc.) that may provide peculiarities to self-renewal, differentiation, and physiologic function that may differ from non-transformed native cells. Tissue engineering approaches to use hMSCs to repair bone defects utilize the growth of hMSCs on three-dimensional scaffolds that can either be a base on which hMSCs can attach and grow or as a means of sequestering growth factors to assist in the chemoattraction and differentiation of native hMSCs. The use of whole native extracellular matrix (ECM) produced by hMSCs, rather than individual ECM components, appear to be advantageous in not only being utilized as a three-dimensional attachment base but also in appropriate orientation of cells and their differentiation through the growth factors that native ECM harbor or in simulating growth factor motifs. The origin of native ECM, whether from hMSCs from young or old individuals is a critical factor in "rejuvenating" hMSCs from older individuals grown on ECM from younger individuals.

Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential.

In vitro mineralization of human mesenchymal stem cells on three-dimensional type I collagen versus PLGA scaffolds: a comparative analysis.

BACKGROUND: Development of a tissue engineered bone graft requires efficient bioactivity screening of biomaterials in clinically relevant three-dimensional systems. The authors analyzed the relative osteogenic potential of two three-dimensional biomaterials--type I collagen and poly(L-lactide-co-glycolide) (PLGA)--to support in vitro mineralization of human mesenchymal stem cells. METHODS: Human mesenchymal stem cells were seeded onto three-dimensional PLGA or type I collagen scaffolds; incubated in osteogenic media; and harvested at 1, 4, and 7 days. Messenger RNA expression was analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction for osteogenic (i.e., alkaline phosphatase, osteocalcin, bone sialoprotein, Runx2/core binding factor α-1) and angiogenic (i.e., vascular endothelial growth factor and interleukin-8) markers. Alkaline phosphatase enzyme activity was measured at 4 and 7 days. Mineralization was detected by alizarin red staining and micro-computed tomographic imaging at 8 and 12 weeks. Mineral composition was analyzed by solid-phase nuclear magnetic resonance spectroscopy. RESULTS: Early osteogenic and angiogenic markers, and alkaline phosphatase enzyme activity, were up-regulated on PLGA versus collagen scaffolds. However, long-term mineralization endpoints favored type I collagen. By 8 weeks, human mesenchymal stem cells on collagen exhibited significantly higher mineral density by micro-computed tomographic and alizarin red staining than PLGA scaffolds. Both biomaterials deposited calcium hydroxyapatite as determined by nuclear magnetic resonance spectroscopy. CONCLUSIONS: The authors' findings suggest that despite early PLGA induction of osteogenic gene expression, long-term mineralization occurs earlier and to a greater extent on type I collagen, highlighting collagen as a potential bone tissue engineering scaffold in the human mesenchymal stem cell niche. When screening the relative osteoinductive profiles of three-dimensional bone tissue engineering scaffolds in vitro, the authors recommend including long-term endpoints of osteogenesis.

CXC receptor knockout mice: characterization of skeletal features and membranous bone healing in the adult mouse.

The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR(-/-) knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR(-/-) mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR(-/-) mice at 6, 12, and 18 weeks of age. Trabecular bone characteristics in mCXCR(-/-) (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by µCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR(-/-). Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR(-/-) long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR(-/-) and WT littermates were not completely healed at 12 weeks; WT animals, however, had significantly more bone in-growth than mCXCR(-/-). New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR(-/-). Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR(-/-) mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular and cortical long bone; (4) decreased femur bone strength; and (5) significantly reduced intramembranous bone formation and number of BlV in new calvarial bone during bone repair.

A synopsis of phosphate disorders in the nursing home.

Elderly patients are at an increased risk of developing both hypophosphatemia and hyperphosphatemia. Renal insufficiency predisposes elderly patients to elevated serum concentrations of phosphate. On the other hand, poor dietary intake and loss of phosphorus in the urine can lead to deficiency states. It is well documented that hyperphosphatemia is correlated with an increase in morbidity and mortality as a result of vascular calcification. This article reviews the etiology, pathophysiology, symptoms, and treatment of hypophosphatemia and hyperphosphatemia.

Extracellular-signal-related kinase 1/2 is responsible for inhibition of osteogenesis in three-dimensional cultured MC3T3-E1 cells.

We have previously demonstrated that osteogenic differentiation is inhibited and angiogenic expression is enhanced in murine preosteoblasts (MC3T3-E1) cultured on three-dimensional (3D) poly-L-lactide-co-glycolide (PLGA) scaffolds when compared to two-dimensional (2D) PLGA films. In the present work we investigated the role of the extracellular signal-related kinase 1/2 (ERK1/2) pathway in modulating osteogenic and angiogenic differentiation in 2D and 3D systems made of two distinct biomaterials-type I collagen and PLGA. The addition of a third dimension, regardless of biomaterials, substantially increased ERK1/2 activation as demonstrated by an increase in phosphorylated ERK1/2. Western blot analysis showed significant increases in phosphorylation of ERK1/2 in cells grown in 3D versus 2D cultures at day 4 (5- and 7.7-fold increases 3D vs. 2D in collagens and PLGA, respectively) and day 7 (4.7- and 4.6-fold increases 3D vs. 2D in collagen and PLGA, respectively). Using an ERK-specific inhibitor, PD 98059, we established a correlation between ERK activation and inhibited osteogenic differentiation. Inhibition of ERK activation in 3D cultures significantly enhanced osteogenic differentiation. It in fact restored osteogenic differentiation to a level equal to that of 2D cultured cells. Inhibition of ERK1/2, however, showed little effect on angiogenic gene expression, indicating that two distinct mechanisms are involved in osteogenic and angiogenic differentiation. Taken together, these results allow us to report a mechanistic model in MC3T3-E1 cells in which distinct activation of ERK1/2 in 3D culture has an inhibitory effect on osteogenic differentiation.

Human and mouse osteoprogenitor cells exhibit distinct patterns of osteogenesis in three-dimensional tissue engineering scaffolds.

BACKGROUND: Understanding interspecies variation between animal models and humans is essential to develop tissue-engineered bone. The authors studied osteogenic and angiogenic marker expression in human and murine osteoblasts and mesenchymal stem cells. METHODS: Three human cells (human mesenchymal stem cells, multilineage progenitor cells, and normal human osteoblasts) and three murine cells (MC3T3-E1, C3H10T1/2, and M2-10B4) were used. Cells were seeded onto poly-lactide-glycolic acid-coated tissue culture plates or three-dimensional poly-lactide-glycolic acid scaffolds, incubated in osteogenic medium, and harvested at 1, 4, and 7 days. mRNA expression was analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction for osteogenic markers, including alkaline phosphatase, osteocalcin, bone sialoprotein, and core-binding factor alpha-1, and angiogenic markers, including vascular endothelial growth factor and interleukin-8. Data were analyzed using analysis of variance. RESULTS: All human cells had significantly increased expression of osteogenic markers in three dimensions compared with two dimensions (alkaline phosphatase by 220 percent, osteocalcin by 323 percent, bone sialoprotein by 534 percent, and core-binding factor alpha-1 by 357 percent). However, all murine cells exhibited significant decreases in the expression of osteogenic markers in three-dimensional compared with two-dimensional cultures (alkaline phosphatase by 89 percent, osteocalcin by 64 percent, bone sialoprotein by 76 percent, and core-binding factor alpha-1 by 73 percent). In contrast, all human and murine cells showed markedly elevated expression of angiogenic factors interleukin-8 and vascular endothelial growth factor in three-dimensional compared with two-dimensional cultures. Measurement of alkaline phosphatase activity confirmed this pattern of osteogenic differentiation. CONCLUSIONS: In three-dimensional versus two-dimensional cultures, osteogenesis increased significantly in human cells but decreased in murine cells; angiogenesis increased regardless of species. Since three-dimensional cultures represent in vivo conditions more closely, this species variation has important translational implications to tissue-engineered bone research.