Paola Manduca

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.

Integrin synthesis and utilization in cultured human osteoblasts.

This study describes the adhesion of human osteoblasts, culturedin vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77–100%, in 2h and at 55nmsubstrata concentration, and it was accompained by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the β1 integrin and antibodies to the α5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against α2, α3, α5, αv, β1 andβ3 integrins we detected synthesis of α3β1, α5β1, αvβ3, and an αvβ1‐like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus α4, α1 and β5 subunits. In cells adhering in the presence of serum we showed organization of β3 and αv integrins in focal contacts. In cells adhering to fibronectin α5 and β1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.

An open letter for the people in Gaza.

We are doctors and scientists, who spend our lives developing means to care and protect health and lives. We are also informed people; we teach the ethics of our professions, together with the knowledge and practice of it. We all have worked in and known the situation of Gaza for years. On the basis of our ethics and practice, we are denouncing what we witness in the aggression of Gaza by Israel. We ask our colleagues, old and young professionals, to denounce this Israeli aggression. We challenge the perversity of a propaganda that justifi es the creation of an emergency to masquerade a massacre, a so-called “defensive aggression”. In reality it is a ruthless assault of unlimited duration, extent, and intensity. We wish to report the facts as we see them and their implications on the lives of the people. We are appalled by the military onslaught on civilians in Gaza under the guise of punishing terrorists. This is the third large scale military assault on Gaza since 2008. Each time the death toll is borne mainly by innocent people in Gaza, especially women and children under the unacceptable pretext of Israel eradicating political parties and resistance to the occupation and siege they impose. This action also terrifi es those who are not directly hit, and wounds the soul, mind, and resilience of the young generation. Our condemnation and disgust are further compounded by the denial and prohibition for Gaza to receive external help and supplies to alleviate the dire circumstances. The blockade on Gaza has tightened further since last year and this has worsened the toll on Gaza’s population. In Gaza, people suff er from hunger, thirst, pollution, shortage of medicines, electricity, and any means to get an income, not only by being bombed and shelled. Power crisis, gasoline shortage, water and food scarcity, sewage outflow and ever decreasing resources are disasters caused directly and indirectly by the siege. People in Gaza are resisting this aggression because they want a better and normal life and, even while crying in sorrow, pain, and terror, they reject a temporary truce that does not provide a real chance for a better future. A voice under the attacks in Gaza is that of Um Al Ramlawi who speaks for all in Gaza: “They are killing us all anyway— either a slow death by the siege, or a fast one by military attacks. We have nothing left to lose—we must fi ght for our rights, or die trying.” Gaza has been blockaded by sea and land since 2006. Any individual of Gaza, including fi shermen venturing beyond 3 nautical miles of the coast of Gaza, face being shot by the Israeli Navy. No one from Gaza can leave from the only two checkpoints, Erez or Rafah, without special permission from the Israelis and the Egyptians, which is hard to come by for many, if not impossible. People in Gaza are unable to go abroad to study, work, visit families, or do business. Wounded and sick people cannot leave easily to get specialised treatment outside Gaza. Entries of food and medicines into Gaza have been restricted and many essential items for survival are prohibited. Before the present assault, medical stock items in Gaza were already at an all time low because of the blockade. They have run out now. Likewise, Gaza is unable to export its produce. Agriculture has been severely impaired by the imposition of a buff er zone, and agricultural products cannot be exported due to the blockade. 80% of Gaza’s population is dependent on food rations from the UN. Much of Gaza’s buildings and infrastructure had been destroyed during Operation Cast Lead, 2008–09, and building materials have been blockaded so that schools, homes, and institutions cannot be properly rebuilt. Factories destroyed by bombardment have rarely been rebuilt adding unemployment to destitution. Despite the difficult conditions, the people of Gaza and their political leaders have recently moved to resolve their confl icts “without arms and harm” through the process of reconciliation between factions, their leadership renouncing titles and positions, so that a unity government can be formed abolishing the divisive factional politics operating since 2007. This reconciliation, although accepted by many in the international community, was rejected by Israel. The present Israeli attacks stop this chance of political unity between Gaza and the West Bank and single out a part of the Palestinian society by destroying the lives of people of Gaza. Under the pretext of eliminating terrorism, Israel is trying to destroy the growing Palestinian unity. Among other lies, it is stated that civilians in Gaza are hostages of Hamas whereas the truth is that the Gaza Strip is sealed by the Israelis and Egyptians. Gaza has been bombed continuously for the past 14 days followed now by invasion on land by tanks and thousands of Israeli troops. More than 60 000 civilians from Northern Gaza were ordered to leave their homes. These internally displaced people have nowhere to go since Central and Southern Gaza are also subjected to heavy artillery bombardment. The whole of Gaza is under attack. The only shelters in Gaza are the schools of the UN Relief and Works Agency for Palestine Refugees in the Near East (UNRWA), uncertain shelters already targeted during Cast Lead, killing many. According to Gaza Ministry of Health and UN Office for the Coordination of Humanitarian Affairs (OCHA), as of July 21, 149 of the 558 killed in Gaza and 1100 of the 3504 wounded are children. Those buried under the rubble are not counted yet. As we write, the BBC reports of the bombing of another hospital, hitting Published Online July 22, 2014 http://dx.doi.org/10.1016/ S0140-6736(14)61044-8

Trimer Carboxyl Propeptide of Collagen I Produced by Mature Osteoblasts Is Chemotactic for Endothelial Cells

During the second phase of osteogenesis in vitro, rat osteoblasts secrete inducer(s) of chemotaxis and chemoinvasion of endothelial and tumor cells. We report here the characterization and purification from mature osteoblast conditioned medium of the agent chemotactic for endothelial cells. The chemoactive conditioned medium specifically induces directional migration of endothelial cells, not affecting the expression and activation of gelatinases, cell proliferation, and scattering. Directional migration induced in endothelial cells by conditioned medium from osteoblasts is inhibited by pertussis toxin, by blocking antibodies to integrins α1, β1, and β3, and by antibodies to metalloproteinase 2 and 9. The biologically active purified protein has two sequences, coincident with the amino-terminal amino acids, respectively, of the α1 and of the α2 carboxyl propeptides of type I collagen, as physiologically produced by procollagen C proteinase. Antibodies to type I collagen and to the carboxyl terminus of α1or α2 chains inhibit chemotaxis. The chemoattractant is the propeptide trimer carboxyl-terminal to type I collagen, and its activity is lost upon reduction. These data illustrate a previously unknown function for the carboxyl-terminal trimer, possibly relevant in promoting endothelial cell migration and vascularization of tissues producing collagen type I.

Role of MT1-MMP in the osteogenic differentiation.

Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.

Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells

BackgroundWe investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU) in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells.MethodsWe analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC), bonesialoprotein (BSP), membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146).ResultsWe report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow.ConclusionsHU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for homeostasis related genes and modulation of mRNAs and proteins for ECM deposition, maturation and remodeling which may be key factors for bone maintenance.

Rat tibial osteoblasts III: Propagation in vitro is accompanied by enhancement of osteoblast phenotype

Postproliferative confluent cultures of primary rat tibial osteoblasts (ROB), cultured in medium supplemented with ascorbic acid and beta-glycerophosphate (AS-bGP, differentiation medium) express, in sequence, specific bone markers which identify a succession of maturation stages, and eventually form mineralized noduli. We report an investigation on the effect of extensive proliferation in vitro in unsupplemented medium on the osteogenic potential of mass cultures of ROB. The growth rates of the populations, derived from two independent primary cultures, was constant throughout 110 cumulative population doublings (CPD) in culture. Propagated cells maintained features similar to osteoblasts in primary cultures with respect to serum and anchorage dependence for growth and to the chemokinetic effect on endothelial cells exerted by their conditioned media (CM). Propagated populations, set at confluence in differentiation medium, were tested for the expression of early [alkaline phosphatase (AP)] and late [osteocalcin (OC); bone sialoprotein (BSP); 45Ca incorporation and mineralization] osteogenic markers. We observed an increase, parallel to the increase in CPD, in both the level of maximal expression of AP (enzyme/microgram cellular DNA) and in the frequency of nodules, reaching five- to sixfold (at 78 CPD) and eightfold (at 60 CPD), respectively, the levels of primary cultures. AP expression (enzyme and mRNA) persisted during mineralization and 45Ca incorporation. The time required by propagated cultures for the formation of nodules decreased with increase of CPD, and was reduced to less than one third at 87 CDP. Nodules became mineralized over a similar lapse of time as in primary cultures and were positive by histochemistry for BSP and OC. We also obtained osteogenic clones from two independent cultures after 72 CPD. 90% of these showed an osteoblast phenotype, expressing AP and forming nodules positive for OC and BSP, which mineralized. Timing of formation and frequency of nodules/plated cells in clones was similar to that found in propagated cultures of equivalent CPD. In summary, propagated ROB populations and derived clones showed enhanced osteoblast phenotype, possibly due to an increase in osteogenic cells and enrichment of proliferating mature osteoblasts, consequent to extended propagation in culture.

Pro-collagen I COOH-terminal Trimer Induces Directional Migration and Metalloproteinases in Breast Cancer Cells

Breast and prostatic carcinomas, melanoma, and endothelial cell lines are chemoattracted by medium conditioned by mature osteoblasts. The chemoattractant for endothelial cells was identified with C3, carboxyl-terminal trimer of pro-collagen type I. We report that C3 induces directional migration and proliferation, the expression of tissue inhibitor of metalloproteinases-2, pro-metalloproteinase-2 and –9, and their activation in MDA MB231 cells, without changing the expression of tissue inhibitor of metalloproteinases-1 and of metalloproteinase-14. Antiserum against metalloproteinase-2 or -9 or -14, tissue inhibitor of metalloproteinases-1, or GM6001 inhibits the C3-induced migration. Urokinase and its receptor are detected and unchanged upon exposure to C3. The antibody against urokinase or addition of plasminogen activator inhibitor inhibits migration. Blocking antibodies to integrins α2, α6, β1, and β3 inhibit chemotaxis and do not change urokinase and urokinase receptor expression. Blockage of α2, β1, and β3 integrins affect differently the induction by C3 of pro-metalloproteinase-2 and -9 and of tissue inhibitor of metalloproteinases-2. Chemotaxis to C3 is also inhibited by genistein, by pertussis toxin, which also inhibits C3-induced pro-metalloproteinase -2 and -9, but not urokinase expression. Wortmannin partially inhibits C3-induced cell migration. Other, but not all, breast carcinoma lines tested responded to C3 with migration and pro-metalloproteinase-2 induction. Presently C3 is the only agent known to induce migration specifically of both endothelial and breast carcinoma cells. The mitogenic and motogenic role of C3in vitro might prefigure a role in in vivo carcinogenesis and in the establishment of metastasis.

FMS*Calciumfluor specifically increases mRNA levels and induces signaling via MAPK 42,44 and not FAK in differentiating rat osteoblasts.

The homeopathic compound of resonance FMS*Calciumfluor (FMS*) reportedly promotes osteogenic differentiation of rat pre‐osteoblasts in vitro. Here, we show that the continuous exposure of differentiating rat osteogenic cells (ROB) to FMS* modulates the level of expression of mRNAs for 7 of the 8 osteogenic markers tested. Alkaline phosphatase (AP), osteocalcin (OC), metalloproteinases (MMP‐2 and −14), procollagenase C (BMP‐1), biglycan (BG) and integrin 1 are expressed at higher levels in FMS*‐treated osteoblasts than in control cultures. MMP‐2 and −14 mRNA are not down‐modulated at mineralization. Also, the pattern of expression induced by FMS* for some of these genes (BMP‐1, BG and integrin 1) is changed, but collagen type I (Coll I) mRNA levels are not affected by treatment with FMS*. This suggests that FMS* modulates mRNA levels and that this is not generalized, but gene(s) specific. We also report that exposure to FMS* rapidly and transiently induces activation of mitogen‐activated protein kinases (MAPKs) 42,44 in populations of early osteoblasts, but not in pre‐osteoblasts, with a cell differentiation stage‐dependent and pertussis toxin (PTX)‐sensitive response. Subsequent to FMS* MAPK signaling activation, an increase in AP and MMP‐14 mRNA is detected, which is also inhibited by PTX, suggesting that FMS* activation of MAPK signaling could be an early event required for the induction of these genes. Exposure to FMS* does not cause changes in the activity of p125 (FAK)‐mediated signaling.

Birth Defects in Gaza: Prevalence, Types, Familiarity and Correlation with Environmental Factors

This is the first report of registration at birth, and of incidence of major structural birth defects (BD) obtained in Gaza at Al Shifa Hospital, where 28% of total births in Gaza Strip occur. Doctors registered 4,027 deliveries, with a protocol comprehensive of clinical, demographic, kin and environmental questions. Prevalence of BD is 14/1,000, without association with intermarriage or gender of the child. Prevalence of late miscarriages and still births are respectively 23.3/1,000 and 7.4/1,000, and of premature births 19.6/1,000. Couples with a BD child have about 10 times higher frequency of recurrence of a BD in their progeny than those with normal children, but none of their 694 siblings and only 10/1,000 of their 1,423 progeny had BD, similar to the frequency in general population. These data suggest occurrence of novel genetic and epigenetic events in determination of BD. Children with BD were born with higher frequency (p < 0 001) in families where one or both parents were under “white phosphorus” attack, that in the general population. Bombing of the family home and removal of the rubble were also frequently reported by couples with BD occurrence. These data suggests a causative/favoring role of acute exposure of parents to the weapons-associated contaminants, and/or of their chronic exposure from their persistence in the environment on the embryonic development of their children.