Extracellular Calcium Deposition Research Articles

Effect of Vitamin D Receptor Activator Therapy on Vitamin D Receptor and Osteocalcin Expression in Circulating Endothelial Progenitor Cells of Hemodialysis Patients

Background: The effects of vitamin D receptor (VDR) and osteocalcin (OC) expression as well as VDR agonist (VDRA) therapy on circulating endothelial progenitor cells (EPCs) has not been elucidated yet. Methods: We therefore analyzed EPCs in 30 healthy controls and 82 patients undergoing dialysis (no VDRA therapy: 28; oral calcitriol: 30, and intravenous paricalcitol, PCTA: 24). The percentage of EPCs (CD34+/CD133-/KDR+/CD45-) expressing VDR or OC, and VDR and OC expression defined by mean fluorescence intensity (MFI) were analyzed using flow cytometry. The in vitro effect of VDRAs was evaluated in EPCs isolated from each patient group. Results: The percentage of VDR+ EPCs correlated positively with VDRA therapy and 25(OH)D, and negatively with diabetes, C-reactive protein, hemoglobin and osteopontin. VDR-MFI correlated positively with VDRA therapy, parathyroid hormone (PTH) and 25(OH)D, and negatively with diabetes and osteopontin. The percentage of OC+ EPCs correlated positively with the calcium score, PTH and phosphate, and negatively with 25(OH)D. OC-MFI correlated positively with calcium score, PTH, phosphate and hemoglobin, and negatively with albumin, 25(OH)D and osteopontin. Cell cultures from patients without VDRA therapy had the highest levels of calcium deposition and OC expression, which both significantly decreased following in vitro VDRA administration: in particular extracellular calcium deposition was only reduced by adding PCTA. Conclusions: Our data suggest that 25(OH)D serum levels and VDRA therapy influence VDR and OC expression on circulating EPCs. Since OC expression may contribute to vascular calcification, we hypothesize a putative protective role of VDRA therapy.

Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith

Gastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85 kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

Specific temporal culturing and microgroove depth influence osteoblast differentiation of human periodontal ligament cells grown on titanium substrata

The purpose of this study is two-fold: to compare differences in the development of osteoblast differentiation processes between human bone marrow mesenchymal stem cells (MSCs) and human periodontal ligament cells (PLCs) cultured on microgrooved titanium (Ti) substrata and to investigate the effects of microgroove depth on PLCs’ osteoblast differentiation. Using photolithography, 60 μm-wide and 10 or 20 μm-deep microgrooves were fabricated on the Ti substrata (NE60/10 or NE60/20). Subsequent acid etching was applied to the fabricated microgrooved Ti to yield the etched microgrooves (E60/10 and E60/20). Smooth and acid-etched Ti were used as the controls (NE0 and E0). Alkaline phosphatase activity and extracellular calcium deposition assays were performed after various timelines of culture on both MSCs and PLCs grown on NE0 and E60/10. For PLCs cultured on NE0, NE60/10, NE60/20, E0, E60/10 and E60/20, cell adhesion, cell proliferation, osteoblast differentiation were determined, followed by the analysis on various osteogenic gene expressions. By comparing the extracellular matrix maturation and mineralization processes on smooth and microgrooved Ti substrata, it was determined that PLCs require more time for osteoblast differentiation than MSCs. Also, E60/10 allowed for the highest levels of adhesion, proliferation, osteoblast differentiation, and osteogenic gene expression by PLCs. PLCs could be an excellent alternative to MSCs for use in future studies investigating cellular processes using microgrooved Ti substrata and other modified surfaces. Optimal Ti microgroove dimensions and osteogenic differentiation timelines are necessary to promote various cellular activities in human PLCs.

Biological Effects of Phosphate on Fibroblast-Like Synoviocytes

This study sought to examine the expression of genes implicated in phosphate transport and pathological calcification in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and investigate the biological effects of phosphate. Results revealed that several genes, which were implicated in phosphate transport and pathological calcification, were differentially expressed in OA FLS and RA FLS. Phosphate stimulated the expression of matrix metalloproteinse-1, matrix metalloproteinse-3, cyclooxygenase-2, and interleukin-1βin a dose-dependent manner. Phosphate also induced OA FLS cell death but not RA FLS cell death at higher concentration. Calcification inhibitors, phosphocitrate (PC), and ethane-1-hydroxy-1,1-diphosphonate (EHDP), effectively inhibited these detrimental biological effects of phosphate. These findings suggest that abnormal expression of genes implicated in phosphate transport and pathological calcification may contribute to the progression of OA through the induction of extracellular matrix-degrading enzymes, proinflammatory cytokines, cell death, and calcium deposits. Calcification inhibitors such as PC and EHDP are potent inhibitors of these detrimental biological effects of phosphate.

Biocompatibility of chitosan-coated iron oxide nanoparticles with osteoblast cells

Background:Bone disorders (including osteoporosis, loosening of a prosthesis, and bone infections) are of great concern to the medical community and are difficult to cure. Therapies are available to treat such diseases, but all have drawbacks and are not specifically targeted to the site of disease. Chitosan is widely used in the biomedical community, including for orthopedic applications. The aim of the present study was to coat chitosan onto iron oxide nanoparticles and to determine its effect on the proliferation and differentiation of osteoblasts.Methods:Nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, x-ray diffraction, zeta potential, and vibrating sample magnetometry. Uptake of nanoparticles by osteoblasts was studied by transmission electron microscopy and Prussian blue staining. Viability and proliferation of osteoblasts were measured in the presence of uncoated iron oxide magnetic nanoparticles or those coated with chitosan. Lactate dehydrogenase, alkaline phosphatase, total protein synthesis, and extracellular calcium deposition was studied in the presence of the nanoparticles.Results:Chitosan-coated iron oxide nanoparticles enhanced osteoblast proliferation, decreased cell membrane damage, and promoted cell differentiation, as indicated by an increase in alkaline phosphatase and extracellular calcium deposition. Chitosan-coated iron oxide nanoparticles showed good compatibility with osteoblasts.Conclusion:Further research is necessary to optimize magnetic nanoparticles for the treatment of bone disease.

Modulation of osteogenic differentiation in hMSCs cells by submicron topographically-patterned ridges and grooves

Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. Four-hundred nanometer pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of expression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants.

The enhanced modulation of key bone matrix components by modified Titanium implant surfaces

Modifications to Titanium (Ti) implant surfaces enhance osseointegration by promoting bone-implant contact and peri-implant bone accrual; which in vitro analyses of osteoblastic cells suggest is due to an enhancement in cellular phenotypic maturation and function. To evaluate these effects on uncommitted cells, this study examined the osteogenic mineralisation and phenotypic marker expression of human marrow derived stromal cells (hBMSCs) from three unrelated donors cultured on tissue culture plastic (TCP), polished (P), rough-hydrophobic (SLA) and rough-hydrophilic (modSLA) Ti surfaces over the course of 21 days. Transcriptional analyses indicated a significant early up-regulation of both Runx2 (p<0.05) and Osteopontin (OP) (p<0.05) but not Bone Sialoprotein 2 (BSP2) (p<0.05) by rough surfaces 1 day post seeding. The phenotypic analyses showed that whilst cellular proliferation was relatively restricted and slower on the rough substrates; osteogenic mineralisation, assessed by quantifying extracellular matrix calcium deposition, collagen formation and the ratio of collagen to mineral deposited were significantly higher (p<0.05); as was alkaline phosphatase (ALP) activity (p<0.05). The rough surfaces caused an increase of secreted osteoblastic markers Osteoprotegrin (OPG) (p<0.05), growth differentiation factor 15 (GDF-15) (p<0.05) and Osteocalcin (OC) (p<0.05). These findings suggest that modified Ti surfaces induce an enhancement in osteogenic commitment and differentiation, which likely underlie the deposition of more stable bone matrix early in the healing process in vivo.

Effects of Heat Stress and Starvation on Clonal Odontoblast-like Cells

Introduction Heat stress during restorative procedures, particularly under severe starvation conditions, can trigger damage to dental pulp. In the present study, we examined effects of heat stress on odontoblastic activity and inflammatory responses in an odontoblast-like cell line (KN-3) under serum-starved conditions. Methods Viability, nuclear structures, and inflammatory responses of KN-3 cells were examined in culture medium containing 10% or 1% serum after exposure to heat stress at 43°C for 45 minutes. Gene expression of extracellular matrices, alkaline phosphatase activity, and detection of extracellular calcium deposition in cells exposed to heat stress were also examined. Results Reduced viability and apoptosis were transiently induced in KN-3 cells during the initial phases after heat stress; thereafter, cells recovered their viability. The cytotoxic effects of heat stress were enhanced under serum-starved conditions. Heat stress also strongly up-regulated expression of heat shock protein 25 as well as transient expression of tumor necrosis factor-alpha, interleukin-6, and cyclooxygenase-2 in KN-3 cells. In contrast, expression of type-1 collagen, runt-related transcription factor 2, and dentin sialophosphoprotein were not inhibited by heat stress although starvation suppressed ALP activity and delayed progression of calcification. Conclusions Odontoblast-like cells showed thermoresistance with transient inflammatory responses and without loss of calcification activity, and their thermoresistance and calcification activity were influenced by nutritional status.

Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.

Oxygen tension differentially influences osteogenic differentiation of human adipose stem cells in 2D and 3D cultures

Skeletal defects commonly suffer from poor oxygen microenvironments resulting from compromised vascularization associated with injury or disease. Adipose stem cells (ASCs) represent a promising cell population for stimulating skeletal repair by differentiating toward the osteogenic lineage or by secreting trophic factors. However, the osteogenic or trophic response of ASCs to reduced oxygen microenvironments is poorly understood. Moreover, a direct comparison between 2D and 3D response of ASCs to hypoxia is lacking. Thus, we characterized the osteogenic and angiogenic potential of human ASCs under hypoxic (1%), normoxic (5%), and atmospheric (21%) oxygen tensions in both 2D and 3D over 4 weeks in culture. We detected greatest alkaline phosphatase activity and extracellular calcium deposition in cells cultured in both 2D and 3D under 21% oxygen, and reductions in enzyme activity corresponded to reductions in oxygen tension. ASCs cultured in 1% oxygen secreted more vascular endothelial growth factor (VEGF) over the 4-week period than cells cultured in other conditions, with cells cultured in 2D secreting VEGF in a more sustained manner than those in 3D. Expression of osteogenic markers revealed temporal changes under different oxygen conditions with peak expression occurring earlier in 3D. In addition, the increase of most osteogenic markers was significantly higher in 2D compared to 3D cultures at 1% and 5% oxygen. These results suggest that oxygen, in conjunction with dimensionality, affects the timing of the differentiation program in ASCs. These findings offer new insights for the use of ASCs in bone repair while emphasizing the importance of the culture microenvironment.

Effect of metformin on bone marrow progenitor cell differentiation: In vivo and in vitro studies

Diabetes mellitus is associated with bone loss. Patients with type 2 diabetes are frequently treated with oral antidiabetic drugs such as sulfonylureas, biguanides, and thiazolidinediones. Rosiglitazone treatment has been shown to increase adipogenesis in bone marrow and to induce bone loss. In this study we evaluated the effect of in vivo and in vitro treatment with metformin on bone marrow progenitor cells (BMPCs), as well as the involvement of AMPK pathway in its effects. The in vitro effect of coincubation with metformin and rosiglitazone on the adipogenic differentiation of BMPCs also was studied. In addition, we evaluated the effect of in vivo metformin treatment on bone regeneration in a model of parietal lesions in nondiabetic and streptozotocin-induced diabetic rats. We found that metformin administration both in vivo and in vitro caused an increase in alkaline phosphatase activity, type I collagen synthesis, osteocalcin expression, and extracellular calcium deposition of BMPCs. Moreover, metformin significantly activated AMPK in undifferentiated BMPCs. In vivo, metformin administration enhanced the expression of osteoblast-specific transcription factor Runx2/Cbfa1 and activation of AMPK in a time-dependent manner. Metformin treatment also stimulated bone lesion regeneration in control and diabetic rats. In vitro, metformin partially inhibited the adipogenic actions of rosiglitazone on BMPCs. In conclusion, our results indicate that metformin causes an osteogenic effect both in vivo and in vitro, possibly mediated by Runx2/Cbfa1 and AMPK activation, suggesting a possible action of metformin in a shift toward the osteoblastic differentiation of BMPCs.

Osteogenic differentiation of human dental pulp stem cells in 3D scaffolds

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D and 3D cultures. DPSCs were isolated by magnetic cell sorting using antibodies against c-Kit, CD34 and STRO-1 surface antigens and then differentiated toward osteogenic lineage on 2D surface of culture flask by using an osteogenic medium. Differentiated cells express specific bone proteins such as Runx-2, Osx, OPN and OCN with a sequential expression analogous to those occurring during osteoblast differentiation and produce extracellular calcium deposits. In a second phase, DPSCs were cultured in MatrigelTM, Collagen and Fibroin 3D scaffolds in order to differentiate cells in a 3D space that mimes the physiological environment. In DPSC-MatrigelTM complexes, cells differentiate in osteoblast phenotype and form calcified nodules. DPSCs differentiated in collagen sponge actively secrete human type I collagen and after implant in immuno-suppressed rats were enveloped in a highly vascularised connectival capsule. DPSC-Collagen complex presents small areas of mineralization but appears infiltrated by inflammatory cells indicating that an immuno-response occurred. Fibroin scaffolds colonized with DPSCs after implant appear enveloped in a connectival capsule highly vascularised similarly to collagen scaffolds. In contrast the immune system invasion did not occur. DPSC are present in the whole scaffold thickness and in intimate contact with the scaffold fibers. After 20 days of implant extended areas of mineralization were stained by alizarin red. Fibroin scaffold appears more suitable for future application in regenerative medicine because it is highly histo-compatible, do not produces immune-response and offers the optimal environment for DPSCs growth and differentiation. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications to resolve pathologies and traumas characterized by critical size bone defects.

The Role of the Extracellular Signal-Related Kinase Signaling Pathway in Osteogenic Differentiation of Human Adipose-Derived Stem Cells and in Adipogenic Transition Initiated by Dexamethasone

Human adipose-derived stem cells (hASCs) offer great promise for bone tissue engineering because of their osteogenic differentiation potential. At molecular levels, this study investigated the contribution of one of the main members of mitogen-activated protein kinases (MAPKs), extracellular signal-related kinase (ERK), to hASC osteogenic differentiation and the regulation of ERK for the balance between osteogenesis and adipogenesis in hASCs in vitro. As analyzed using western blot, ERK activation in osteo-induced hASCs was initiated at day 7, peaked at day 10, and declined from day 14 to basal levels. As detected using histochemical and biochemical methods, alkaline phosphatase (ALP) activity in hASCs experienced a process similar to that of ERK activation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as revealed by an ALP activity assay, extracellular calcium deposition detection, osteocalcin (OCN) secretion examination, and real-time polymerase chain reaction (PCR) analysis for expression of osteogenesis-relative genes: core binding factor alpha 1, collagen type I, ALP, and OCN. Blockage of ERK phosphorylation in osteo-induced hASCs by PD98059 supplemented with dexamethasone (Dex) led to adipogenic differentiation, as confirmed by Nile Red staining to detect intracellular lipid droplets and real-time PCR analysis for expression of adipogenesis-relative genes: peroxisome proliferator-activated receptor gamma 2 and fatty acid-binding protein. These findings indicated a potential mechanism for the function of ERK in hASC osteogenic differentiation, especially the regulation of ERK in association with Dex for the balance between osteogenesis and adipogenesis, pointing out the significance of ERK signaling pathway for ASCs as a promising cell source for bone tissue engineering.

Isolation, characterization and osteogenic differentiation of adipose-derived stem cells: from small to large animal models

One of the most important issues in orthopaedic surgery is the loss of bone resulting from trauma, infections, tumours or congenital deficiency. In view of the hypothetical future application of mesenchymal stem cells isolated from human adipose tissue in regenerative medicine, we have analysed and characterized adipose-derived stem cells (ASCs) isolated from adipose tissue of rat, rabbit and pig. We have compared their in vitro osteogenic differentiation abilities for exploitation in the repair of critical osteochondral defects in autologous pre-clinical models. The number of pluripotent cells per millilitre of adipose tissue is variable and the yield of rabbit ASCs is lower than that in rat and pig. However, all ASCs populations show both a stable doubling time during culture and a marked clonogenic ability. After exposure to osteogenic stimuli, ASCs from rat, rabbit and pig exhibit a significant increase in the expression of osteogenic markers such as alkaline phosphatase, extracellular calcium deposition, osteocalcin and osteonectin. However, differences have been observed depending on the animal species and/or differentiation period. Rabbit and porcine ASCs have been differentiated on granules of clinical grade hydroxyapatite (HA) towards osteoblast-like cells. These cells grow and adhere to the scaffold, with no inhibitory effect of HA during osteo-differentiation. Such in vitro studies are necessary in order to select suitable pre-clinical models to validate the use of autologous ASCs, alone or in association with proper biomaterials, for the repair of critical bone defects.

The impact of diamond nanocrystallinity on osteoblast functions

Nanocrystalline diamond has been proposed as an anti-abrasive film on orthopedic implants. In this study, osteoblast (bone forming cells) functions including adhesion (up to 4 h), proliferation (up to 5 days) and differentiation (up to 21 days) on different diamond film topographies were systematically investigated. In order to exclude interferences from changes in surface chemistry and wettability (energy), diamond films with nanometer and micron scale topographies were fabricated through microwave plasma enhanced chemical-vapor-deposition and hydrogen plasma treatment. Scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy and water contact angle measurements verified the similar surface chemistry and wettability but varied topographies for all of the diamond films prepared on silicon in this study. Cytocompatibility assays demonstrated enhanced osteoblast functions (including adhesion, proliferation, intracellular protein synthesis, alkaline phosphatase activity and extracellular calcium deposition) on nanocrystalline diamond compared to submicron diamond grain size films for all time periods tested up to 21 days. An SEM study of osteoblast attachment helped to explain the topographical impact diamond had on osteoblast functions by showing altered filopodia extensions on the different diamond topographies. In summary, these results provided insights into understanding the role diamond nanotopography had on osteoblast interactions and more importantly, the application of diamond films to improve orthopedic implant lifetimes.

O.615 Co-cultivation of oral keratinocytes and osteoblasts

Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. Four-hundred nanometer pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of expression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants.

Familial calcific band-shaped keratopathy: Report of two new cases with early recurrence

We report two siblings with the rare entity of familial calcific band-shaped keratopathy (BSK). Detailed ophthalmic and systemic investigations failed to reveal any underlying causative pathology. Topical disodium ethylenediamine-tetraacetate (EDTA) was applied for 30 min to all four eyes. In addition the right eye of the younger sibling required a superficial keratectomy. An improvement in corneal clarity was seen in the immediate postoperative period in both siblings. Histopathology of the keratectomy specimen revealed linear extracellular sub-epithelial granular calcium deposits. However, an early recurrence was noted in all four eyes at four weeks postoperatively. We report the second instance in the English literature of this entity. Band-shaped keratopathy presenting without an obvious etiology merits a complete systemic and ophthalmic workup. Patients with familial idiopathic BSK could be cases with poor prognosis for treatment with EDTA due to an early recurrence of the disease.

Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells

The objective of this study was to isolate osteoprogenitor cells (OPC) from BM mesenchymal stromal cells (MSC) and test their capacity to proliferate and differentiate into osteoblasts. Human MSC were separated on a Percoll gradient and cultured in DMEM supplemented with 15% human serum, and characterized by flow cytometric analyzes for CD34, CD13, CD90, CD105 and CD117. To induce differentiation, cultured cells were exposed to 10(-7) m dexamethasone (dexa) and/or 10(-3) m sodium beta-glycerophosphate (beta-GlyP) and 1,25-dihydroxyvitamin D3 (calcitriol) or 9-cis-retinoic acid (9-RA). alkaline phosphatase (AP) activity was detected in cells irrespective of the dexa and/or beta-GlyP treatment. Antigenic phenotypes of MSC were CD34- (more than 99%) and CD13+ CD90+ CD105+ CD117+ (c. 50%). The treatment induced extracellular calcium deposition and gene and protein expression of osteonectin (ON) and bone sialoprotein (BSP): beta-GlyP induced an increase (c. 2.2-fold) of the ON gene and dexa augmented (c. 2.7-fold) the gene expression of BSP II. Gene expression of BSP I reached a maximum at 3 weeks of combined treatment. Osteocalcin gene expression was induced only after additional treatment with calcitriol or 9-RA. Ultrastructural analysis revealed the secretory phenotype of OPC. Under appropriate treatment, MSC can give rise to OPC that have the capacity to differentiate into osteoblasts characterized by the expression of osteogenic markers, osteoblastic properties and stromal BM cells phenotypes. These cells may represent a promising material to be utilized in orthopedic cellular therapy.

Bilateral Recurrent Calcareous Degeneration of the Cornea

To report clinical, histologic, and electron microscopic findings in several consecutive keratoplasties with recurrent pancorneal calcification in a patient with chronic graft versus host disease and severe keratoconjunctivitis sicca following bone marrow transplantation for chronic myelogenous leukemia. Altogether 5 penetrating keratoplasties were performed in both eyes for descemetocele formation and corneal perforation as well as pancorneal calcification associated with severe visual loss. Histologic examination and electron microscopy were performed on the corneal buttons obtained. Histology and electron microscopy confirmed pancorneal extracellular calcium deposition with increasing severity in each consecutive keratoplasty. Calcification was associated with necrosis and inflammation of the corneal stroma. Clinically unsuspected fungal keratitis was observed in 1 specimen. Keratoconjunctivitis sicca, epithelial defects, corneal inflammation, and infection are most probably responsible for the recurrent pancorneal calcification seen in our patient. The role of cytokines in this complication is discussed.

Transient Changes in Oxygen Tension Inhibit Osteogenic Differentiation and Runx2 Expression in Osteoblasts

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.