Douglas M. Burns

Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism.

Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site (K(D)=405+/-29 pM; 13,100+/-223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP(1)) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP(2)) receptor, [Cys(Acm)(2,7)]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP(1) receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP(2) receptor agonist than are typical CGRP(1) receptors. These receptor proteins were not exactly matched with the known components of a CGRP(1) receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP(1) receptor.

Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RS™): In vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo

We have previously demonstrated that multilayered periosteal sheets prepared from the explant culture of alveolar periosteum serve as a promising osteogenic grafting material in periodontal tissue regeneration. For the preparation of more potent periosteal sheets, we examined the applicability of stem-cell culture media. Compared to the control medium (Medium 199+10% FBS), periosteal sheets expanded with MesenPRO-RS™ medium exhibited these features: Cells grew three-dimensionally and deposited collagen in the extracellular spaces to form thicker multilayers of cells. Chondrocytic markers were not significantly upregulated. Contractile force was generated in proportion with the increased thickness of the periosteal sheets and the formation of cytoplasmic α-smooth muscle actin fibers. However, myofibroblastic markers were not significantly upregulated. The surface marker CD146 was substantially upregulated, while both CD73 and CD105 were downregulated. Alkaline phosphatase, a representative osteoblastic marker, was not upregulated by osteogenic induction. However, these expanded periosteal sheets exhibited substantially stronger osteogenic differentiation when implanted in nude mice. Therefore, despite our reservations, MesenPRO medium effectively expanded the cells contained in periosteal sheets to promote the formation of thicker multilayers of cells in vitro, and these enhanced periosteal sheets expressed increased osteogenic potential at implantation sites in vivo. In conjunction with data indicating that CD146-positive cells were notably expanded and the recently proposed concept that CD146 is a marker for osteogenic progenitor cells found in the bone marrow stroma, our findings suggest that MesenPRO medium improves the preparation of highly osteogenic periosteal sheets suitable for clinical application largely through the induction of CD146-positive cells.

Improved adhesion of human cultured periosteal sheets to a porous poly(L-lactic acid) membrane scaffold without the aid of exogenous adhesion biomolecules.

Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 μm diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane.

Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentrations in UMR 106 osteoblastic cells

Calcitonin gene-related peptide (CGRP) was examined for its effects on intracellular free Ca2+ concentration ([Ca2+]i) in UMR 106 osteoblast-like cells. Cells loaded with the Ca2+ dye FURA-2 dose-dependently responded to CGRP (1-100 nM) with transient two-fold increases in [Ca2+]i. An intracellular source for this Ca2+ transient was suggested by the failure of membrane depolarization with high extracellular K+ or acute depletion of extracellular Ca2+ ([Ca2+]e) with EGTA to attenuate this response. After cells were incubated for 45 min with 0.1 mM extracellular Ca2+ to deplete intracellular Ca2+ stores, CGRP produced a 25-30% decrease in [Ca2+]i rather than a transient increase. This calcium decrease was mimicked by membrane depolarization or by pinacidil, a specific activator of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, and blocked by glybenclamide, a specific blocker of KATP channels. Our data suggest that CGRP has diverse Ca2+ regulatory effects in UMR 106 cells, mobilizing Ca2+ from intracellular stores via classical signaling while possibly promoting cellular Ca2+ efflux or inhibiting uptake through voltage-dependent Ca2+ channels via KATP-mediated hyperpolarization.

Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets.

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66GPa)≫dispersed (9.99kPa)>central region (5.20kPa)>peripheral regions (3.67kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed>central region>peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site.

1,25-Dihydroxyvitamin D3 promotes prostaglandin E1-induced differentiation of HL-60 cells

Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E1 (PGE1) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can induce monocytic differentiation. We have now examined the effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE1 (1 μg/ml) or 1,25(OH)2D3 (10 nM) each inhibited cell proliferation over 48–96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE1 treatment, and 1,25(OH)2D3 produced a fivefold stimulation. However, combined treatment (PGE1 plus 1,25(OH)2D3) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric oxide (NO) before 48 hours in culture, and treatment with PGE1 or 1,25(OH)2D3 did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE1, but not 1,25(OH)2D3, markedly stimulated cellular claboration of interleukin (IL)-1α, IL-6, and tumor necrosis factor (TNF)-α, and 1,25(OH)2D3 cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)2D3 plus PGE1 generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE1 induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)2D3 had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE1 treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE1, but not by 1,25(OH)2D3; however, the combination of 1,25(OH)2D3 and PGE1 again produced the strongest stimulation. Similarly, only PGE1 significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE1, not 1,25(OH)2D3, is sufficient to promote rapid in vitro differentiation of HL-60 cells along the granulocytic pathway; however, the PGE1-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)2D3. In addition, these converted HL-60 cells preferentially utilize the glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.

Protein tyrosine phosphorylation induced by epidermal growth factor and insulin-like growth factor-I in a rat clonal dental pulp-cell line

Both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) produce a dose-dependent stimulation in the rate of cell division in a rat clonal dental pulp-cell line (RDP 4-1). To elucidate the initial mitogen-induced cellular events that may mediate mitogenic action, the effects of EGF and IGF-I on cellular protein tyrosine phosphorylation were examined. In a dose-dependent manner, EGF (1-100 ng/ml) transiently stimulated tyrosine phosphorylation in four major proteins with apparent molecular weights of 220, 180, 140 and 120 kDa, and in five other more minor proteins (90, 80, 65, 55 and 44 kDa). IGF-I (1-100 ng/ml) dose-dependently stimulated the tyrosine phosphorylation of 160- and 140-kDa proteins, and had a smaller effect on the 80-, 65- and 44 kDa proteins. In contrast to the action of EGF, IGF-I-induced tyrosine phosphorylation was sustained for more than 60 min, particularly that of the 160-kDa phosphoprotein. From the results of specific immunoprecipitation/Western-blot analyses, the 180-kDa EGF-sensitive protein could be identified as the EGF receptor (EGF-R). Among the IGF-I-sensitive pulp cell proteins, the 160-kDa protein was identified as insulin-receptor substrate-1. Both mitogenic treatments stimulated the tyrosine phosphorylation of a weak, 44-kDa protein, which we have identified as the extracellular signal-regulated kinase-1. Despite the presence of phosphoproteins of the correct size, neither the IGF-I receptor (IGF-I-R) nor the phospholipase C gamma-isoform could be identified as tyrosine kinase substrates in either treatment. Pretreatment with the tyrosine kinase inhibitor genistein (20 micrograms/ml) significantly inhibited EGF- and IGF-I-induced tyrosine phosphorylation in permeabilized RDP 4-1 cells, and the tyrosine phosphatase inhibitor orthovanadate (1 mM) significantly prolonged the duration of the mitogen-stimulated tyrosine phosphorylation in both intact or permeabilized cells. Phorbol 12-myristate 13-acetate (100 nM), which activates protein kinase C (PKC), inhibited the tyrosine phosphorylation induced by either growth factor. This action was blocked by pretreatment with staurosporine (200 nM, 15 min), a selective PKC inhibitor. However, neither removing external Ca2+ with EGTA (1 mM) nor inducing Ca2+ influx with A23187 ionophore (2 microM) significantly altered EGF- or IGF-I-induced phosphorylation. These findings strongly suggest that authentic EGF-R and IGF-I-R on RDP 4-1 cells are coupled to complex, tyrosine kinase-mediated, intracellular signalling systems that are sensitive to a PKC-dependent mechanism. EGF- and IGF-I-induced tyrosine phosphorylation cascades may have important roles in vivo in the regulation of dental pulp-cell proliferation and ultimately may affect dentine formation.

An osteogenic grafting complex combining human periosteal sheets with a porous poly(l-lactic acid) membrane scaffold: Biocompatibility, biodegradability, and cell fate in vivo

In this in vitro study, novel porous poly(l-lactic acid) membranes were developed to improve periosteal sheets by promoting initial adhesion of periosteal tissue segments and stimulating the formation of a viable multilayered cellular sheet. The biocompatibility, biodegradability, and osteogenicity were evaluated using human periosteal tissue segments cultured on porous poly(l-lactic acid) membranes; the periosteal sheets were osteogen induced and were then implanted in the dorsal subcutaneous tissue of nude mice. In vivo, the membrane degraded into clusters of membrane particles separated by wide cracks; fibroblastic cells invaded along with small blood vessels from the surrounding mouse connective tissue. In osteoinduced periosteal sheets, the membrane clusters were surrounded by numerous capillaries and a number of tartrate-resistant acid phosphatase–positive, multinucleated cells. Neither severe inflammation nor fibrous encapsulation was observed throughout the implantation (~12 weeks). These porous poly(l-lactic acid) membranes were highly biocompatible and functioned well as biodegradable scaffolds that could enhance the use of osteogenic periosteal sheets in therapy.

Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins in chemically permeabilized rat UMR106 cells

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways in UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like cells that contrasts with the simpler regulatory mechanisms found in A431 cells.

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199+10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75°C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37°C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.