M. J. O. Francis’s research while affiliated with University of Bari Aldo Moro and other places

The effects of retinol (vitamin A) and retinoic acid on primary cultures of isolated chicken osteoclasts have been studied. The experiments were performed to establish the direct actions of these two agents on the organization of cytoskeletal structures, on the acid phosphatase contents, and on the bone resorption activities of these cells. The results showed that by treating the cultures with retinol or retinoic acid, from 10(-8) to 10(-5) M, there were dose-related responses of the osteoclasts. Adhesion to the substratum was stimulated by increasing the number of cells exhibiting the specialized dot-like adhesion structures, or podosomes, which represent the active part of the sealing zone. The treatments also induced rearrangement of the microtubular patterns with reversible depolymerization of microtubules. Acid phosphatase activity was significantly higher both in vitamin A-treated osteoclasts and in their media. When [3H]proline-labeled bone particles were added to the retinoid-treated osteoclasts, the release of [3H]proline was increased significantly compared to controls. These results suggest that the two vitamin A metabolites cause several modifications of the metabolic status of isolated osteoclasts that result in augmented rates of bone resorption.

Osteoblasts, osteocytes and osteoclasts are specialised cells of bone that play crucial roles in the formation, maintenance and resorption of bone matrix. Bone formation and resorption critically depend on optimal intracellular calcium and phosphate homeostasis and on the expression and activity of plasma membrane transport systems in all three cell types. Osteotropic agents, mechanical stimulation and intracellular pH are important parameters that determine the fate of bone matrix and influence the activity, expression, regulation and cell surface abundance of plasma membrane transport systems. In this paper the role of ATPase pumps is reviewed in the context of their expression in bone cells, their contribution to ion homeostasis and their relation to other transport systems regulating bone turnover.

The presence of osteogenic progenitors in human skeletal muscle is suggested by the formation of ectopic bone in clinical and experimental conditions, but their direct identification has not yet been demonstrated. The aims of this study were to identify osteogenic progenitor cells in human skeletal muscle tissue and to expand and characterize them in culture. Specimens of gracilis and semitendinosus muscle were obtained from young adults and digested to separate the connective tissue and satellite cell fractions. The cells were cultured and characterized morphologically and immunohistochemically using antibodies known to be reactive with primitive osteoprogenitor cells, pericytes, intermediate filaments, and endothelial cells. Alkaline phosphatase activity and osteocalcin gene expression were also determined. In the early stages of culture, the connective tissue cells obtained were highly positive for primitive osteoprogenitor cell and for pericyte markers. Alkaline phosphatase activity was detectable at early stages of culture and rose as a function of time, whereas primitive osteoprogenitor cell markers declined and osteocalcin mRNA expression became detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). It is shown that human skeletal muscle connective tissue contains osteogenic progenitor cells. Their identification as pericytes, perivascular cells with established osteogenic potential, suggests a cellular link between angiogenesis and bone formation in muscle tissue. These cells are easily cultured and expanded in vitro by standard techniques, providing an alternative source of osteogenic progenitor cells for possible cell-based therapeutic use in certain conditions.

Na+, K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where it serves as the principal regulator of intracellular ion homeostasis. Na+, K+-ATPase is responsible for generating and maintaining transmembrane ionic gradients that are of vital importance for cellular function and subservient activities such as volume regulation, pH maintenance, and generation of action potentials and secondary active transport. The diversity of Na+, K+-ATPase subunit isoforms and their complex spatial and temporal patterns of cellular expression suggest that Na+, K+-ATPase isozymes perform specialized physiological functions. Recent studies have shown that the alpha subunit isoforms possess considerably different kinetic properties and modes of regulation and the beta subunit isoforms modulate the activity, expression and plasma membrane targeting of Na+, K+-ATPase isozymes. This review focuses on recent developments in Na+, K+-ATPase research, and in particular reports of expression of isoforms in various tissues and experiments aimed at elucidating the intrinsic structural features of isoforms important for Na+, K+-ATPase function.

Osteoblasts play a critical role in bone formation and mineralization, a process that depends on optimal calcium and phosphate homeostasis. Transcellular transport of free calcium [Ca2+], uptake of inorganic phosphate (P(i)) and numerous other transport systems in osteoblasts depend on a low intracellular Na+:K+ ratio furnished by (Na++K+)-stimulated adenosine triphosphatase (Na+,K+-ATPase), an enzyme embedded in the plasma membrane. In this study, we have examined, for the first time, the expression of the catalytic α and regulatory β subunit isoforms of Na+,K+-ATPase in primary human bone derived osteoblasts using isoform specific monoclonal and polyclonal antibodies. Immunofluorescence was used to detect the α1, β1 and β2 isoforms of Na+,K+-ATPase in dispersed osteoblasts. Laser scanning confocal microscopy also revealed an abundance of Na+,K+-ATPase isoforms in subcellular compartments. The existence of α1, β1 and β2 suggests that at least two major isozyme combinations of Na+,K+-ATPase are present in human bone (α1β1,α1β2).

Chondrocytes exist in an unusual and variable ionic and osmotic environment in the extracellular matrix of cartilage and are responsible for maintaining the delicate equilibrium between extracellular matrix synthesis and degradation. The mechanical performance of cartilage relies on the biochemical properties of the matrix. Alterations to the ionic and osmotic extracellular environment of chondrocytes have been shown to influence the volume, intracellular pH and ionic content of the cells, which in turn modify the synthesis and degradation of extracellular matrix macromolecules. Physiological ion homeostasis is fundamental to the routine functioning of cartilage and the factors that control the integrity of this highly evolved and specialized tissue. Ion transport in cartilage is relatively unexplored and the biochemical properties and molecular identity of membrane transport mechanisms employed by chondrocytes in the control of intracellular ion concentrations and pH is not fully defined and this review focuses on these processes. Chondrocytes have been shown to express voltage and stretch activated ion channels, passive exchangers and ATP dependent ion pumps. In addition, recent studies of transport systems in chondrocytes have demonstrated the presence of isozyme diversity that includes Na+/H+ exchange (NHE1, NHE3), Na+, K(+)-ATPase (several isoforms) and others each of which possess considerably different kinetic properties and modes of regulation. This multitude of isozyme diversity indicates the highly specialized handling of ions and protons in order to accomplish a fine regulation of their transmembrane fluxes. The complexities of these transport systems and their patterns of isoform expression underscore the subtlety of ion homeostasis and pH regulation in normal cartilage. Perturbations in these mechanisms may affect the physiological turnover of cartilage and thus increase the susceptibility to degenerative joint disease.

The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied. Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100μ m l ‐ascorbic acid‐2‐phosphate (Asc‐2‐P) and 10n m dexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21% p O 2 in the presence of 100μ m asc‐2‐P and 10n m dex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5mCi/ml [ ³ H]proline after 3, 6 and 12 days in culture. At 21% p O 2 , the presence of asc‐2‐P and dex induced significantly greater ( P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc‐2‐P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10% p O 2 and 5% p O 2 was associated with increased collagen synthesis. These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro . Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O 2 . Variation of p O 2 in ligaments during healing may therefore be an important modulator of successful repair.

Human bone derived osteoblasts were cultured on collagen-calcium phosphate composites. The ability of the substrates to support cell attachment, proliferation and bone formation was assessed using histochemical staining for alkaline phosphatase activity and immunolocalisation of transforming growth factor- β1and type 1 collagen. The effect of calcium phosphate phase and crystal size was investigated and the calcified samples compared with uncalcified collagen. Osteoblasts adhere to the collagen-calcium phosphate composites and express a mature osteoblast phenotype in vitro. Cell adhesion was greater on unmineralised collagen than on the mineralised composites, however, these cells were less differentiated. The presence of larger crystals seemed to have a detrimental effect on the cells, reducing proliferation and alkaline phosphatase activity. There was no discernible difference between the effect of hydroxyapatite and octacalcium phosphate on the cells.