Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

Summary We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)₂ vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)₂D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.     

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.     

The growth and phenotypic expression of human osteoblasts

The care of patients with a skeletal deficiency currently involves the use of bone graft or a non-biologic material such as a metal or polymer. There are alternate possibilities in development which involve the growth of bone cells (osteoblasts) on degradable polymer scaffolds. These tissue engineering strategies require production of the polymeric scaffold, cellular harvest followed by either ex vivo or in vivo growth of the cells on the scaffold, and exploration of the interaction between the cell and scaffold. Research into these strategies utilizes cells from a variety of species, but clinical applications will likely require human osteoblasts. This study explores the process whereby human osteoblasts are harvested under sterile conditions during joint replacement surgery from normally discarded cancellous bone, transported from the operating room to the lab, and grown in culture. This process is feasible, and the cells express their phenotype via the production of alkaline phosphatase and collagen in culture.     

A novel spectrofluorometric technique for specific biocompatibility testing of implantable materials by cell culture. Report on use for multiparameter analysis of human osteoblasts cultured on commercially pure titanium and hydroxyapatite

The authors describe a novel spectrofluorometric technique based on double-labelled fluorescence imaging using immunoconjugates labelled with fluorochromes. Following isolation and characterization, cells are seeded on the surface of disks of the material(s) to be tested. After application of a primary antibody and an antibody bearing a fluorochrome, the signal emitted by the molecules in the extracellular matrix on the surface of the test disks is measured by spectrofluorimetry. Measurement is thus independent of the surface characteristics of the test material. Measured values are compared with pre-established standard curves. This technique facilitates determination of the characteristic molecules expressed by a given cell type,thus allowing accurate evaluation of the response of pertinent biological samples to implantable biomaterials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures.

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-beta1, TGF-beta2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.     

Characterization of newly established human osteosarcoma cell line,LM-1

Summary The characteristics of Cell Line LM-1, established from a human osteosarcoma, have been studied extensively. The cell produced both bone-specific and placental-like alkaline phosphatases when treated with hydrocortisone 21-phosphate; they had specific membrane antigens that reacted with sera from osteosarcoma patients. Injection of LM-1 cells into newborn hamsters treated with antilymphocyte serum produced nodular tumors. The characteristics of LM-1 suggest that this tumor cell line has unique features that may be useful in a variety of studies of human and animal osteosarcoma. This research is supported in part by USPHS Grants HD-09938 and CA-25746.     

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2

We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of β-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and α1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1–7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts. J. Cell. Biochem. 67:386–398, 1997. © 1997 Wiley-Liss, Inc.     

In vitro behaviour of human osteoblasts on dentin and bone

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines.     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Characterization of human osteoblastic cells: Influence of the culture conditions

Summary Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)₂ vitamin D₃. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D₃. 10 mM βGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.     

Expression and characterization of alkaline phosphatases during differentiation of human pancreatic cancer (Capan-1) cells in culture.

Human pancreatic cells of the Capan-1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan-1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan-1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four-bodied structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP-positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan-1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.     

Hormone-responsive cells derived from human dental papilla: Characterization in vitro and in vivo in diffusion chambers

Summary Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E₂ there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.     

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy

Background. In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. Methods. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. Results. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. Conclusion. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16: 163-9.)     

Phosphatidylcholine metabolism and choline kinase in human osteoblasts

There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment

Summary A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were separately minced into approximately 1-mm³ pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing 5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 μm) and the cells collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture. This work was supported by research grants from the National Institute of Environmental Health Sciences, Bethesda, MD (ES3101) and the United States Environmental Protection Agency, Washington, D. C. (R810146).     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.