Application of multiple parallel perfused microbioreactors and three-dimensional stem cell culture for toxicity testing.

In this study, a multiple parallel perfused microbioreactor platform, TissueFlex, was developed which can be used to perform cell and tissue culture under almost uniform and precisely controlled environment in a mid-throughput and parallel manner. These microbioreactors were used to culture human bone marrow cells (hBMCs) in three-dimensional (3D) scaffolds and also in two-dimensional (2D) monolayer for comparison for upto 7 days. Several scaffolding materials were evaluated for this purpose in terms of easiness in handling, ability to support the hBMC growth, and feasibility for non-destructive optical assays. The feasibility and efficacy of using the developed 3D-hBMCs-based model tissue-constructs cultured in TissueFlex microbioreactors for drug evaluation and toxicity testing was then studied. As a demonstration case study, the cultured cells were challenged with two chemicals, trimethoprim and pyrimethamine, both known to be harmful to cellular activities, with different protocols. Cytotoxicity in terms of cell viability and growth was determined using the AlamarBlue assay. The 3D spatial variations in cell morphology and cell survival were also monitored using 3D optical imaging using non-linear multiphoton microscopy. The results show that (i) the data obtained from 3D hBMCs culture and from (2D) monolayer cultures on the effect of the tested chemicals on cell growth are significantly different, and that (ii) the perfused microbioreactor technology could provide a highly controlled and prolonged cell culture environment for testing of various drugs and chemicals. The outcome of this study demonstrated the feasibility and potentials of the using 3D stem cell based model tissues in TissueFlex microbioreactors for drug evaluation and toxicity testing of chemicals as an efficient and standardized alternative testing method.

The fate of implanted autologous chondrocytes in regenerated articular cartilage.

Autologous chondrocyte implantation (ACI) is used to treat some articular cartilage defects. However, the fate of the cultured chondrocytes after in-vivo transplantation and their role in cartilage regeneration remains unclear. To monitor the survival and fate of such cells in vivo, the chondrocytes were labelled with a lipophilic dye and the resultant regenerated tissue in dogs examined. It was found that, 4 weeks after implantation, the osteochondral defects were filled with regenerative tissue that resembled hyaline cartilage. Fluorescence microscopy of frozen sections of the regenerated tissue revealed that the majority of cells were derived from the DiI-labelled implanted chondrocytes. From these results, it was concluded that a large population of implanted autologous chondrocytes can survive at least 4 weeks after implantation and play a direct role in cartilage regeneration. However, it remains unknown whether other cells, such as periosteal cells or bone marrow stromal stem cells, are involved in the regeneration of cartilage after ACI.

Prostaglandin E1 increases in vivo and in vitro calcitriol biosynthesis in rabbits.

INTRODUCTION: Prostaglandins have an anabolic effect on bone. Possible mediation of this effect is via calcitriol. This study determines in vivo and in vitro effects of PGE(1) on calcitriol synthesis. METHODOLOGY: In vivo: rabbits received intravenous vehicle or prostaglandin E(1) (50 microg/day) for 20 days before measurements of serum total and ionic calcium, magnesium and phosphorus levels, total and bone-specific alkaline phosphatases, 25(OH)D(3), calcitriol, parathyroid hormone and calcitonin. In vitro: rabbit proximal renal tubules were incubated with 25(OH)D(3) (8 microM) together with PGE(1) (2.82 x 10(-6) M) and the prostaglandin receptor inhibitor AH6809 (10(-4) M) in selected samples. After 5 or 30 min incubation, calcitriol production was measured by radioimmunoassay and data analysed statistically. RESULTS: In vivo, in groups receiving PGE(1), levels of total Ca, Mg and calcitriol increased significantly and 25 dihydroxyvitamin D(3), parathyroid hormone and calcitonin remained unchanged. In vitro, PGE(1) increased calcitriol biosynthesis and the prostaglandin inhibitor AH6809 reduced calcitriol levels significantly after prolonged incubation. CONCLUSIONS: In vivo and in vitro results demonstrate that PGE(1) stimulates calcitriol synthesis. This study represent a major advancement in knowledge of bone metabolism.

A tissue-specific protein in rat osteogenic tissues.

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.

Effect of vitamin A on bone resorption: evidence for direct stimulation of isolated chicken osteoclasts by retinol and retinoic acid.

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.

Interferon-inhibited human osteosarcoma xenografts induce host bone in nude mice.

The growth of human osteosarcoma xenografts in nude mice can be inhibited by human interferon-alpha (IFN-alpha). Histologic examination of growth-inhibited tumors has revealed mineralization and partial replacement of the tumor by normal bone tissue. We have investigated whether the normal bone tissue was formed by differentiated tumor cells or by induction of host stroma to differentiate into bone tissue. Employing antibodies to both murine and human type I collagen, it was found that the normal bone produced in IFN-inhibited osteosarcomas was host derived. These results suggest that IFN induced the osteosarcoma cells to produce a bone-inductive agent that interacts with the host cells, and leads to the formation of mature normal bone tissue in a heterotopic site.

A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: implications for the identification of cells of the osteoblastic lineage in vitro.

Rabbit antisera to bovine osteocalcin were produced independently in two laboratories and their specificities established by western blot analysis. By immunohistochemistry each of the five polyclonal antisera produced an intense cytoplasmic staining in human bone-derived cells. Staining intensity was strongly attenuated by preabsorption of the antisera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for osteocalcin synthesis yielded an unexpected positive staining pattern similar to that seen with the bone-derived cells over a range of antiserum dilutions. This was not caused by the uptake of exogenous osteocalcin from the culture medium because a similar pattern of staining was observed when medium was supplemented with osteocalcin-depleted fetal calf serum. Treatment with 1,25-dihydroxyvitamin D3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone-derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other proteins synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone-derived cells, skin cells, and osteosarcoma cells (MG63). These observations indicate that the shared epitope residues in the central region of osteocalcin and are consistent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromise immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.

Modulation of osteogenesis and adipogenesis by human serum in human bone marrow cultures.

Knowledge of the controlling mechanisms of human osteoprogenitor cell differentiation has important implications for understanding bone turnover. The in vitro differentiation of human bone marrow fibroblasts into adipogenic and osteogenic cells and the interaction of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone in this process has been investigated together with the effects of human serum. Marrow fibroblasts cultured in human serum and dexamethasone for 28 days, generated lipid containing cells as confirmed by morphology, Oil red O staining and immunocytochemistry using antiserum to the adipocyte-specific protein, adipocyte P2 (aP2). In cultures containing 1,25(OH)2D3 and dexamethasone, adipogenesis was stimulated within 21 days. Osteocalcin expression, as assessed by in situ hybridization, was dependent on the presence of 1,25(OH)2D3 and was decreased in cultures treated with dexamethasone. Northern analysis confirmed the decrease in osteocalcin expression and increase in lipoprotein lipase expression with the appearance of the adipogenic phenotype in these cultures. Marrow cultures maintained for 14 days in human serum and osteotropic agents before switching to fetal calf serum indicated the continuous requirement of human serum in these cultures for adipogenesis. These results demonstrate that human serum contains factors that exert dramatic effects on human bone marrow cell differentiation to augment the osteogenic and adipogenic activity of 1,25(OH)2D3 and dexamethasone.

Localization of estrogen receptor-alpha in human and rabbit skeletal tissues.

Estrogen is essential for the development and maintenance of optimal bone mass in women and men, and acts through activation of estrogen receptors (ER). We have examined the pathways of estrogen action on the skeleton by seeking to localize the "classical" estrogen receptor, ER alpha, to particular cells to test the hypotheses that 1) estrogen directly influences growth plate chondrocytes; and 2) estrogen has a principal action on bone tissue via osteoblasts. ER alpha messenger ribonucleic acid (mRNA) was localized by in situ hybridization in human specimens from five males (11-15 yr old), two females (9 and 11 yr old), and three growing rabbits. In all of the human material examined, ER alpha mRNA was consistently identified in chondrocytes. In all of the rabbit tissue studied, ER alpha mRNA was localized in chondrocytes of the growth plate and the subarticular epiphyseal growth center. ER alpha mRNA signals were readily observed in both active osteoblasts and lining cells on trabecular surfaces of all samples. No clear evidence of positive staining was detectable in osteoclasts or osteocytes in either species. The distribution of ER alpha mRNA coincided with immunolocalization of the ER protein in the human specimens. These data suggest a direct action of estrogen on growth plate chondrocytes that may affect longitudinal growth and subsequent fusion of the growth plate and also on osteoblasts to affect bone formation at trabecular sites.

The localization of androgen receptors in human bone.

Androgens have important effects on the human skeleton, and deficiency has been associated with bone loss in both males and females. The skeletal actions of androgens may be mediated directly via the androgen receptor (AR) or indirectly via the estrogen receptor after aromatization to estrogens. The presence of androgen receptors has been demonstrated in bone cells and chondrocytes in vitro, but their presence in human bone in situ has not been reported. In order to provide further evidence for a direct action of androgens on bone via androgen receptors, we have used specific monoclonal antibodies to investigate the expression of human AR in normal developing and osteophytic bone of both sexes. In the growth plates from the developing bone, androgen receptors were predominantly expressed in hypertrophic chondrocytes and in osteoblasts at sites of bone formation. They were also observed in osteocytes in the bone, and in mononuclear cells and endothelial cells of blood vessels within the bone marrow. In the osteophytes, androgen receptors were widely distributed at sites of endochondral ossification in proliferating, mature, and hypertrophic chondrocytes and at sites of bone remodeling in osteoblasts. They were also expressed in osteocytes and mononuclear cells within the bone marrow. The pattern and number of cells expressing the receptor was similar in both sexes. Our results show for the first time the presence and distribution of androgen receptors in normal developing human and osteophytic bone in situ and further provide evidence for a direct action of androgens on bone and cartilage cells.

The localization of the functional glucocorticoid receptor alpha in human bone.

Glucocorticoids have well-documented effects on the skeleton, although their mechanism of action is still poorly understood. The actions of glucocorticoids on bone cells are mediated, in part, directly via specific receptors. The presence of these receptors has been demonstrated in both rodent and human osteoblastic cells in vitro, but their presence in human bone in vivo has not been reported. In this study, we have used specific affinity purified polyclonal antibodies to the functional glucocorticoid receptor alpha (GRalpha) to investigate its expression in both developing and adult human bone using sections of neonatal rib, calvarial, and vertebral bones, tibial growth plates from adolescents, and iliac crest biopsies from adults who were to undergo liver transplantation. In the tibial growth plates, GRalpha was predominantly expressed in the hypertrophic chondrocytes within the cartilage. In the primary spongiosa, the receptor was highly expressed by osteoblasts at sites of bone modeling. Within the bone marrow, receptors were also detected in mononuclear cells and in endothelial cells of blood vessels. In the neonatal rib and vertebrae, GRalpha was widely distributed at sites of endochondral bone formation in resting, proliferating, mature, and hypertrophic chondrocytes. They were also highly expressed in osteoblasts at sites of bone modeling. At sites of intramembranous ossification in neonatal calvarial bone and rib periosteum, GRa was widely expressed in cells within the fibrous tissue and in osteoblasts at both the bone-forming surface and at modeling sites. In the iliac crests from adults, GRalpha was predominantly expressed in osteocytes. The receptors were not detected in osteoclasts. Our results show for the first time the presence of the functional GRalpha in human bone in situ and suggest that the actions of glucocorticoids on bone may be mediated, in part, directly via the GR at different stages of life. The absence of receptor expression in osteoclasts also suggests that the effects of glucocorticoids on bone resorption may be mediated indirectly.

Future potentials for using osteogenic stem cells and biomaterials in orthopedics.

Ideal skeletal reconstruction depends on regeneration of normal tissues that result from initiation of progenitor cell activity. However, knowledge of the origins and phenotypic characteristics of these progenitors and the controlling factors that govern bone formation and remodeling to give a functional skeleton adequate for physiological needs is limited. Practical methods are currently being investigated to amplify in in vitro culture the appropriate autologous cells to aid skeletal healing and reconstruction. Recent advances in the fields of biomaterials, biomimetics, and tissue engineering have focused attention on the potentials for clinical application. Current cell therapy procedures include the use of tissue-cultured skin cells for treatment of burns and ulcers, and in orthopedics, the use of cultured cartilage cells for articular defects. As mimicry of natural tissues is the goal, a fuller understanding of the development, structures, and functions of normal tissues is necessary. Practically all tissues are capable of being repaired by tissue engineering principles. Basic requirements include a scaffold conducive to cell attachment and maintenance of cell function, together with a rich source of progenitor cells. In the latter respect, bone is a special case and there is a vast potential for regeneration from cells with stem cell characteristics. The development of osteoblasts, chondroblasts, adipoblasts, myoblasts, and fibroblasts results from colonies derived from such single cells. They may thus, theoretically, be useful for regeneration of all tissues that this variety of cells comprise: bone, cartilage, fat, muscle, tendons, and ligaments. Also relevant to tissue reconstruction is the field of genetic engineering, which as a principal step in gene therapy would be the introduction of a functional specific human DNA into cells of a patient with a genetic disease that affects mainly a particular tissue or organ. Such a situation is pertinent to osteogenesis imperfecta, for example, where in more severely affected individuals any improvements in long bone quality would be beneficial to the patient. In conclusion, the potentials for using osteogenic stem cells and biomaterials in orthopedics for skeletal healing is immense, and work in this area is likely to expand significantly in the future.

Identification and enrichment of human osteoprogenitor cells by using differentiation stage-specific monoclonal antibodies.

A major problem in developmental bone biology is the inability to clearly identify early progenitor cells of the osteogenic and related lineages. Identification of these cells is important for the study of their normal development and for determination of potential changes in skeletal diseases. The objective of the present study was to obtain specific markers for early progenitor cells. Monoclonal antibodies were raised against human marrow stromal fibroblastic cell cultures, known to be rich in progenitors for the stromal lineages. Antibodies were selected initially by their reactivity with these marrow cultures and their immunohistochemical localization in human fetal tissues, in progenitor cell regions adjacent to osteoblastic cells. Antibody HOP-26 was strongly reactive with cells in marrow stromal colonies at early stages of differentiation, before the induction of alkaline phosphatase activity, and decreased dramatically after the cells reached confluence. In sections of human fetal limb, binding of HOP-26 was restricted to cells in close proximity to the developing bone, in periosteum, and between the developing bone trabeculae. In adult trabecular bone tissue, HOP-26 was reactive with occasional cells present within the marrow spaces with osteoblasts, adipocytes, and fibrous tissue unreactive. No antibody binding was detected in sections of skin, muscle, appendix, brain, tonsil, or liposarcoma, or cultured SaOS II, MG63, or skin cells. In primary cell suspensions, HOP-26 was unreactive with blood cells but strongly reactive with 0.59 +/- 0.27% of nucleated marrow cells. The antigen associated with these cells was detectable both intracellularly and on the cell surface, and by using immunopanning, HOP-26 selected the marrow stromal fibroblastic colony-forming units (CFU-F). HOP-26 provides the means to identify osteogenic progenitor cells directly and with high specificity. The present studies demonstrate the value of this antibody in providing enriched populations of progenitor cells for experimental studies of osteogenic differentiation and in histopathology.

Human bone tissue formation in diffusion chamber culture in vivo by bone-derived cells and marrow stromal fibroblastic cells.

Direct grafts of human cells into immunocompromised or cortisone-treated animals, either alone or within carrier materials, have been used with some success to assess the developmental capability of the grafted cells. However, identification of the donor or host origins of the generated tissue in such direct grafts is essential. In an alternative and extensively used experimental system, cells are cultured within the isolated environments of diffusion chambers, which are surgically implanted in appropriate hosts. This system allows the direct study of the cellular potentials for differentiation as host tissues are excluded. In the present study, human osteoprogenitor cell populations derived from trabecular bone explants or marrow suspensions of 3 patients (2 females aged 14 years and 1 male aged 27 years) were cultured in the absence or the continuous presence of dexamethasone (10 nmol/L). Cells were impregnated into porous hydroxyapatite ceramics before subcutaneous implantation, or placed within diffusion chambers before intraperitoneal implantation, in athymic mice. All subcutaneous implants of cells in ceramic showed morphological evidence for the formation of bone tissue. In the diffusion chambers it was found that both marrow- and bone-derived fibroblastic cells cultured in the absence of dexamethasone generally produced fibrous tissue only. When cultured in the continuous presence of dexamethasone (10 nmol/L), these cell populations produced similar osteogenic tissues with active osteoblasts, wide osteoid seams, and mineralized tissue, with cartilage toward the interior of the chamber. These results validate the diffusion chamber as an experimental system to study human osteogenesis using appropriately primed cell populations.

Interconversion potential of cloned human marrow adipocytes in vitro.

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.

Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro study.

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.

A biodegradable fibrin scaffold for mesenchymal stem cell transplantation.

A potential therapy to enhance healing of bone tissue is to deliver isolated mesenchymal stem cells (MSCs) to the site of a lesion to promote bone formation. A key issue within this technology is the development of an injectable system for the delivery of MSCs. Fibrin gel exploits the final stage of the coagulation cascade in which fibrinogen molecules are cleaved by thrombin, convert into fibrin monomers and assembled into fibrils, eventually forming fibers in a three-dimensional network. This gel could have many advantages as a cell delivery vehicle in terms of biocompatibility, biodegradation and hemostasis. The objective of this study was to explore the possibility of using fibrin gel as a delivery system for human MSCs (HMSCs). To this end we have determined the optimal fibrinogen concentrations and thrombin activity for loading HMSCs in vitro into the resultant fibrin gels to obtain cell proliferation. We found that a concentration of 18 mg/ml of fibrinogen and a thrombin activity of 100 IU/ml was optimal for producing fibrin scaffolds that would allow good HMSCs spreading and proliferation. In these conditions, cells were able to proliferate and expressed alkaline phosphatase, a bone marker, in vitro. When implanted in vivo, HMSCs were able to migrate out of the fibrin gel and invade a calcium carbonate based ceramic scaffold suggesting that fibrin gel could serve as a delivery system for HMSCs.

Evaluation of sodium alginate for bone marrow cell tissue engineering.

Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.

Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.

Materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications for bone regeneration and for use in skeletal reconstruction and joint replacement. We have studied the growth and interactions of human bone marrow cells on a variety of new cement composites in vitro. These cement materials are composed of calcium-deficient hydroxyapatites, carbonated apatite and amorphous calcium phosphate. Cell proliferation was significantly reduced and cell differentiation increased in the presence of these cements compared with cells cultured on tissue culture plastic. Alkaline phosphatase, one of the markers of the osteoblast phenotype, was dramatically stimulated by 3 of the 4 cements examined between day 4 and day 10, above levels observed following culture of human osteoblasts on plastic alone. Photomicroscopic examination demonstrated growth and close integration of bone marrow cells and 3 of the composites. Longer term marrow cultures (15 day) on the cements confirmed the stimulation of cell differentiation over proliferation. From these studies, enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas, cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro culture system demonstrates the use of human bone marrow cells for the potential evaluation of new biomaterials and the development of a novel carbonated apatite that may be of potential use in orthopaedic implants.

Effects of novel calcium phosphate cements on human bone marrow fibroblastic cells.

The identification and characterization of biocompatible materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications in skeletal reconstruction and joint replacement. In the present study, we have examined the effects of three biocements, biocement H, calcium-deficient apatite; biocement F, apatite + CaHPO(4); biocement D, carbonated apatite + CaHPO(4) + CaCO(3) and an amorphous calcium phosphate (ACP) proposed as implant fixing materials, on the growth, differentiation, and cell surface interaction of human bone marrow fibroblastic cells. These cells are known to be progenitors of osteoblasts, chondroblasts, adipocytes, myoblasts, and reticulocytes. Alkaline phosphatase enzyme activity, a marker of the osteoblast phenotype, was increased by a factor of two- to sixfold on carbonated apatite, one- to sixfold on apatite and three- to 10-fold on calcium-deficient apatite, over levels observed on plastic. Cell proliferation was significantly reduced. Photomicroscopic examination indicated high biocompatibility with close adhesion of the bone marrow fibroblastic cells to composites D, F, and H. Longer term marrow cultures (15 days) confirmed the stimulation of cell differentiation, as assessed by collagen production, over cell proliferation, of cells grown on carbonated apatite. Enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro human bone marrow fibroblast culture system provides a simple and effective method for the evaluation of new biomaterials. The development of these novel cements may be of potential use in orthopedic implants.