Primary Bovine Osteoblasts Research Articles

CK2.3, a Mimetic Peptide of the BMP Type I Receptor, Increases Activity in Osteoblasts over BMP2.

Bone is one of the most important organs in the human body. It provides structure, function, and protection for other vital organs; therefore, bone maintenance and homeostasis are critical processes. As humans age, their bone mineral density decreases, which leads to diseases like osteoporosis. This disease affects one in two women and one in five men aged 50 and over. As the aging population increases, the interest and significance of studying this debilitating bone disease becomes more relevant. Current therapeutic products for osteoporosis have many side effects and can be taken for a limited number of years. Most therapeutic products only focus on decreasing bone resorption, not increasing bone formation. Bone morphogenetic protein 2 is an essential growth factor that drives osteoblast differentiation and activity and is essential for bone formation. However, usage in the clinic is unsuccessful due to several side effects. Recently, a signaling disparity in bone marrow stromal cells within the bone morphogenetic protein pathway that led to decreased bone morphogenetic protein 2 responsiveness was identified in patients diagnosed with osteoporosis. However, it is unclear how other cell populations, especially osteoblasts, which are key players in bone remodeling, are affected and whether the bone morphogenetic protein pathway is affected during osteoporosis. Our research group designed a novel peptide, casein kinase 2.3, that acts downstream of the bone morphogenetic receptor type Ia and increases bone mineralization in murine cells and primary bovine osteoblasts. The aim of the study presented here was to compare the responsiveness of osteoblasts to bone morphogenetic protein 2 and casein kinase 2.3, especially in patients diagnosed with osteoporosis. Mature osteoblasts were extracted from patients diagnosed with osteoporosis or osteoarthritis from Christiana Care Hospital in Newark, Delaware. They were stimulated with either bone morphogenetic protein 2 or casein kinase 2.3, and their effect on osteoblast activity was determined. The osteoporotic patients showed no mineralization response to bone morphogenetic protein 2 stimulation, while the osteoarthritis patients significantly responded to bone morphogenetic protein 2 stimulation. Furthermore, markers for osteoblast activity were increased by casein kinase 2.3, which was in sharp contrast to bone morphogenetic protein 2. This further supports a major bone morphogenetic protein signaling disparity in both the elderly and those suffering with osteoporosis. Both patient types did significantly respond to casein kinase 2.3. Further analysis of the bone morphogenetic protein pathway could lead to new therapeutic products for osteoporosis.

Eggshells as natural calcium carbonate source in combination with hyaluronan as beneficial additives for bone graft materials, an in vitro study.

IntroductionIn bone metabolism and the formation especially in bone substitution, calcium as basic module is of high importance. Different studies have shown that the use of eggshells as a bone substitute material is a promising and inexpensive alternative. In this in vitro study, the effects of eggshell granulate and calcium carbonate towards primary bovine osteoblasts were investigated. Hyaluronan (HA) was used as artificial extracellular matrix (ECM) for the used cells to facilitate proliferation and differentiation and to mimic the physiological requirements given by the egg in vivo.MethodsHyaluronan, eggshells, a combination of hyaluronan and eggshells and CaCO3 were applied to the cells as additive to the used standard medium (modified High Growth Enhancement Medium) in a concentration of 0,1 g/l. The effect of the additives in the culture medium was examined by proliferation tests, immunohistochemical staining (anti-collagen type I, anti-osteopontin, anti-osteonectin and anti-osteocalcin) and kinetic oxygen measurements.ResultsOur investigations revealed that all investigated additives show beneficial effect on osteoblast activity. Cell proliferation, differentiation and the metabolic activity of the differentiated cells could be influenced positively. Especially in the case cell cultures treated with eggshells the strongest effects were detected, while for the hyaluronan compared with eggshells, a weaker increase in cell activity was observed.ConclusionIn summary, it can be stated that the investigated components come into consideration as beneficial supplements for bone graft materials especially for maxillo facial surgery application.

Bone morphogenetic protein 7 (BMP-7) influences tendon-bone integration in vitro.

IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.Materials and MethodsTo study the biological effects of BMP-7 on the process of tendon-bone-integration, two independent in vitro models were used. The first model involved the mono- and coculture of bovine tendon specimens and primary bovine osteoblasts with and without BMP-7 exposure. The second model comprised the mono- and coculture of primary bovine osteoblasts and fibroblasts. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), lactate and osteocalcin (OCN) were analyzed by ELISA. Histological analysis and electron microscopy of the tendon specimens were performed.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.

Effect of Bone Morphogenetic Protein-2 on Tendon–Bone Integration in an In Vitro Cell Culture

The goal of this study was to evaluate the influence of bone morphogenetic protein-2 (BMP-2) on tendon-bone integration in a bovine in vitro cell culture. Seventy-two bovine tendons were cultivated over 3 months. The effects of BMP-2 were evaluated by generation in 4 subgroups. The groups differed in 2 parameters: the application of BMP-2 and the application of primary bovine osteoblasts. Results were analyzed biochemically by determining alkaline phosphatase activity and histologic tendon calcification, both markers for graft incorporation. Histological analysis demonstrated a positive effect of BMP-2 on the production of extracellular matrix and therefore the induction of osteogenesis. In addition, the results showed a superior cell ingrowth on the tendon in the BMP-2-stimulated groups. Calcium carbonate-like structures and organized ossification zones could only be detected in the BMP-2-stimulated tendons. The histological results matched those of the biochemical alkaline phosphatase analysis. The highest alkaline phosphatase activity was detected using BMP-2 stimulation in the first month (P<.001). High alkaline phosphatase values suggest high osteoblast activity and a high potential for mineralization. Furthermore, a positive effect of BMP-2 on fibroblasts existed with regard to the overall integration process. These results confirm the positive influence and triggering effect of BMP-2 on the mineralization process. Bone morphogenetic protein-2 seems to accelerate and optimize tendon-bone integration in the early process of graft incorporation. Besides the influence of BMP-2 on bovine osteoblasts, an additional positive effect of BMP-2 on bovine fibroblasts was detected; therefore, graft incorporation may be carried out by osteoblasts and fibroblasts.

An in vitro study of osteoblast vitality influenced by the vitamins C and E

Vitamin C and vitamin E are known as important cellular antioxidants and are involved in several other non-antioxidant processes. Generally vitamin C and vitamin E are not synthesized by humans and therefore have to be applied by nutrition. The absence or deficiency of the vitamins can lead to several dysfunctions and even diseases (e.g. scurvy). The main interest in this study is that vitamin C and E are known to influence bone formation, e.g. vitamin C plays the key role in the synthesis of collagen, the major component of the extracellular bone matrix.In the present study we evaluate the effect of ascorbic acid (vitamin C) and α-tocopherol (vitamin E) on the proliferation and differentiation of primary bovine osteoblasts in vitro. Starting from standard growth medium we minimized the foetal calf serum to reduce their stimulatory effect on proliferation.An improved growth and an increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin was observed while increasing the ascorbic acid concentration up to 200 μg/ml. Furthermore the effects of α-tocopherol on cell growth and cell differentiation were examined, whereby neither improved growth nor increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin were detected.Further investigations are necessary to target at better supportive effect of vitamins on bone regeneration, and healing.

Dynamic redistribution of paxillin in bovine osteoblasts stimulated with adenosine 5′-triphosphate

Exposure to extracellular 5′-adenosine triphosphate (ATP) is known to induce membrane blebbing. In this study, we investigated the subcellular distribution of the cytoskeletal adaptor protein paxillin in primary bovine osteoblasts upon stimulation with ATP. Cells expressing a fusion protein of green fluorescent protein (GFP) and paxillin were followed by time-lapse video-microscopy after stimulation with 100 μM ATP. Within 100 s, GFP-paxillin became incorporated in numerous de novo formed focal aggregates localized at the cell periphery. The assembly of individual paxillin-containing aggregates occurred with a mean half-life time of <60 s, whereas their disassembly lasted twice as long. Despite the ongoing presence of ATP, the formation of paxillin aggregates was self-limiting within 25 min. Paxillin clustering was preceded by a transient rise in cytoplasmic calcium transients, which peaked already 20 s after adding ATP. The high mobility of paxillin was confirmed by measuring the dissociation rate of GFP-paxillin at mature focal adhesions, demonstrating the presence of a highly mobile fraction with a mean recovery half-life of 8.2 ± 1.2 s, followed by a slower phase (53 ± 20 s). Thus, both the exchange of paxillin at mature focal adhesions and the increase in intracellular calcium concentrations upon ATP stimulation are very rapid processes, which override the time course of ATP-induced paxillin membrane clustering by one to two orders of magnitude. Our data demonstrate that the transient recruitment of paxillin in membrane protuberances is based on the high intracytoplasmic mobility of unbound paxillin molecules and their rapid focal accumulation.

Prim�re Osteoblastenreaktionen auf SLA- und mikrostrukturierten Implantatoberfl�chen

The osseointegration process of dental implants depends on the tissue reaction at the tissue-implant interface. Osteoblasts are the main cells responsible for the regulation of osteoinduction. The manner and kinetics of the tissue reaction crucially depend on the interaction between osteoblasts and the morphology of the implant surface. The aim of this study was to investigate osteoblast behaviour on different implant surfaces (smooth, microgrooved, SLA) under standardized conditions. For this in vitro investigation we used primary bovine osteoblasts. Attachment kinetics, proliferation rate and synthesis of bone-associated proteins were used as parameters for cell reaction. The results demonstrate that both attachment and adhesion strength of the primary cell surface interaction was higher on the microgrooved surfaces than on SLA surfaces. The proliferation rate of cells and the synthesis of bone-specific proteins were higher on microgrooved surfaces in contrast to SLA surfaces. Ultrastructural analysis revealed phenotypic osteoblast-like cells on smooth and microgrooved surfaces, whereas cells on SLA surfaces showed a more fibroblastic appearance. This study demonstrates that the morphology of the implant surface determined the subsequent osteoblast reaction. An optimal cell reaction was found at surfaces which are smooth in the microenvironment of osteoblasts.

Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone.

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride-containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mof the proteoglycan was 85, 000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin alphavbeta3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts.

Attachment kinetics, proliferation rates and vinculin assembly of bovine osteoblasts cultured on different pre-coated artificial substrates.

Primary bovine osteoblasts were used to study in-vitro effects of attachment on vinculin assembly in cells cultured on various artificial substrates. Materials coated with fibronectin and bovine serum albumin (BSA) as well as untreated materials (tissue culture polystyrene and Aclar foils) were chosen to investigate substrate-dependent proliferation during the first 3 days of culture. Proliferation was highest on fibronectin-coated substrates, followed by BSA-coated and untreated substrates. During the first 24 h of cultivation, cell attachment kinetics revealed no significant difference between the various substrates. After 24 h detachment rates obtained by calcium depletion with ethylenediaminetetraacetic acid were highest on uncoated materials, followed by BSA- and fibronectin-coated substrates. Phase contrast microscopy revealed typical osteoblast morphology after cell adhesion for 24 h. The dynamic attachment process was concomitant with the reassembly of vinculin into streak-like focal contacts clustered on the ventral side of cells. The kinetics of vinculin reassembly were independent of the underlying coating. Thus, fibronectin coating of artificial substrates increased the attachment strength and proliferation rate of osteoblasts. While the reassembly of vinculin in focal contacts seems to be a prerequisite of osteoblast attachment in vitro, it does not seem to have profound effects on the subsequent cell behaviour on artificial substrates.

No mechanical role for vinculin in strain transduction in primary bovine osteoblasts

Vinculin is thought to play a central role in linking the actin cytoskeleton to the integrin adhesion proteins of focal contacts and also in cell-cell adhesion sites. We have investigated aspects of attachment and the transduction of mechanical stimulation on the function of vinculin and its assembly in primary bovine osteoblasts. The dynamic attachment process was concomitant with the reassembly of vinculin from a soluble cytoplasmic pool into Triton-insoluble focal adhesions. Immunoblotting experiments demonstrated a slight increase in the amount of vinculin concentrated in focal contacts that was paralleled by a discrete decrease in the Triton-soluble fraction of cytoplasmic vinculin. Sixty cycles of 1 Hz deformations at 0.02% (hypophysiological, no change in cell division rate), 0.2% (within the normal physiological range), and 1% (hyperphysiological) resulted in a rapid and reversible disassembly of vinculin from focal contacts to a homogeneous cytoplasmic localization, although alterations in the shape and morphology of cells could not be detected. Although all mechanical loading protocols dramatically depleted vinculin from focal contacts, its initial distribution in adhesion plaques was fully restored within the next 60 min, demonstrating the highly dynamic and reversible shift of this protein from a membrane-associated pool to the cytoplasm. By first depleting vinculin from the focal adhesion sites by applying 0.02% strain, waiting 60 s, and then applying 0.2% strain, we show that the transduction of mechanical deformation was identical with controls that had not been pretreated. This indicates that vinculin does not play a role in mechanical transduction, that significant mechanical forces are not transmitted through vinculin, and also that loss of vinculin at the focal adhesion site does not have significant effects, in the short term, on cell adhesion.

No mechanical role for vinculin in strain transduction in primary bovine osteoblasts

Vinculin is thought to play a central role in linking the actin cytoskeleton to the integrin adhesion proteins of focal contacts and also in cell-cell adhesion sites. We have investigated aspects of attachment and the transduction of mechanical stimulation on the function of vinculin and its assembly in primary bovine osteoblasts. The dynamic attachment process was concomitant with the reassembly of vinculin from a soluble cytoplasmic pool into Triton-insoluble focal adhesions. Immunoblotting experiments demonstrated a slight increase in the amount of vinculin concentrated in focal contacts that was paralleled by a discrete decrease in the Triton-soluble fraction of cytoplasmic vinculin. Sixty cycles of 1 Hz deformations at 0.02% (hypophysiological, no change in cell division rate), 0.2% (within the normal physiological range), and 1% (hyperphysiological) resulted in a rapid and reversible disassembly of vinculin from focal contacts to a homogeneous cytoplasmic localization, although alterations in the shape and morphology of cells could not be detected. Although all mechanical loading protocols dramatically depleted vinculin from focal contacts, its initial distribution in adhesion plaques was fully restored within the next 60 min, demonstrating the highly dynamic and reversible shift of this protein from a membrane-associated pool to the cytoplasm. By first depleting vinculin from the focal adhesion sites by applying 0.02% strain, waiting 60 s, and then applying 0.2% strain, we show that the transduction of mechanical deformation was identical with controls that had not been pretreated. This indicates that vinculin does not play a role in mechanical transduction, that significant mechanical forces are not transmitted through vinculin, and also that loss of vinculin at the focal adhesion site does not have significant effects, in the short term, on cell adhesion.