In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 μm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.     

Type XII collagen regulates osteoblast polarity and communication during bone formation

Differentiated osteoblasts are polarized in regions of bone deposition, demonstrate extensive cell interaction and communication, and are responsible for bone formation and quality. Type XII collagen is a fibril-associated collagen with interrupted triple helices and has been implicated in the osteoblast response to mechanical forces. Type XII collagen is expressed by osteoblasts and localizes to areas of bone formation. A transgenic mouse null for type XII collagen exhibits skeletal abnormalities including shorter, more slender long bones with decreased mechanical strength as well as altered vertebrae structure compared with wild-type mice. Col12a(-/-) osteoblasts have decreased bone matrix deposition with delayed maturation indicated by decreased bone matrix protein expression. Compared with controls, Col12a(-/-) osteoblasts are disorganized and less polarized with disrupted cell-cell interactions, decreased connexin43 expression, and impaired gap junction function. The data demonstrate important regulatory roles for type XII collagen in osteoblast differentiation and bone matrix formation.     

Role of N-cadherin in bone formation

Cell-cell adhesion mediated by cadherins is essential for the function of bone forming cells during osteogenesis. Here, the evidence that N-cadherin is an important regulator of osteoblast differentiation and osteogenesis is reviewed. Osteoblasts express a limited number of cadherins, including the classic N-cadherin. The expression profile of N-cadherin in osteoblasts during bone formation in vivo and in vitro suggests a role of this molecule in osteogenesis. Functional studies using neutralizing antibodies or antisense oligonucleotides indicate that N-cadherin is involved in the control the expression of osteoblast marker gene expression and differentiation. Cleavage of N-cadherin during osteoblast apoptosis also suggests a role of N-cadherin-mediated-cell-cell adhesion in osteoblast survival. Hormonal and local factors that regulate osteoblast function also regulate N-cadherin expression and subsequent cell-cell adhesion associated with osteoblast differentiation or survival. Signaling mechanisms involved in N-cadherin-mediated cell-cell adhesion and osteoblast gene expression have also been identified. Alterations of N-cadherin expression are associated with abnormal osteoblast differentiation and osteogenesis in pathological conditions. These findings indicate that N-cadherin plays a role in normal and pathological bone formation and provide some insight into the process involved in N-cadherin-mediated cell-cell adhesion and differentiation in osteoblasts.     

Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways

Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression, although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss.     

Influence of Enamel Matrix Derivative on Cells at Different Maturation Stages of Differentiation

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2–5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.     

Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: Altered expression of collagenase,cell growth-related,and mineralization-associated genes

Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1–10), addition of acidic FGF (100 μg/ml) to actively proliferating cells increased (P < 0.05) ³H-thymidine uptake (2,515 ± 137, mean ± SEM vs. 5,884 ± 818 cpm/10⁴ cells). During the second stage of maturation (days 10–15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16–29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14–29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7–8) enhanced histone H4, osteopontin, type 1 collagen, and TGF-β mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14–15) reactivated H4, osteopontin, type I collagen, and TGF-β gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 μg/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-β and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence. © 1996 Wiley-Liss, Inc.     

Hypothermia inhibits osteoblast differentiation and bone formation but stimulates osteoclastogenesis

It has long been known that core body temperature declines with age, with temperatures of 35.5°C or below common in the elderly. However, the effects of temperature reduction on bone cell function and skeletal homeostasis have been little studied. We investigated the effects of mild hypothermia (35.5°C) and severe hypothermia (34°C) on bone-forming osteoblasts, and bone-resorbing osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by 75% at 35.5°C and by 95% at 34°C after 14-16 days culture, compared to 37°C. In addition to reductions in osteoblast cell number, expression of mRNAs for Runx2, alkaline phosphatase, osteocalcin and type I collagen were also down-regulated in hypothermia. In contrast, formation of osteoclasts in mononuclear cell cultures derived from mouse marrow, showed a 1.5 to 2-fold stimulation in hypothermia; resorption pit formation was similarly increased. Taken together, these data show that hypothermia exerts reciprocal effects on bone cell function by retarding osteoblast differentiation and bone formation, whilst increasing osteoclastogenesis and thus resorption. These results suggest the possibility that hypothermia in the elderly could potentially have a direct, negative impact on bone metabolism.     

High-dose retinoic acid modulates rat calvarial osteoblast biology

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.     

Simulated microgravity suppresses osteoblast phenotype,Runx2 levels and AP-1 transactivation

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood but involve the suppression of osteoblast function. To elucidate the influence of mechanical unloading on osteoblasts, a rotating wall vessel (RWV) was employed as a ground based model of simulated microgravity. Mouse MC3T3-E1 osteoblasts were grown on microcarrier beads for 14 days and then placed in the RWV for 24 h. Consistent with decreased bone formation during actual spaceflight conditions, alkaline phosphatase and osteocalcin expression were decreased by 80 and 50%, respectively. In addition, runx2 expression and AP-1 transactivation, key regulators of osteoblast differentiation and bone formation, were reduced by more than 60%. This finding suggests that simulated microgravity could promote dedifferentiation and/or transdifferentiation to alternative cell types; however, markers of adipocyte, chondrocyte, and myocyte lineages were not induced by RWV exposure. Taken together, our results indicate that simulated microgravity may suppress osteoblast differentiation through decreased runx2 and AP-1 activities.     

在海藻酸钠凝胶上诱导骨髓间充质干细胞分化为成骨细胞

通过在海藻酸钠凝胶上诱导bMSCs向成骨细胞分化,探讨其对骨髓间充质干细胞（bone mesenchymal stem cells, bMSCs）的生物学效应。采用MTT、甲苯胺蓝染色、von Kossa染色和RT-PCR分别检测细胞的增殖、生长形态、诱导后细胞的钙化结节和成骨相关基因的表达。实验组bMSCs生长状况良好、细胞增殖迅速,与对照组的增殖无差异;bMSCs成集落样生长明显,集落中央细胞重叠生长形成钙化结节;培养至12d,实验组和对照组的成骨相关基因,包括碱性磷酸酶、I型胶原和骨钙素,均为阳性表达,但实验组的表达量高于对照组。海藻酸钠凝胶能够促进bMSCs向成骨细胞的分化,是良好的骨组织工程支架材料。     

Prolactin decreases the expression ratio of receptor activator of nuclear factor kappaB ligand/osteoprotegerin in human fetal osteoblast cells

Prolactin (PRL) enhanced bone remodeling leading to net bone loss in adult and net bone gain in young animals. Studies in PRL-exposed osteoblasts derived from adult humans revealed an increase in the expression ratio of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG), thus supporting the previous finding of PRL-induced bone loss in adults. This study thus investigated the effects of PRL on the osteoblast functions and the RANKL/OPG ratio in human fetal osteoblast (hFOB) cells which strongly expressed PRL receptors. After 48h incubation, PRL increased osteocalcin expression, but had no effect on cell proliferation. However, the alkaline phosphatase activity was decreased in a dose-response manner within 24h. The effect of PRL on alkaline phosphatase was abolished by LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor. PRL also decreased the RANKL/OPG ratio by downregulating RANKL and upregulating OPG expression, implicating a reduction in the osteoblast signal for osteoclastic bone resorption. It could be concluded that, unlike the osteoblasts derived from adult humans, PRL-exposed hFOB cells exhibited indices suggestive of bone gain, which could explain the in vivo findings in young rats. The signal transduction of PRL in osteoblasts involved the PI3K pathway.     

Effects of cAMP on intercellular coupling and osteoblast differentiation

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca(2+) waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers.     

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.     

Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions.

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.     

Induction of a program gene expression during osteoblast differentiation with strontium ranelate

Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.