Dependence of mesenchymal cell responses on duration of exposure to bone morphogenetic protein-2 in vitro

Bone morphogenetic proteins (BMPs) induce osteoblastic responses in cultures of pluripotent mesenchymal cells. The effects of chronic treatment of these cells with BMPs and of withdrawal following exposure, however, have not been fully elucidated. Thus, the aim of this study was to obtain information about the duration of exposure to recombinant human BMP-2 (rhBMP-2) required for expression and retention of osteoblastic characteristics with subsequent formation of a mineralized extracellular matrix in mesenchymal cell cultures. C3H10T1/2 cells and bone marrow stromal cells were cultured with 1 μg/ml rhBMP-2 for either 0, 7, 14, 21, or 28 days, with the remainder of the 4 week total culture period in the absence of rhBMP-2. Growth and expression of osteoblastic characteristics were examined at the end of each week. C3H10T1/2 cells responded to increasing duration of exposure to rhBMP-2 with increased cell growth. Additionally, the longer the cells were exposed to rhBMP-2, the more fully they expressed and sustained osteoblastic traits, i.e., they exhibited duration of exposure-dependent higher levels of alkaline phosphatase and osteocalcin and larger total amounts of mineral in the matrix. In comparison, exposure of bone marrow stromal cells to rhBMP-2 for at least 14 days restrained cell growth and prevented detachment. With respect to osteoblastic traits, stromal cells exposed to rhBMP-2 also exhibited a dependence on the duration of exposure, however, cultures treated for 14, 21, or 28 days exhibited similar levels of alkaline phosphatase activity and comparable amounts of calcium in the mineralizing matrix. J. Cell. Physiol. 173:93–101, 1997. © 1997 Wiley-Liss, Inc.     

Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.     

Effects of Bone Morphogenetic Protein-2 and Transforming Growth Factor-β1 on Gene Expression of Decorin and Biglycan by Cultured Osteoblastic Cells

The influence of bone morphogenetic protein-2 (BMP-2) and transforming growth factor (TGF-) on the expression of small proteoglycans, decorin and biglycan was investigated in a clonal rat osteoblastic cell line, ROS-C26 (C26) cells, which is a potential osteoblast precursor cell line and capable of differentiating into mature osteoblasts after treatment with recombinant BMP-2 (rhBMP-2). Following the culture of C26 cells for 3, 6, and 9 days in the presence or absence of rhBMP-2, alkaline phosphatase activity increased in the rhBMP-2 treated cells in direct proportion to their differentiation into more mature osteoblastic cells, whereas decorin mRNA decreased in the cells, when compared to control cells without rhBMP-2 treatment. These results were evident 6 days after treatment. However, rhBMP-2 treatment had no effect on biglycan mRNA expression in the cells. Subsequently, after removal of rhBMP-2 from the culture media, the cells were further cultured for 24h with graded concentrations of TGF-1 (0, 0.1, 1.0, 5.0, and 10ng/ml). TGF-1 decreased decorin mRNA expression in the cells dose dependently, but did not affect their biglycan mRNA expression. Furthermore, either removal of rhBMP-2 from the culture media or addition of TGF-1 significantly decreased alkaline phosphatase activity of rhBMP-2-induced cells. These results indicate that osteoblastic differentiation is accompanied by increased alkaline phosphatase activity and decreased expression of decorin mRNA, but continuous expression of biglycan mRNA. Both rhBMP-2 and TGF-1 inhibit decorin mRNA expression in osteoblasts at varying stages of differentiation, but their effects on biglycan mRNA expression and alkaline phosphatase are different.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells

For bone development, remodeling, and repair; the recruitment of mesenchymal progenitor cells (MPC) and their differentiation to osteoblasts is mandatory. The process of migration is believed to be regulated in part by growth factors stored within the bone matrix and released by bone resorption. In this study, primary human MPCs and to osteoblasts differentiated progenitor cells were examined for chemotaxis in response to human basic fibroblast growth factor (rhbFGF), human transforming growth factor beta 1 (rhTGF-beta1), human platelet derived growth factor bb (rhPDGF-bb), human bone morphogenetic protein-2 (rhBMP-2), and recombinant bone morphogenetic protein-4 of Xenopus laevis (rxBMP-4) from 0.001 to 1.0 ng/ml each. The results of migration were expressed as a chemotactic index (CI). Migration of primary human progenitor cells was stimulated by rhBMP-2, rxBMP-4, and rhPDGF-bb in a dose-dependent manner. The increase of CI was up to 3.5-fold for rhBMP-2, 3.6-fold for rxBMP-4, and up to 22-fold for rhPDGF-bb, whereas rhTGF-beta1 and rhbFGF did not stimulate cell migration in the concentration range tested. In contrast differentiated progenitor cells behave similar to primary human osteoblasts. RhBMP-2, rhPDGF-bb, and rhTGF-beta1 stimulated the migration from 2.2 to 2.4-fold each, while rxBMP-4 and rhbFGF reached only a CI of 1.7-1.6. The effect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary human MPC, including the change in response to growth factors after differentiation suggests a functional role for recruitment of MPCs during bone development and remodeling, as well as fracture healing.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.     

Recombinant human bone morphogenetic protein-2 enhances expression of interleukin-6 and transforming growth factor-β1 genes in normal human osteoblast-like cells

The process of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced endochondral ossification involves (1) the proliferation and differentiation of mesenchymal cells into chondroblasts and osteoblasts; (2) the production and maturation of cartilage and bone matrix; and (3) the differentiation of circulating osteoclast precursor cells into osteoclasts. Currently the molecular mechanisms of these complex sequential events are unknown. It seemed reasonable to us to assume that communication between cells through soluble mediators during bone induction by rhBMP-2 may play an important role in the sequential differentiation of chondroblasts, osteoblasts, and osteoclasts. We have therefore used a human osteoblast-like initial transfectant cell line (HOBIT) to study the effect of rhBMP-2 on gene expression of interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1), both of which affect osteogenesis and ostoeclastogenesis. Our results have demonstrated that rhBMP-2 acts on HOBIT cells to stimulate expression of IL-6 and TGF-β1 genes and the production of IL-6. Enhancement of gene expression of IL-6 and TGF-β1 by rhBMP-2 was both sensitive (half maximal effect at approximately 10 ng/ml) and potent (maximum induction was approximately four and threefold greater than controls, respectively). Time course studies showed that the induction of TGF-β1 and IL-6 mRNA occurs within short periods—4 and 8 hours after exposure to rhBMP-2, respectively. Interestingly, these effects, however, were not accompanied by the mitogenic action of rhBMP-2. It suggests that rhBMP-2 enhances IL-6 and TGF-β1 production during osteogenesis and at least in part mediates the complex sequential differentiation of chondroblasts, osteoblasts, and osteoclasts during rhBMP-2-induced endochondral ossification. © 1994 wiley-Liss, Inc.     

Recombinant human bone morphogenetic protein-2 promotes Indian hedgehog-mediated osteo-chondrogenic differentiation of a human chondrocytic cell line in vivo and in vitro

We examined osteo-chondrogenic differentiation of a human chondrocytic cell line (USAC) by rhBMP-2 in vivo and in vitro. USAC was established from a transplanted tumor to athymic mouse derived from an osteogenic sarcoma of the mandible. USAC usually shows chondrocytic phenotypes in vivo and in vitro. rhBMP-2 up-regulated not only the mRNA expression of types II and X collagen, but also the mRNA expression of osteocalcin and Cbfa1 in USAC cells in vitro. In vivo experimental cartilaginous tissue formation was prominent in the chamber with rhBMP-2 when compared with the chamber without rhBMP-2. USAC cells implanted with rhBMP-2 often formed osteoid-like tissues surrounded by osteoblastic cells positive for type I collagen. rhBMP up-regulated Ihh, and the expression of Ihh was well correlated with osteo-chondrogenic cell differentiation. These results suggest that rhBMP-2 promotes chondrogenesis and also induces osteogenic differentiation of USAC cells in vivo and in vitro through up-regulation of Ihh.     

Comparing the Effects of Chitosan Scaffolds Containing Various Divalent Metal Phosphates on Osteogenic Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth

Inducing the differentiation of stem cells from human exfoliated deciduous teeth (SHEDs) proceeds with low efficiency, which greatly limits clinical applications. Divalent metal elements play an important role in osteoinductivity for bone remodeling because they can simulate bone formation and decrease bone resorption. The purpose of this study was to investigate the effect of some divalent metal phosphates on osteogenic differentiation from human exfoliated deciduous teeth. These divalent metal ions can be gradually released from the scaffold into the culture medium and continually induce osteoblastic differentiation. Experimental results revealed that SHEDs cultured in chitosan scaffolds containing divalent metal phosphates had notably increased osteoblastic differentiation compared with cells cultured without divalent metal phosphates. This effect was due to the high activity of alkaline phosphatase, as well as the bone-related gene expression of collagen type I, Runx2, osteopontin, osteocalcin, VEGF, and Ang-1, shown through RT-PCR and bone-related protein immunocytochemistry stains. A calcium-content assay further revealed significant enhancement of deposited minerals on the scaffolds after 21 days of culture, particularly for magnesium phosphate and zinc phosphate. Thus, divalent metals, except for barium phosphate, effectively promoted SHED cell differentiation and osteoblastic cell maturation. This study demonstrated that the divalent metal elements magnesium, strontium, and zinc could effectively induce SHED osteoblastic differentiation for use in tissue engineering and bone repair.     

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells.

Mesenchymal progenitor cells derived from cord blood (unrestringated somatic stem cells, USSC) and bone marrow (mesenchymal stem cells, MSC) are able to differentiate under defined culture conditions into at least bone, cartilage, adipose and muscle cells in vitro. The culture media and other in vitro conditions influence the osteogenic differentiation potency of both cell types. To increase and expand the number of osteoblasts in vitro an optimization of culture conditions is required. The aim of this study was to evaluate different culture media toward their osteogenic promoting capacity on human USSCs and MSCs in vitro. Immunohistochemical stainings against osteonectin (ON), osteopontin (OP) served as markers for an osteoblastic differentiation. Cellular morphology was analysed by light microscopy technique. We found significant differences between bone marrow and cord blood derived stem cells towards an osteoblastic differentiation. Considering the number of osteoblasts MesenCult seems to have advantages in bone marrow progenitor cells, whereas low glucose DMEM and HAMS-F12 promoted an osteoblastic differentiation in cord blood derived cells more than other tested media.     

Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells

Proper bone remodeling requires an active process of angiogenesis which in turn supplies the necessary growth factors and stem cells. This tissue cooperation suggests a cross‐talk between osteoblasts and endothelial cells. This work aims to identify the role of paracrine communication through vascular endothelial growth factor (VEGF) in co‐culture between osteoblastic and endothelial cells. Through a well defined direct contact co‐culture model between human osteoprogenitors (HOPs) and human umbilical vein endothelial cells (HUVECs), we observed that HUVECs were able to migrate along HOPs, inducing the formation of specific tubular‐like structures. VEGF₁₆₅ gene expression was detected in the HOPs, was up‐regulated in the co‐cultured HOPs and both Flt‐1 and KDR gene expression increased in co‐cultured HUVECs. However, the cell rearrangement observed in co‐culture was promoted by a combination of soluble chemoattractive factors and not by VEGF₁₆₅ alone. Despite having no observable effect on endothelial cell tubular‐like formation, VEGF appeared to have a crucial role in osteoblastic differentiation since the inhibition of its receptors reduced the co‐culture‐stimulated osteoblastic phenotype. This co‐culture system appears to enhance both primary angiogenesis events and osteoblastic differentiation, thus allowing for the development of new strategies in vascularized bone tissue engineering. J. Cell. Biochem. 106: 390–398, 2009. © 2009 Wiley‐Liss, Inc.     

Pleiotrophin regulates bone morphogenetic protein (BMP)-induced ectopic osteogenesis

We previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2 microg) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100 microg) or rhPTN (5 and 10 microg) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10 microg of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100 microg of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100 microg of PTN to 1.2 microg of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.     

Growth behaviour of human mononuclear cells derived from bone marrow and cord blood on a collagen carrier for osteogenic regeneration]

We investigated the biocompatibility and osteogenetic potency of a porcine collagen I/III carrier in a human bone marrow and cord blood cell culture system. METHODS: Human mesenchymal mononuclear cells were isolated from cord blood and iliac crest bone marrow and cultivated in various cell densities on a semipermeable porcine collagen I/III carrier. After 14 days of in vitro cultivation both cultures were subjected to osteogenic stimulation by dexamethasone, ascorbic acid and beta-glycerol phosphate (DAG) until day 40. Semiquantitative immunochemical evaluation based on osteoblastic and progenitor cell markers was then done. RESULTS: With regard to the minimal local cell density required for growth and osteogenic differentiation, cord blood derived progenitor cells showed lower tolerance in comparison with bone marrow derived cells. For both cell culture systems three-dimensional growth and calcification within the collagen fibres were seen after osteogenic stimulation. CONCLUSION: Human cord blood and bone marrow derived mesenchymal stem cell are capable of differentiating into osteoblasts after incubation with a collagen I/III biomaterial.     

Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different biomaterials

The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.