Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

Bi, Lian Xiang; Simmons, David J.; Mainous, Elgene G.

doi:10.1007/s002239900580

Cited by 52 publications

(35 citation statements)

References 47 publications

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“…BMP-2 has been reported to be expressed by BMSC in vitro, particularly when stimulated with OM [Bi et al, 1999], and in turn to stimulate BMSC to generate bone tissue in vivo [Yamagiwa et al, 2001]. BSP has been proposed as the main nucleator of hydroxyapatite crystal formation and correlates with the initial phase of matrix mineralization [Bianco et al, 1993].…”

Section: Discussionmentioning

confidence: 99%

Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

Frank

Heim

Jakob

et al. 2002

J of Cellular Biochemistry

336

223

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We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.

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Section: Discussionmentioning

confidence: 99%

Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

Frank

Heim

Jakob

et al. 2002

J of Cellular Biochemistry

336

223

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“…Some researchers use calvarial cells 4,15,16 to examine the osteogenic capacity of the scaffold material, while others use bone marrow cells. 4,17,18 Besides, different types of rats are used for cell retrieval, that is, Wistar 18,19 or Sprague-Dawley. 4,20 Also, the rats used varied in age.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Various Seeding Techniques for Culturing Osteogenic Cells on Titanium Fiber Mesh

Dolder

Spauwen²,

Jansen³

2003

Tissue Engineering

112

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The objective of the present study was to learn more about the effect of seeding and loading techniques on the osteogenic differentiation in vitro of rat bone marrow cells into titanium fiber mesh. This material was used as received or subjected to glow discharge treatment (RFGD). The seeding methods that were used included a so-called droplet, cell suspension (high and low cell density), and rotating plate method. Osteogenic cells were cultured for 4, 8, and 16 days into titanium fiber mesh. DNA, osteocalcin, scanning electron microscopy (SEM) analysis, and calcium measurements were used to determine cellular proliferation and differentiation. DNA analysis of the differently seeded specimens showed that proliferation proceeded faster in the first versus second run for droplet and cell suspension samples. No clear and distinct additional effect was found when RFGD treatment was used. Statistical analyses revealed that high cell density and low rotational speed resulted always in a significantly higher DNA content. Calcium measurements and osteocalcin analysis showed that using high cell densities during inoculation of the scaffolds prevented the occurrence of differences between experimental runs. SEM examination showed that for droplet and cell suspension samples cells were present at only one side of the mesh. The mesh side where the cell sheet was observed depended on the additional use of glow discharge treatment. On these materials, the cells had penetrated through the meshes and formed a cell sheet at the bottom side. When rotation was used, no cell sheet was formed and cells had invaded the meshes and were growing around the titanium fibers. On the basis of our results, we conclude that (1). titanium fiber mesh is indeed suitable to support the osteogenic expression of bone marrow cells, and (2). changing the initial cell density as well as the use of dynamic seeding methods can influence the osteogenic capacity of the scaffold.

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“…[11][12][13] Also, the addition of factors such as dexamethasone and BMP is known to direct the differentiation of mesenchymal stromal cells into the osteoblast lineage. [14][15][16][17] Finally, the use of scaffold material and surface modifications, such as a calcium phosphate coating, can play an important role in stimulating the osteogenic potential of stromal cells. [18][19][20] In view of the aforementioned, our previous studies with titanium fiber mesh indicated that culture time can be important for the final bone formation.…”

Section: Introductionmentioning

confidence: 99%

Bone formation by rat bone marrow cells cultured on titanium fiber mesh: Effect of in vitro culture time

Dolder

Vehof

Spauwen³

et al. 2002

J. Biomed. Mater. Res.

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The objective of this study was to examine the effect of cell culture time on bone formation by rat bone marrow cells seeded in titanium fiber mesh. As a seeding technique, a high cell suspension was used (3 x 10(6) cells/mL). Therefore, 30 meshes were repeatedly rotated in a 10 mL tube (containing 30 x 10(6) cells) on a rotation plate (2 rpm) for 3 h. Osteogenic cells were cultured for 1, 4, and 8 days on titanium fiber mesh and finally implanted subcutaneously in rats. Meshes without cells were also implanted subcutaneously in rats. DNA and scanning electron microscopy (SEM) analyses and calcium measurements determined cellular proliferation and differentiation during the in vitro incubation period of the mesh implants. Four weeks after implant insertion, the animals were sacrificed. The implants, with their surrounding tissue, were retrieved and prepared for histologic evaluation and calcium measurements. DNA analysis of the in vitro experiment showed a lag phase from day 1 through day 4, but a 42% increase in DNA between days 4 and 8. SEM and calcium measurements indicated an increase in calcium from day 1 to day 4, yet only a small but significant increase from days 4 to 8. Histologic analysis demonstrated that bone was formed in all day 1 and day 4 implants, and that the bone-like tissue was present uniformly through the meshes. The bony tissue was morphologically characterized by osteocytes embedded in a mineralized matrix, with a layer of osteoid and osteoblasts at the surface. The day 8 implants showed only calcium phosphate deposition in the titanium fiber mesh. Calcium measurements of the implants revealed that calcification in day 1 implants was significantly higher (p < 0.05) compared to day 4 and day 8 implants. No significant difference in calcium content existed between day 4 and day 8 implants. On the basis of our results, we conclude that 1) bone formation was generated more effectively in osteogenic cells by a short culture time after seeding in titanium fiber mesh; 2) dynamic cell seeding is probably more effective than static cell seeding; and 3) selection of the right cells from the heterogenous bone marrow population remains a problem.

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Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

Cited by 52 publications

References 47 publications

Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

Real‐time quantitative RT‐PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

Evaluation of Various Seeding Techniques for Culturing Osteogenic Cells on Titanium Fiber Mesh

Bone formation by rat bone marrow cells cultured on titanium fiber mesh: Effect of in vitro culture time

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