Juliette van den Dolder

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

Bone tissue reconstruction using titanium fiber mesh combined with rat bone marrow stromal cells

The study aim was to evaluate the effect of bone marrow stromal cells (BMSCs) cultured in titanium fiber mesh and implanted in a rat cranial defect. A total of 24 titanium meshes were placed in a tube containing 10 ml BMSC suspension (3 x 10(6)cells/ml) and the tube was rotated on a rotation plate (2 rpm) during 3 h. Thereafter, meshes with cells were subcultured for 1 day under standard conditions. Cell-loaded implants and non-cell-loaded controls were placed in a 8 mm cranial defect and retrieved after 3, 15 and 30 days of implantation. Histology showed that after 3 days of implantation, the mesh porosity of both implant groups was mainly invaded with blood cells. On the other hand, at 15 days of implantation, the cell-loaded implants were filled for 15 +/- 10% of their volume with bone, while the controls showed 1.5 +/- 3.5% of bone. The 30-day cell-loaded implants showed 40 +/- 12.5% of bone and the 30-day control implants 17 +/- 14.5%. At both implantation times the differences were statistically significant. Therefore, we conclude that inoculation of titanium fiber mesh with BMSCs can improve the bone healing capacity of this material.

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

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.

Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow

The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic scaffold material, and then either cultured in an osteogenic medium or subcutaneously implanted into nude mice. For cell culture, samples were collected at weeks 0, 1, 3, and 5. Results were analyzed by measuring cell proliferation rate and alkaline phosphatase activity, scanning electron microscopy, and real-time PCR. Samples from the implantation study were retrieved after 5 and 10 weeks and evaluated by histology and real-time PCR. The results indicated that in vitro abundant cell growth and mineralization of extracellular matrix was observed for all types of cells. However, in vivo matured bone formation was found only in the samples seeded with rat bone marrow stromal cells. Real-time PCR suggested that the expression of Runx2 and the expression osteocalcin were important for the differentiation of bone marrow stromal cells, while dentin sialophosphoprotein contributed to the odontogenic differentiation. In conclusion, the limited hard tissue regeneration ability of dental pulp stromal cells questions their practical application for complete tooth regeneration. Repeated cell passaging may explain the reduction of the osteogenic ability of both bone- and dentinal-derived stem cells. Therefore, it is essential to develop new cell culture methods to harvest the desired cell numbers while not obliterating the osteogenic potential.

Evaluation of the biocompatibility of calcium phosphate cement/PLGA microparticle composites.

In this study, the biocompatibility of a calcium phosphate (CaP) cement incorporating poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles was evaluated in a subcutaneous implantation model in rats. Short-term biocompatibility was assessed using pure CaP discs and CaP discs incorporating PLGA microparticles (20% w/w) with and without preincubation in water. Long-term biocompatibility was assessed using CaP discs incorporating varying amounts (5, 10, or 20% w/w) and diameter sizes (small, 0-50 mum; medium, 51-100 mum, or large, 101-200 mum) of PLGA microparticles. The short-term biocompatibility results showed a mild tissue response for all implant formulations, irrespective of disc preincubation, during the early implantation periods up to 12 days. Quantitative histological evaluation revealed that the different implant formulations induced the formation of similar fibrous tissue capsules and interfaces. The results concerning long-term biocompatibility showed that all implants were surrounded by a thin connective tissue capsule (<10 layers of fibroblasts). Additionally, no significant differences in capsule and interface scores were observed between the different implant formulations. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth throughout the implants, indicating PLGA degradation and interconnectivity of the pores. The results demonstrate that CaP/PLGA composites evoke a minimal inflammatory response. The implants containing 20% PLGA with medium- and large-sized microparticles showed fibrous tissue ingrowth after 12- and 24-weeks indicating PLGA degradation and interconnectivity of the pores. Therefore, CaP/PLGA composites can be regarded as biocompatible biomaterials with potential for bone tissue engineering and advantageous possibilities of the microparticles regarding material porosity.

Effect of Fibronectin- and Collagen I-Coated Titanium Fiber Mesh on Proliferation and Differentiation of Osteogenic Cells

The objective of this study was to evaluate the effects of fibronectin and collagen I coatings on titanium fiber mesh on the proliferation and osteogenic differentiation of rat bone marrow cells. Three main treatment groups were investigated in addition to uncoated titanium fiber meshes: meshes coated with fibronectin, meshes coated with collagen I, and meshes coated first with collagen I and then subsequently with fibronectin. Rat bone marrow cells were cultured for 1, 4, 8, and 16 days in plain and coated titanium fiber meshes. In addition, a portion of each of these coating treatment groups was cultured in the presence of antibodies against fibronectin and collagen I integrins. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, osteocalcin, and calcium content and alkaline phosphatase activity. There were no significant effects of the coatings on cellular proliferation as indicated by the DNA quantification analysis. When antibodies against fibronectin and collagen I integrins were used, a significant reduction (p < 0.05) in cell proliferation was observed for the uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. The different coatings also did not affect the alkaline phosphatase activity of the cells seeded on the coated meshes. However, the presence of antibodies against fibronectin or collagen I integrins resulted in significantly delayed expression of alkaline phosphatase activity for uncoated titanium meshes, meshes coated with collagen, and meshes coated with collagen and fibronectin. Calcium measurements did not reveal a significant effect of fibronectin or collagen I coating on calcium deposition in the meshes. Also, no difference in calcium content was observed in the uncoated titanium meshes and meshes coated with fibronectin when antibodies against fibronectin or collagen I integrins were present. Meshes coated with both collagen I and fibronectin showed significantly higher calcium content when cultured in the presence of antibodies to collagen and fibronectin integrins. A similar phenomenon was also observed for collagen-coated meshes cultured in the presence of antibodies to fibronectin integrins. No significant differences in osteocalcin content were observed between the treatment groups. However, all groups exposed to antibodies against fibronectin integrins showed a significant decrease in osteocalcin content on day 16. These results show that a fibronectin or collagen I coating does not stimulate the differentiation of rat bone marrow cells seeded in a titanium fiber mesh.

Observations on the effect of BMP-2 on rat bone marrow cells cultured on titanium substrates of different roughness

The objective of this study was to examine the osteoinductive capacity of different concentrations of BMP-2 on bone marrow stromal cells in vitro. Further, we intended to determine whether titanium provided with an increased surface roughness is more efficient in osteoblast differentiation than machined titanium. Therefore, 20,000 cells/ml were seeded and cultured on machined and grit-blasted titanium discs for 4, 8 and 16 days. Different concentrations of rhBMP-2 (0, 10, 100, 1000 ng/ml) were supplemented to the medium for 8 days of culturing. To evaluate cellular proliferation and differentiation, specimens were examined for DNA, alkaline phosphatase activity, and calcium content. Morphological appearance of the specimens at 8 and 16 days of incubation was evaluated using scanning electron microscopy. Two separate experimental runs were performed. Evaluation of the DNA and alkaline phosphatase data revealed that a significant difference existed for these data between both experimental runs. Further analysis of the DNA figures learned that roughening of the titanium surface and addition of BMP-2 had no effect on cell proliferation. The alkaline phosphatase analysis and calcium measurements revealed that BMP-2 stimulated the early differentiation of osteogenic cells on machined titanium substrates in a dose-dependent manner. After 16 days of culture, no significant differences in calcium content could be observed anymore between machined and roughened titanium surfaces. Further, the data revealed that the machined surfaces showed a significant increase in calcium deposition when 100 and 1000 ng/ml BMP-2 were supplemented to the medium. However, the roughened surfaces showed this significant enhancement in calcium content only with 1000 ng/ml BMP-2. In addition, SEM evaluation revealed a dose-dependent response to BMP-2. Increasing BMP-2 concentrations resulted in more calcified globular accretions on bone surfaces than when no BMP-2 was added. On the basis of our results, we conclude that (1) due to the heterogeneous nature of bone marrow, experimental results with primary rat bone marrow cells are difficult to reproduce from one experiment to the other, and (2) addition of rhBMP-2 in the medium stimulates the early differentiation and matrix mineralization of osteogenic cells on machined titanium surfaces in a dose-responsive manner. Further, we concluded that our roughened titanium surfaces had no effect on proliferation and differentiation of primary derived rate bone marrow cells.

Bone regenerative properties of injectable PGLA-CaP composite with TGF-beta1 in a rat augmentation model.

The aim of this study was to examine the bone augmentation properties of an injectable composite consisting of PLGA microspheres/CaP cement (20/80), and the additional effect of loading PLGA microspheres with TGF‐β1 (200 ng). For this purpose, PLGA/CaP composites (control) and PLGA/CaP composites loaded with TGF‐β1 (test group) were injected on top of the skulls of 24 Wistar rats. Each rat received 2 materials from the same experimental group, and in total 48 implants were placed (n = 8). After 2, 4, and 8 weeks the results were evaluated histologically and histomorphometrically. The contact length between the implants and newly formed bone increased in time, and was significantly higher for the TGF‐β1‐loaded composites after 2 weeks. Also, bone formation was significantly higher for the TGF‐β1‐loaded composites (18.5% ± 3) compared to controls (7.21% ± 5) after 8 weeks of implantation. Immunohistochemical staining demonstrated massive inflammatory infiltrates in both groups, particularly at 2 weeks, which decreased substantially at 4 and 8 weeks. In conclusion, injectable PLGA/CaP composites stimulated bone augmentation in a rat model. The addition of TGF‐β1 to the composite significantly increased bone contact at 2 weeks and enhanced new bone formation at 8 weeks. Copyright

Evaluation of an orthotopically implanted calcium phosphate cement containing gelatin microparticles

This study focused on the degradation properties of gelatin microparticles incorporated in calcium phosphate (CaP) cement and the subsequent effect of these composites on bone formation. Positively charged alkaline gelatin (type A) microparticles or negatively charged acidic gelatin (type B) microparticles were incorporated in CaP cement, which was implanted in critical-sized cranial defect in rats and left in place for 2, 4, and 8 weeks. The degradation of the gelatin was monitored using radioiodinated microparticles. After 4 and 8 weeks of implantation, a significantly faster degradation of type A gelatin over type B gelatin was found. Light microscopic analysis of the specimens showed similar bone response concerning implants containing either type A or B gelatin microparticles. At 2 weeks of implantation, a minimal amount of bone formation was observed from the cranial bone toward the implant, while after 8 weeks of implantation an entire layer of newly formed bone was present from the cranial bone toward the implant periphery. Bone ingrowth into the implant was observed at sites of gelatin microparticle degradation, predominantly at the implant periphery. Histomorphometrical evaluation did not reveal significant differences in bone formation between CaP cement incorporated with either type A or B gelatin microparticles during implantation periods up to 8 weeks. In conclusion, this study demonstrates that gelatin type influences the degradation of gelatin microparticles incorporated in CaP cements. However, this difference in degradation and the concomitant subsequent macroporosity did not induce differences in the biological response.