Biomedical subjects

J D De Bruijn

Publications and source records attributed to J D De Bruijn.

12 recordsLinked to original sources

PubMed2004

Genetic marking with the DeltaLNGFR-gene for tracing goat cells in bone tissue engineering.

The use of bone marrow derived stromal cells (BMSC's) for bone tissue engineering has gained much attention as an alternative for autologous bone grafting. Little is known however, about the survival and differentiation of the cells, especially in the clinical application. The aim of this study was to develop a method to trace goat BMSC's in vivo. We investigated retroviral genetic marking, which allows stable expression of the label with cell division. Goat BMSC's were subjected to an amphotropic envelope containing a MoMuLV-based vector expressing the human low affinity nerve growth factor receptor (DeltaLNGFR). Labeling efficiency and effect on the cells were analyzed. Furthermore, transduced cells were seeded onto porous ceramic scaffolds, implanted subcutaneously in nude mice and examined after successive implantation periods. Flow cytometry indicated a transduction efficiency of 40-60%. Immunohistochemistry showed survival and subsequent bone formation of the gene-marked cells in vivo. Besides, marked cells were also found in cartilage and fibrous tissue. These findings indicate the maintenance of the precursor phenotype following gene transfer as well as the ability of the gene to be expressed following differentiation. We conclude that retroviral gene marking with DeltaLNGFR is applicable to trace goat BMSC's in bone tissue engineering research.

Animals↗

PubMed2003

Bone formation by mesenchymal progenitor cells cultured on dense and microporous hydroxyapatite particles.

Hydroxyapatite (HA) microparticles, varying in size and microporosity, were evaluated in vitro and in vivo on their suitability to be used as a carrier in an injectable tissue engineered bone filler. Depending on their manufacturing method, either dense (HA-s) or microporous (HA-r) particles were produced in diameter ranges of 212-300 microm (HA-s and HA-r) and 500-706 microm (HA-s). After seeding and culturing goat mesenchymal progenitor cells on the various particles for 1 week, sheets were produced in which multilayers of cells and extracellular matrix held the particles together. Subcutaneous implantation of the constructs in nude mice for 4 weeks revealed abundant bone formation with the 212 to 300-microm diameter particle range. Up to 30% bone was formed in the available areas between the individual microparticles, while bone marrow was present in the samples containing microporous particles. Surprisingly, no bone or bone marrow formation was apparent with the 500 to 706-microm diameter range particles. These results show that size and microporosity of HA microparticles affect the osteogenic potential of cultured cells and indicate that particles in a diameter range of 212-300 microm may be used toward the development of injectable formulations of tissue-engineered bone.

Animals↗

PubMed2002

A cultured living bone equivalent enhances bone formation when compared to a cell seeding approach.

The development of cell therapy methods to confer osteogenic potential to synthetic bone replacement materials has become common during the last years. At present, in the bone tissue engineering field, two different approaches use patient own cultured osteogenic cells in combination with a scaffold material to engineer autologous osteogenic grafts. One of the approaches consists of seeding cells on a suitable biomaterial, after which the construct is ready for implantation. In the other approach, the seeded cells are further cultured on the scaffold to obtain in vitro formed bone (extracellular matrix and cells), prior to implantation. In the present study, we investigated the in vivo osteogenic potential of both methods through the implantation of porous hydroxyapatite (HA) scaffolds coated with a layer of in vitro formed bone and porous HA scaffolds seeded with osteogenic cells. Results showed that as early as 2 days after implantation, de novo bone tissue was formed on scaffolds in which an in vitro bone-like tissue was cultured, while it was only detected on the cell seeded implants from 4 days onwards. In addition, after 4 days of implantation statistical analysis revealed a significantly higher amount of bone in the bone-like tissue containing scaffolds as compared to cell seeded ones.

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PubMed2002

Evaluating 3D bone tissue engineered constructs with different seeding densities using the alamarBlue assay and the effect on in vivo bone formation.

Bone tissue engineering using patient derived cells seeded onto porous scaffolds has gained much attention in recent years. Evaluating the viability of these 3D constructs is an essential step in optimizing the process. The alamarBlue (aB) assay was evaluated for its potential to follow in vitro cell proliferation on architecturally standardized hydroxyapatite scaffolds. The impact of the aB assayed and seeding density on subsequent in vivo bone formation was investigated. Twelve scaffolds were seeded with various densities from 250 to 2.5x10(6) cells/scaffold and assay by aB at 5 time points during the 7-day culture period. Twelve additional scaffolds were seeded with 2.5x10(5) cells/scaffold. Two control and 2 aB treated scaffolds were subcutaneously implanted into each of 6 nude mice for 6 weeks. Four observers ranked bone formation using a pair wise comparison of histological sections form each mouse. The aB assay successfully followed cell proliferation, however, the diffusion kinetics of the 3D constructs must be considered. The influence of in vitro aB treatment on subsequent in vivo bone formation cannot be ruled out but was not shown to be significant in the current study. The aB assay appears to be quite promising for evaluating a maximum or end-point viability of 3D tissue engineered constructs. Finally, higher seeding densities resulted in more observed bone formation.

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PubMed2002

A comparison of the osteoinductive potential of two calcium phosphate ceramics implanted intramuscularly in goats.

The osteoinductive potential, or bone induction potency, of two calcium phosphate ceramics was evaluated after intramuscular implantation in goats. The ceramics were comprised of hydroxyapatite (HA) and biphasic calcium phosphate (BCP), the later of which contained a 85/15 mixture of hydroxyapatite and tricalcium phosphate (TCP). Both ceramics had a similar macroporosity of around 55% and a pore distribution between 100 and 800 microm. Besides the difference in chemistry, BCP was also microporous and hence had a different surface microstructure. After implantation in the back muscles of four goats for 12 weeks, all 8 BCP samples (7x7x7 mm(3)) showed the presence of bone formation in the macropores (1+/-1%), while no bone was found in any of the HA samples. The used BCP can therefore be characterized as an osteoinductive material. Having the ability to induce bone formation in soft tissues, the BCP presented herein may be a useful biomaterial for bone repair when combined with cultured osteogenic cells, growth factors or both.

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PubMed2001

Use of an osteoinductive biomaterial as a bone morphogenetic protein carrier.

A porous calcium phosphate ceramic, which induced bone formation in soft tissues of dogs, was termed as osteoinductive biomaterial and studied as a carrier of bone morphogenetic protein (rhBMP-2). Cylinder implants (laser spot 4 x 5 mm) impregnated with 0, 1, 10 and 40 microg rhBMP-2 were implanted in dorsal muscles of rabbits for five weeks. Histological observation and histomorphometric analysis were performed on thin un-decalcified sections. No bone formation was detected in the implants without rhBMP-2, while mature lamellar bone was found inside the implants with 1 microg rhBMP-2, both on the outer surface and inside the implants with 10 microg and 40 microg rhBMP-2. Little difference in formed bone was found between 1 microg and 10 microg rhBMP-2, but no difference was found between 10 microg and 40 microg rhBMP-2. A significant difference in bone marrow formation was found among 1, 10 and 40 microg rhBMP-2. The more rhBMP-2, the more bone marrow formed. The present results indicate that osteoinductive biomaterial is a good carrier of BMP and high dose of BMP is not necessary for bone formation in clinic.

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PubMed2001

Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous alpha-TCP and beta-TCP.

Two kinds of tri-calcium phosphate ceramics (Ca/P = 1.50), alpha-TCP and beta-TCP, which has the same macrostructure and microstructure, but different phase composition, were implanted in dorsal muscles of dogs. The samples were retrieved at 30, 45 and 150 days, respectively, after implantation, and were analyzed histologically. There were critically different tissue responses between alpha-TCP ceramic and beta-TCP ceramic. Higher cell populations were observed inside the pores of beta-TCP than those of alpha-TCP, bone tissue was found in beta-TCP at 45 and 150 days, but no bone formation could be detected in any alpha-TCP implants in this study. On the other hand, the bone tissue in beta-TCP seemed to degenerate at 150 days. The results indicate that porous beta-TCP can induce bone formation in soft tissues of dogs; while the rapid dissolution of the ceramic and the higher local Ca2+, PO(4)3- concentration due to the rapid dissolution of alpha-TCP may resist bone formation in alpha-TCP and the less rapid dissolution of beta-TCP may be detrimental to already formed bone in beta-TCP.

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PubMed1998

In vivo bone formation by human bone marrow cells: effect of osteogenic culture supplements and cell densities.

Bone marrow is known to contain a population of osteoprogenitor cells that can go through complete differentiation when cultured in a medium containing appropriate bioactive factors. In this study, porous particles of a calcium phosphate material were seeded with adult human bone marrow cells in the second passage. After an additional culture period of 1 wk in the particles, these hybrid constructs were subcutaneouslly implanted in nude mice with a survival period of 4 wk. The cell seeding densities range from 0-200 000 cells per particle and the cell culture system was designed to investigate the single and combined effects of dexamethasone and recombinant human bone morphogenetic protein 2 (rhBMP-2). The hybrid "material/tissue" constructs were processed for histology and the amount of de novo bone formation was quantified, for each culture condition, by histomorphometric techniques. The relative percentage of mineralized bone formation reached a maximal value of 19.77+/-5.06, for samples cultured in the presence of rhBMP-2 and with a seeding density of 200 000 cells/particle, compared to 0.52+/-0.45 for samples in which no cells had been cultured and had been incubated in culture medium supplemented with Dex and rhBMP-2. For the tested conditions and for the low cell numbers used in this study, rhBMP-2 proved to be an essential bioactive factor to obtain in vivo bone formation by our culture system. The results from this study prove the potential of cultured adult human bone marrow cells to initiate and accelerate de novo bone formation after transplantation into an ectopic site.

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PubMed1998

Comparison of the cytotoxicity of molybdenum as powder and as alloying element in a niobium-molybdenum alloy.

Commercially pure metal niobium (c.p. Nb) as well as niobium-molybdenum (Nb-Mo) alloys were produced following several powder metallurgical routes. In brief, niobium and molybdenum powders were blended and milled in order to form Nb-Mo alloys. The alloy powders and the c.p. Nb were then either pressed and sintered, or cold isostatically pressed followed by hot isostatically pressing. In order to assess the cytotoxicity of the c.p. Nb and c.p. Mo powders, a 72 h minimal essential medium-extraction test was performed according to ISO/EN 10993-5. The cytotoxicity of the c.p. Nb metal and the Nb-Mo alloys was tested in a 72 h direct contact test. Compared to a negative control (UHMWPE), c.p. Nb was non-toxic, but c.p. Mo was moderately toxic. None of the powder metallurgically produced materials were toxic. Neither differences in molybdenum concentration, nor in porosity of the samples, due to different production routes, had any influence on the toxicity of the materials. Rat bone marrow cultures showed that only on c.p. Nb was a mineralized extracellular matrix formed, while on the more porous Nb-Mo alloys, cell growth was observed, but no mineralization. In conclusion, c.p. Mo powder is moderately toxic, however, as an alloying element it is non-toxic. Material porosity seems to influence differentiation of bone tissue in vitro.

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PubMed1998

Osteoinduction by calcium phosphate biomaterials.

Different materials were implanted in muscles of dogs to study the osteoinduction of calcium phosphate biomaterials. Bone formation was only seen in calcium phosphate biomaterials with micropores, and could be found in hydroxyapatite (HA) ceramic, tricalcium phosphate/hydroxyapatite ceramic (BCP), beta-TCP ceramic and calcium phosphate cement. The osteoinductive potential was different in different materials. The results indicate that osteoinduction can be a property of calcium phosphate biomaterials when they exhibit specific chemical and structural characteristics.

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PubMed1998

Bone tissue engineering on calcium phosphate-coated titanium plates utilizing cultured rat bone marrow cells: a preliminary study.

The use of osteoinductive in vitro tissue-coated implants in orthopaedic and dental surgery (e.g. revision hip arthroplasty), could result in a better fixation of these implants. However, this tissue engineering technology has only proved to be effective in porous materials and not on flat implant surfaces. In this study we have demonstrated that calcium phosphate-coated titanium plates with a layer of cultured osteogenic cells and their extracellular matrix can initiate bone formation in vivo. Both primary and subcultured rat bone marrow cells were grown on to biomimetic calcium phosphate-coated titanium plates. After 7 d of culture, in the presence or absence of dexamethasone, the implants were subcutaneously implanted in nude mice for 4 wk. Control samples, which consisted of calcium phosphate-coated plates without cultured cells and porous calcium phosphate particles with or without cultured cells, were also implanted subcutaneously. At autopsy, no bone formation could be detected on any of the control samples without cells and samples with subcultured cells, which were primary cultured in medium without dexamethasone. In contrast, clear de novo bone formation could be observed on the calcium phosphate-coated plates and in the porous calcium phosphate particles with primary or subcultured cells, which had been continuously cultured in medium with dexamethasone. These results indicate that this hybrid technology offers great potential for the fixation of flat bone replacement implants (e.g. artificial hips) in inferior bone in the future.

Journal Article↗

PubMed1998

Bone morphogenetic protein and ceramic-induced osteogenesis.

To investigate the relationship between bone morphogenetic proteins (BMP) and calcium phosphate ceramic-induced osteogenesis in soft tissues, in vitro and in vivo experiments were performed. In an in vitro study, the ability of different calcium phosphate ceramics to absorb bovine BMP (bBMP) from a bBMP solution was tested. In vivo studies included immunohistochemical BMP staining before bone formation in the ceramics was detected, and the enhancement of bone formation in calcium phosphate ceramics by bBMP. The results were: (1) calcium phosphate ceramics have a strong ability to absorb bBMP; (2) a high BMP concentration reaches inside the ceramic implants before bone formation in soft tissues of domestic pig occurs; (3) by 56% at 50 d and by 23% at 100 d, bBMP enhances bone formation in calcium phosphate ceramics implanted in soft tissues of dogs. The results indicate the BMP plays an important role in calcium phosphate ceramic-induced osteogenesis and that adsorption of native BMP from the body fluids to ceramic implants may be a key step in osteoinduction by calcium phosphate ceramics.

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