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L B Creemers

Publications and source records attributed to L B Creemers.

11 recordsLinked to original sources
PubMed2006

Altered in vitro chondrogenic properties of chondrocytes harvested from unaffected cartilage in osteoarthritic joints.

OBJECTIVE: In vitro models of chondrogenesis often depart from chondrocytes harvested from less-affected areas of osteoarthritic joints. However, there are indications that these chondrocytes are phenotypically different from chondrocytes from healthy joints and thus might differ in their capacity to generate hyaline cartilage. The goal of this study was to compare the chondrogenic capacity of chondrocytes from healthy and OA joints. DESIGN: Chondrocytes isolated from nine healthy and nine OA knee joints were expanded in monolayer for two passages. Chondrocytes from passages 1 and 2 were analyzed for expression of (de)differentiation and hypertrophy markers and were seeded at passage 2 on collagen-coated filters for redifferentiation culture to study cartilage matrix formation. RESULTS: The collagen II/I mRNA ratio, reflecting differentiation, decreased from passage 1 to 2 in both chondrocytes from OA joints and chondrocytes from healthy joints (P<0.05), without a significant difference between the two donor types. At passage 1, levels of the cartilage transcription factors Sox-5, Sox-6 and Sox-9 appeared to be higher in chondrocytes from OA joints (n.s.), but this was not seen at passage 2. However, a clear difference was observed in collagen type X expression, which was high in chondrocytes from OA joints at both passages, while undetectable in chondrocytes from healthy joints (P<0.01). Tissue generated by chondrocytes from healthy joints redifferentiated for 28 days, showed a significantly better morphology, as assessed by histological scoring (P<0.01) and higher proteoglycan content (P<0.05), compared to chondrocytes from OA joints. Matrix turnover parameters, i.e., proteoglycan synthesis and degradation rate, were not significantly affected by donor tissue origin. CONCLUSIONS: These results suggest that clear differences between chondrocytes from healthy and OA joints exist and that these are not completely abolished during the process of de- and redifferentiation. Therefore, in vitro cartilage regeneration models, which use chondrocytes from OA joints, should be interpreted with care.

Aged↗
PubMed2006

Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes.

Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.

Cartilage, Articular↗
PubMed2002

Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation.

With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals. Successful depletion of endogenous spermatogenesis was achieved using fractionated ionizing irradiation. A dose of 1.5 Gy followed by a dose of 12 Gy after 24 h reduced the percentage of tubule cross-sections displaying endogenous spermatogenesis to approximately 3% and 10% as evidenced by histologic evaluation of testes at 12 and 21 wk, respectively, after irradiation. At this dose, no apparent harmful side effects were noted in the animals. Upon transplantation, GDNF-overexpressing germ cells were found to be able to repopulate the irradiated testes and to form clusters of spermatogonia-like cells resembling those found in the overexpressing donor mice. The cluster cells in transplanted host testes expressed human GDNF, as had been shown previously for clusters in donor animals, and both were strongly positive for the tyrosine kinase receptor Ret. Thus, we devised an efficient method for depleting the seminiferous epithelium of host mice without appreciable adverse effects. In these host mice, GDNF-overexpressing cells reproduced the aberrant phenotype found in the donor transgenic mice.

Animals↗
PubMed2002

Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture.

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.

Animals↗
PubMed2002

Isolation and purification of type A spermatogonia from the bovine testis.

The aim of this study was to isolate and purify bovine type A spermatogonia. Testes from 5-7-month-old calves were used to isolate germ cells using a two-step enzymatic digestion. During the isolation and purification steps, the viability of cells was determined using live/dead staining. The identity of type A spermatogonia during isolation and purification was determined under a light microscope equipped with a Nomarski lens. Isolated cells were characterized further by using specific markers for type A spermatogonia, including Dolichos biflorus agglutinin (DBA) and c-kit. The cell suspension was transplanted into immunodeficient recipient mouse testes and the colonization was assessed 1-3 months after transplantation, to assess the stem cell population among the isolated cells. After isolation, a cell suspension was obtained containing about 25% type A spermatogonia, which was enriched further by differential plating and separation on a discontinuous Percoll gradient. Finally, fractions containing 65-87% pure type A spermatogonia were obtained. Large and small type A spermatogonia with different numbers and sizes of nucleoli were found. DBA stained both large and small type A spermatogonia and its application in fluorescence-activated cell sorting (FACS) resulted in comparable percentages of type A spermatogonia to those determined by morphological examination under a light microscope equipped with a Nomarski lens. Nearly all of the large type A spermatogonia showed strong c-kit immunoreactivity, indicating that these cells had undergone at least an initial differentiation step. In contrast, approximately half of the small type A spermatogonia were negative for c-kit, indicating the presence of the spermatogonial stem cells in this population. At 3 months after transplantation, groups of bovine type A spermatogonia were found in most tubule cross-sections of the recipient mouse testes, showing the presence of spermatogonial stem cells among the isolated cells.

Animals↗
PubMed2000

Spermatogonial stem cell transplantation.

The development of the spermatogonial transplantation technique has given new impetus to research on spermatogonial stem cells. Possibilities opened by this technique include: (a) New ways to study fundamental aspects of spermatogenesis; (b) Generation of transgenic large domestic animals; (c) Protection of (young) male cancer patients from infertility due to chemotherapy or radiotherapy. Spermatogonial stem cell transplantation for the above purposes encompasses a number of steps. First, the stem cells have to be isolated and possibly purified. Second, it should be possible to cryopreserve the stem cells, for example till the children have reached puberty. Third. it should be possible to culture spermatogonial stem cells for a prolonged period of time which would also allow transfection and subsequent selection of stably transfected cells. Fourth, in case of animal studies. the host testis should be emptied from endogenous stem cells. This is probably best done by local irradiation. Finally, the stem cells will have to be transplanted.

Animals↗
PubMed1998

Involvement of V-ATPases in the digestion of soft connective tissue collagen.

The contribution of vacuolar H+-ATPases (V-ATPases) to collagen degradation was investigated in soft connective tissue explants (periosteum). Immunolocalisation showed faint to intense staining of cells throughout the periosteum. The V-ATPase inhibitors, bafilomycin A1 and folimycin, decreased overall collagen degradation by 40 and 50% after 24 and 48 h, respectively. The participation of V-ATPases in intracellular degradation of collagen was demonstrated by the decrease of the amount of phagocytosed collagen in fibroblasts upon inhibition of pump activity. The inhibition of degradation was not due to a reduction in activity of gelatinase A, an enzyme previously found to mediate collagen degradation, as assessed by zymographic analysis of tissue and conditioned medium. Bafilomycin A1 even induced an increase of gelatinase A and B levels in both fractions. In conclusion, acidification by V-ATPases may represent an important mechanism in extracellular and intracellular collagen degradation in soft connective tissue.

Animals↗
PubMed1998

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants.

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects. Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Me. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.

Animals↗
PubMed1998

Gelatinase A (MMP-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue.

The degradation of soft connective tissue collagen is considered to depend on the activity of various proteolytic enzymes, particularly those belonging to the group of matrix metalloproteinases and cysteine proteinases. In the present study, we investigated the contribution of these enzymes to this process. Using a general inhibitor of MMPs (SC44463), collagen degradation was strongly inhibited, by about 40% after 24 h and up to 80% after 72 h of culturing. Blockage of cysteine proteinase activity (with leupeptin or E-64) reduced breakdown at these time intervals by 50% and 20%, respectively. Given the abundant presence of gelatinases--in particular gelatinase A (MMP-2)--in the tissue, the effect of an inhibitor selective for gelatinases (CT1166) was studied. Gelatinase inhibition resulted in a dose-dependent decrease of collagen breakdown up to 90% after 48 h. The ability of gelatinase A to degrade collagens was demonstrated by the induction of breakdown in devitalized explants by addition of activated gelatinase A, or by activation of endogenous enzyme with 4-aminophenylmercuric acetate. This latter effect was not found with plasmin, an activator of MMPs other than gelatinase A. Finally, the relevance of gelatinase A to the in vivo degradation of soft connective tissue collagen was implicated by the significant correlation found between its activity and the collagen turnover rates of four soft connective tissues (tooth pulp, periodontal ligament, molar gingiva and skin). We conclude that collagen degradation in soft connective tissue is mediated by MMPs and to a lesser extent by cysteine proteinases. Our data are the first to attach a key role to gelatinase A in this process.

Animals↗
PubMed1991

Prolactinoma and body weight: a retrospective study.

Body weight and weight history in association with hormone levels were studied retrospectively in 47 patients with prolactinoma; macroprolactinoma was diagnosed in 36, microprolactinoma in 11 patients. At the time of diagnosis a weight history could be traced in 14 patients, 9 patients having gained weight (11.8 +/- 2 kg), 1 with a weight loss of 5 kg, and 4 reporting unaltered body weight. Body weight and Body Mass Index before treatment were 83 +/- 2.4 kg and 27.3 +/- 0.6 kg/m2, respectively. In the male patients the prevalence of BMI greater than or equal to 25 was greater than in the average Dutch male population (p less than 0.001). After 6 months of treatment, mainly with bromocriptine, patients with macroprolactinoma had lost 5.5 +/- 1.6% (p less than 0.002) of initial body weight. No significant weight change occurred in patients with microprolactinoma. Weight loss did not correlate with degree of hyperprolactinemia, nor with decrease of prolactin levels during treatment, in either group of patients. Thyroid or gonadal function were not associated with weight loss either. It appears that prolactinoma is associated with a higher frequency of overweight, as far as patients with macroprolactinoma are concerned.

Adolescent↗