Growth in patients with mucopolysaccharidosis type III (Sanfilippo disease).

BACKGROUND: Mucopolysaccharidosis III (MPS III), known as Sanfilippo disease, is a lysosomal storage disorder mainly characterized by progressive neurodegeneration with cognitive decline and relatively attenuated somatic signs and symptoms. Although short stature is invariably present in patients with the other mucopolysaccharidoses, it has not been sufficiently addressed in MPS III. The aim of this study was to investigate growth data of a large Dutch MPS III cohort in order to construct growth charts for MPS III patients. METHODS: Height, weight, head circumference (HC), and body mass index (BMI) data from 118 MPS III patients were used to construct reference curves, using the lambda, mu, sigma (LMS) method. Genotype-group comparisons for height standard deviation scores (SDS) were performed by Kruskal-Wallis analysis for different age groups. RESULTS: Birth weight and length were within normal ranges for gestational age and showed a significantly stunted growth from age 6 years onward. Mean final heights were 169.7 cm (-2.0 SDS) and 165.4 cm (-0.84 SDS) for adult male and female, patients, respectively. Phenotypic severity, as assessed by genotyping, correlated with growth pattern and final height. In addition, mean BMI and HC SDS were significantly higher when compared with Dutch standards for both boys and girls. CONCLUSIONS: Growth in MPS III is stunted mainly in patients with the severe phenotype. We provide disease-specific growth references that can be used for clinical management of MPS III patients and may be of value for future treatment studies.

Heparan sulfate derived disaccharides in plasma and total urinary excretion of glycosaminoglycans correlate with disease severity in Sanfilippo disease.

BACKGROUND: Sanfilippo disease (Mucopolysaccharidosis III) is a neurodegenerative lysosomal disorder characterized by accumulation of the glycosaminoglycan heparan sulfate (HS). MPS III has a large phenotypic variability and early assessment of disease severity is difficult. We investigated the correlation between disease severity and the plasma concentration of HS (pHS, defined by the sum of the heparan sulfate derived disaccharides obtained after enzymatic digestion) and urinary total GAGs level (uGAGs, measured by the dimethylene blue test) in a cross-sectional cohort of 44 MPS III patients. METHODS: Disease severity was established on the basis of the age of complete loss of independent walking and of full loss of speech in all patients. Hazard ratios (HR) were obtained with cox-regression analysis. In order to allow prediction of a severe phenotype based on a cut-off value for pHS, patients were divided in two groups (severely affected and less severely affected) based on predictive mutations or on the age of full loss of speech. Receiver operator characteristics (ROC) were obtained for pHS. RESULTS: pHS and uGAGs were independently and linearly associated with an increased risk of speech loss with a HR of 1.8 (95 % CI 1.3-2.7) per 500 ng/ml increase of HS in plasma (p = 0.002), and a HR of 2.7 (95 % CI 1.6-4.4) per 10 mg/mmol creatinine increase of uGAGs (p < 0.001). pHS and uGAGS were less strongly associated with loss of walking. The area under the ROC curve for pHS was 0.85, indicating good discrimination. CONCLUSION: pHS and uGAGs may be useful biomarkers for prediction of severity in MPS III.

Tissue reaction to Dacron velour and titanium fibre mesh used for anchorage of percutaneous devices.

Dacron velour is often used to anchor a percutaneous device, like the catheter used in peritoneal dialysis. However, exit-site infections complicate this method of dialysis and are supposed to be related to the design of the catheter. In animal experiments, a percutaneous device provided with a titanium fibre mesh to anchor the implant was not affected by infectious complications. The purpose of this study was to compare the differences in soft tissue reaction to Dacron velour and titanium fibre mesh under the same experimental conditions. Therefore, we placed implants, provided with either Dacron or titanium mesh, subcutaneously in the dorsum of goats. The implants were left in situ for 4 months. Histological and histomorphological evaluations were performed. It was found that the soft tissue response inside the Dacron was mainly inflammatory, while the titanium mesh evoked good biocompatible behaviour. We concluded that the limited fibrous tissue ingrowth into the Dacron cuff has to be the reason for the observed high failure incidence of a percutaneous device.

Wound healing phenomena in titanium fibre mesh: the influence of the length of implantation.

In previous experiments a new type of percutaneous device for implantation in soft tissue was designed, containing a sintered titanium fibre mesh. The devices are inserted by a so-called "two-phase' surgical technique with an intervening healing period of 3 months between insertion of the subcutaneous and percutaneous parts. From a clinical point of view, this time interval is too long. The aim of this study was to investigate whether it was possible to reduce the intervening healing period. The implants were inserted in the backs of nine goats. In each goat, six implants were placed with intervals of 1 week. Consequently, at the end of the experiment, in each goat six implants were present with implantation periods ranging from 1 to 6 weeks. After 6 weeks, the animals were killed and the implants with surrounding tissue were processed histologically. Analysis demonstrated that during the first 2 weeks an inflammatory response was present. Thereafter, no difference in tissue response was found between the various implantation periods. In conclusion, the experiment suggests that for titanium mesh percutaneous devices a 3-week healing period is sufficient between the installation of the subcutaneous and percutaneous parts.

Histological evaluation of a biodegradable Polyactive/hydroxyapatite membrane.

Guided tissue regeneration (GTR) is a technique which is used for the treatment of bone defects associated with periodontal disease or enossal dental implants. In most experimental studies on GTR, non-degradable membranes are used. A drawback inherent to such devices is that at the end or in the course of the wound healing they have to be removed. Therefore, the aim of the present study was to investigate a new biodegradable membrane material for use in GTR, which also has excellent mechanical properties and is biocompatible. The material is a composite consisting of poly(ethyleneglycol terephthalate) and poly(butylene terephthalate) segmented copolymer (PEG/PBT), which for the experiments was used in pure form and also mixed with hydroxyapatite (HA) grains. Subcutaneous and subgingival implantation studies in goats were performed to determine the biocompatibility and biodegradability characteristics of several of these materials. Differences between materials were introduced in the production process, PEG/PBT ratio, material thickness and presence of HA. Implantation periods were 3, 6 and 12 wk. The histological results indicated that all investigated materials were biocompatible with the surrounding tissue. Degradation of the membranes was attended by a mild cellular reaction. The degradation process was mainly influenced by the PEG/PBT ratio. A higher PBT content resulted in a decreased degradation.

The effect of poly-L-lactic acid with parallel surface micro groove on osteoblast-like cells in vitro.

In this study we evaluated the behavior of rat bone marrow (RBM) cells on microgrooved poly-L-lactic acid (PLA) and polystyrene (PS) surfaces. The applied groove depth was 0.5, 1.0 or 1.5 microns, with a groove and ridge width of 1, 2, 5 or 10 microns. Scanning electron microscopical examination showed that a collagen-rich mineralized layer of extracellular matrix (ECM) was deposited. Alignment of the cells and matrix to the surface grooves was observed as described before. Quantitative evaluation, using a tetracycline labeling assay, revealed that more mineralized ECM was formed on the PLA than on the PS. Further, PLA surfaces with a groove depth of 1.0 micron and groove widths of 1 and 2 microns induced most mineralized ECM. Finally, alkaline phosphatase activity was also higher on most microgrooved PLA surfaces, compared with the other materials. On the basis of these observations, we concluded that microtextured surfaces are able to influence the differentiation of osteoblast-like cells and the deposition of mineralized matrix. Probably, this phenomenon can be used to increase the bone regeneration around oral implants.

Ectopic bone formation in rats: the importance of vascularity of the acceptor site.

Bone graft substitutes (BGS) can be fabricated by the combination of three key ingredients: (1) competent bone-forming cells, (2) a suitable framework or scaffold, and (3) the presence of biological stimulants. Although much research has been done to develop the ideal BGS, still the results are not very consistent. In view of this, the cellularity and vascularity of the recipient site are supposed to be important for the osteoinductive capacity of BGS. Therefore, we hypothesized that a muscle recipient site could favor bone formation in a cell-based BGS compared to a subcutaneous recipient site due to the higher vascularity of muscle tissue. To prove this hypothesis, 48 titanium fiber mesh implants were seeded with rat bone marrow stromal cells (RBM) and implanted subcutaneously and intramuscularly in the adductor thigh muscle of rats. The amount of bone formation after 1, 3 and 6 weeks was evaluated by histology and histomorphometry as well as by calcium content. Analysis revealed that the bone formation increased during implantation. However, bone formation did not exceed 12% of the implant surface, both for the intramuscular and subcutaneous recipient site. Also, no significant differences in bone amount between these two sites existed. Consequently, our hypothesis could not be confirmed.

Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves.

In order to quantify the effect of the substrata surface topography on cellular behaviour, planar and micro-textured silicon substrata were produced and made suitable for cell culture by radio frequency glow discharge treatment. These substrata possessed parallel surface grooves with a groove and ridge width of 2.0 (SilD02), 5.0 (SilD05) and 10 microns (SilD10). Groove depth was approximately 0.5 micron. Rat dermal fibroblasts (RDFs) were cultured on these substrata and a tissue culture polystyrene control surface for 1, 2, 3, 5 and 7 days. After incubation the cell proliferation was quantified with a Coulter Counter, and RDF size, shape and orientation with digital image analysis. Cell counts proved that neither the presence of the surface grooves nor the dimension of these grooves had an effect on the cell proliferation. However, RDFs on SilD02, and to a lesser extent on SilD05 substrata, were elongated and aligned parallel to the surface grooves. Orientation of the RDFs on SilD10 substrata proved to be almost comparable to the SilD00 substrata. Finally, it was observed that the cells on the micro-textured substrata were capable of spanning the surface grooves.

Quantitative analysis of fibroblast morphology on microgrooved surfaces with various groove and ridge dimensions.

Fibroblasts have been shown to respond to substratum surface roughness. The change in cell size, shape and orientation of rat dermal fibroblasts (RDF) was therefore studied using smooth and microtextured silicone rubber substrata. The microtextured substrata possessed parallel surface microgrooves that ranged in width from 1.0 to 10.0 microns, and were separated by ridges of 1.0 to 10.0 microns. The grooves were either 0.45 or 1.00 microns deep. Prior to incubation, the substrata were cleaned and given a radio frequency glow discharge treatment. After surface evaluation with scanning electron microscopy and confocal laser scanning microscopy, RDF were incubated on these substrata for 5 days. During this period of incubation, the RDF were photographed on days 1, 2, 3, 4, and 5, using phase contrast microscopy. Digital image analysis of these images revealed that on surfaces with a ridge width < or = 4.0 microns, cells were highly orientated (< 10 degrees) and elongated along the surface grooves. Protrusions contacting the ridges specifically could be seen. If the ridge width was larger than 4.0 microns, cellular orientation was random (approximately 45 degrees) and the shape of the RDF became more circular. Furthermore, results showed that the ridge width is the most important parameter, since varying the groove width and groove depth did not affect the RDF size, shape, nor the angle of cellular orientation.

Osteoblast differentiation of bone marrow stromal cells cultured on silica gel and sol-gel-derived titania.

Primary cultures of osteogenic precursor cells derived from rat bone marrow stroma were performed on commercially available pure titanium discs (Ti c.p.) and surface modified Ti c.p.using a sol-gel technique (Ti sol). In separate repeated experimental runs, cell behavior and in vitro mineralization were compared with cultures on silica gel bioactive glass discs (S53P4). All substrates were incubated in simulated body fluid prior to the experiment. Overall, variable effects between experimental runs were seen. Apparently, this was due to the heterogeneous nature of the used cell population. Therefore, only careful conclusions can be made. Initial cell adhesion and growth rates between 3 and 5 days of culture--analyzed by cell numbers--were in general comparable for the two titanium substrates, while initial growth up to day 3 is suggested to be higher in Ti c.p. compared to Ti sol. Although initial cell adhesion on the S53P4 glass discs was lower than the titanium substrates, cell growth rates appeared to be higher on the silica gel compared to the two titanium substrates. Further, there were some indications that the early and late osteoblast differentiation markers, alkaline phosphatase and osteocalcin, monitored up to day 24, were elevated in Ti c.p cultures compared to Ti sol cultures. There were no differences observed in in vitro mineralization between the titanium groups. S53P4 seemed to display a substantially higher differentiating capacity for both osteogenic cell markers as well as in vitro mineralization compared to the two titanium substrates.

Modulation of integrin expression on rat bone marrow cells by substrates with different surface characteristics.

Biomaterials have been shown to be able to influence the growth and differentiation of osteogenic cells cultured on the surface. Although the precise mechanisms by which the materials influence osteogenic cells are unclear, it is possible that the materials manipulate the expression of integrins by the cells. We therefore studied the expression of a number of integrins by rat bone marrow (RBM) cells, after culture on culture polystyrene, on machined and grit-blasted titanium, and on calcium phosphate-coated titanium. Integrin expression was studied by FACS analysis. We found a large variation in the expression of integrins by cells in replicate experiments. After culture on polystyrene for 7 days, cells expressed alpha1, alpha2, alpha3, alpha5, alpha6, beta1, and beta3, although some of the subunits were expressed only occasionally. The cells did not express the alpha4 subunit. After culture of RBM cells for 8 days on coated and noncoated titanium substrates, cells always expressed alpha3, alpha5, alpha6, and beta1. The alpha1 and beta3 subunits were only expressed in some of the experiments. Frequently, the expression of alpha5, alpha6, and beta1 was higher on the coated than on the noncoated titanium substrates. Based on our results, we conclude that the studied materials are capable of influencing the expression of integrins by RBM cells cultured on relevant implant materials.

Analysis of integrin expression in U2OS cells cultured on various calcium phosphate ceramic substrates.

Earlier we observed that calcium phosphate (Ca-P)-coated implant substrates stimulated the differentiation of osteoblast-like cells compared to uncoated substrates. This suggests that this difference in osteogenic induction is due to the chemical composition of the substratum. We hypothesized that Ca-P coatings modulate integrin expression patterns, because those receptors are the sensors of the cell. Therefore, in the present study we quantitatively analyzed integrin expression of osteosarcoma cells and their proliferation behavior on various well-defined Ca-P substrates. For this study we used the osteosarcoma cell line U2OS. Five groups of substrates were used: thermanox (Th), uncoated titanium (Ti), dense sintered hydroxyapatite (HA), and two Ca-P-coated titanium discs (TiHA-O% and TiHA-5%). At day 5, cell numbers were significantly lower (p < 0.05) for both types of Ca-P-coated titanium substrates compared to the other substrates. There were no significant differences between HA and uncoated titanium. From day 5 to 8, accumulated cell number was ranking highest to lowest HA > Th = Ti > TiHA-0% > TiHA-5%. Integrin expression at day 5 and day 8 of incubation was analyzed by flow cytometry for integrin subunits beta 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v. Fluorescence-activated cell sorting (FACS) analysis showed that the cells express high levels of beta 1, low levels of alpha 4, alpha 5, and alpha 6, and moderate levels of alpha 3 and alpha v integrin subunits on the various biomaterial substrates. Minor differences in integrin expression between the various substrates were seen. Therefore, the observed differences in proliferation between the coatings may reside in modulating the functional properties of integrins.

The applicability of a keratin 7 monoclonal antibody in routinely Papanicolaou-stained cytologic specimens for the differential diagnosis of carcinomas.

The monoclonal antibody OV-TL 12/30, which detects keratin 7, was tested for its usefulness in cytologic diagnosis by reincubating previously Papanicolaou-stained slides. For this purpose malignant effusions of 73 patients with histologically confirmed cancers of the colon, ovary, mesothelium, breast, lung, esophagus, pancreas, urinary bladder, stomach, kidney, and prostate were used. All malignant cells from ovarian adenocarcinomas were positive, whereas malignant cells from colonic adenocarcinomas and malignant mesotheliomas were negative. Adenocarcinomas of gastric, renal, pancreatic, esophageal, and mammary origin demonstrated variable staining. Transitional cell carcinomas were positive, whereas squamous and small cell lung carcinomas were negative. Because OV-TL 12/30 does not react with normal and atypical mesothelial cells in these preparations, this reagent is a valuable tool in distinguishing benign mesothelial cells and adenocarcinoma cells. The authors' results demonstrate that this antibody is an excellent tool in the differential diagnosis of malignant cells in effusions and can be used in routinely stained cytologic specimens to determine primary tumor localization. In addition to its ability to distinguish between ovarian and colonic adenocarcinomas, its negativity in mesotheliomas may prove helpful in several diagnostic considerations.

Bone formation in CaP-coated and noncoated titanium fiber mesh.

The osteogenic activity of calcium phosphate (CaP)-coated and noncoated porous titanium (Ti) fiber mesh loaded with cultured syngeneic osteogenic cells after prolonged in situ culturing was compared in a syngeneic rat ectopic assay model. Rat bone marrow (RBM) cells were loaded onto the CaP-coated and noncoated Ti scaffolds using either a droplet or a suspension loading method. After loading, the RBM cells were cultured for 8 days in vitro. Thereafter, implants were subcutaneously placed in 39 syngeneic rats. The rats were euthanized and the implants retrieved at 2, 4, and 8 weeks postoperatively. Further, in the 8 week group fluorochrome bone markers were injected at 2, 4, and 6 weeks. Histological analysis demonstrated that only the CaP-coated meshes supported bone formation. The amount of newly formed bone varied between single and multiple spheres to filling a significant part of the mesh porosity. In the newly formed bone, osteocytes embedded in a mineralized matrix could be observed clearly. On the other hand, in the noncoated titanium implants, abundant deposition of calcium-containing material was seen. This deposit lacked a bonelike tissue organization. Further analysis revealed that the cell-loading method did not influence the final amount of bone formation. In CaP-coated implants the accumulation sequence of the fluorochrome markers showed that bone formation started on the mesh fibers. In conclusion, our results prove that the combination of a thin CaP coating, Ti-mesh, and RBM cells can indeed generate ectopic bone formation after prolonged in vitro culturing. No effect of the loading method was observed on the final amount of bone.

An infectious cDNA clone of porcine reproductive and respiratory syndrome virus.

A plasmid containing a full-length cDNA copy of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was constructed. When RNA that was transcribed in vitro from this full-length cDNA clone was transfected to BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. The infectious clone of LV enables us to mutagenize the viral genome at specific sites and thus will be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

Mucopolysaccharidosis type III (Sanfilippo Syndrome): emerging treatment strategies.

Mucopolysaccharosis III (MPS III) is a lysosomal storage disorder and belongs to the group of mucopolysaccharidoses. MPS III is caused by a deficiency of one of the four enzymes catalyzing the degradation of the glycosaminoglycan heparan sulfate. MPS III is clinically characterized by progressive dementia with distinct behavioral disturbances and relatively mild somatic disease. This review will summarize and discuss the available and potential future therapeutic options for patients with MPS III. This includes enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), substrate reduction therapy (SRT), chaperone-mediated therapy, and gene therapy. Although clinical efficacy has not yet been fully demonstrated for any of these therapies, it is likely that future developments will lead to disease-modifying treatment for this devastating disease.

The effect of a subcutaneous silicone rubber implant with shallow surface microgrooves on the surrounding tissues in rabbits.

It has been suggested that during wound healing microtextured surfaces can alter events at the interface between implant surface surface and surrounding tissues. To investigate this phenomenon, smooth and microtextured silicone rubber implants were implanted subcutaneously in rabbits for 3, 7, 42, and 84 days. The textured implants possessed parallel surface microgrooves and ridges with a width of 2.0, 5.0, and 10.0 microns. All grooves had a depth of approximately 0.5 microns. SEM observation showed fibroblasts, erythrocytes, lymphocytes, macrophages, fibrin, and collagen on all implant surfaces after 3 and 7 days. After 42 and 84 days only little collagen, a small number of fibroblasts, but no inflammatory cells were seen on the implant surfaces. The fibroblasts were not oriented along the surface grooves on all textured surfaces. Three-dimensional reconstruction of CLSM images and LM images showed no significant differences between the thickness of the capsules surrounding the smooth and those surrounding the microgrooved implants. In contrast LM did show a significantly lower number of inflammatory cells and a significantly higher number of blood vessels in the capsules surrounding the microgrooved implants. Differences between the 2.0, 5.0, and 10.0 microns grooved implants were not detected. Our results concerning the capsule thickness suggest that the depth of our grooves was not sufficient to facilitate mechanical interlocking, but the cause for the observed differences in inflammatory response and number of blood vessels remains unclear.

Soft tissue and epithelial models.

The applicability of a biomaterial for the manufacturing of oral implants is determined by its physicochemical and geometric surface properties. Research, therefore, is concerned with the cellular reactions that occur when an implant material comes into contact with body tissues. For permucosal oral implants, this involves both the reaction of bone and gingival cells. In vitro cell culturing--including the use of various analytical techniques like light microscopy, scanning and transmission electron microscopy, confocal laser scanning microscopy, and digital image analysis--is a good tool whereby investigators can obtain more insight into the relevant components of implant-tissue adhesion. In the current overview, the role of cell models in oral implant research is discussed, specifically with reference to responses of epithelial cells and fibroblasts.

Titanium fiber mesh anchorage for percutaneous devices applicable for peritoneal dialysis.

In previous experiments a new type of percutaneous device for implantation in soft tissue was designed. The subcutaneous component of the new device consists of a sintered titanium fiber web. The percutaneous devices are inserted by a so-called "two-phase" surgical technique with an intervening healing period of 3 months between the insertion of the subcutaneous flange and the placement of the percutaneous part. From a clinical point of view, this time interval is too long. Therefore, the aim of this study was to investigate a possible reduction of the intervening healing period. The implants were inserted in the backs of 18 rabbits. In each rabbit, six implants were placed with intervals of 1 week. Consequently, at the end of the experiment, in each rabbit six implants were present with implantation periods ranging from 5-10 weeks. After 10 weeks, the animals were sacrificed and the implants with their surrounding tissues were processed histologically. Light microscopic and histomorphometric analysis demonstrated that there was no difference in tissue response between the various implantation periods. Furthermore, it was found that the fiber mesh material showed good biocompatible behavior. In conclusion, the experiment has demonstrated that for titanium mesh percutaneous devices a 5-week healing period is sufficient between the installation of the subcutaneous and percutaneous parts.