Accumulation Of Sulfated Glycosaminoglycans Research Articles

Impact of Molecular Dynamics of Polyrotaxanes on Chondrocytes in Double-Network Supramolecular Hydrogels under Physiological Thermomechanical Stimulation.

Hyaline cartilage, a soft tissue enriched with a dynamic extracellular matrix, manifests as a supramolecular system within load-bearing joints. At the same time, the challenge of cartilage repair through tissue engineering lies in replicating intricate cellular-matrix interactions. This study attempts to investigate chondrocyte responses within double-network supramolecular hybrid hydrogels tailored to mimic the dynamic molecular nature of hyaline cartilage. To this end, we infused noncovalent host-guest polyrotaxanes, by blending α-cyclodextrins as host molecules and polyethylene glycol as guests, into a gelatin-based covalent matrix, thereby enhancing its dynamic characteristics. Subsequently, chondrocytes were seeded into these hydrogels to systematically probe the effects of two concentrations of the introduced polyrotaxanes (instilling different levels of supramolecular dynamism in the hydrogel systems) on the cellular responsiveness. Our findings unveiled an augmented level of cellular mechanosensitivity for supramolecular hydrogels compared to pure covalent-based systems. This is demonstrated by an increased mRNA expression of ion channels (TREK1, TRPV4, and PIEZO1), signaling molecules (SOX9) and matrix-remodeling enzymes (LOXL2). Such outcomes were further elevated upon external application of biomimetic thermomechanical loading, which brought a stark increase in the accumulation of sulfated glycosaminoglycans and collagen. Overall, we found that matrix adaptability plays a pivotal role in modulating chondrocyte responses within double-network supramolecular hydrogels. These findings hold the potential for advancing cartilage engineering within load-bearing joints.

Tracheal Reconstruction with the Scaffolded Cartilage Sheets in an Orthotopic Animal Model.

Tracheal reconstruction remains challenged in clinical. We aimed to fabricate scaffolded cartilage sheets with rigid and elastic supports for tracheal reconstruction. The chondrocyte cell infiltration activity was examined in poly-caprolactone sheet scaffolds with various thicknesses and pore sizes after seeding cells on the top surface of the sheet scaffolds. The expression of cartilage-related genes and accumulation of sulfated glycosaminoglycans were elevated in the cell-scaffold composites upon chondrogenic induction. The thicker cartilage sheets represented stronger mechanical properties than the thinner cartilage sheets. Two different cartilage sheets were orthotopically implanted into a trachea in a rabbit model for 2, 4, and 16 weeks. Cartilage-related sulfated glycosaminoglycans and type II collagen macromolecules were stably expressed in the tracheal implants. However, the invasive migration of fibrous tissue and profibrotic collagen fibers into cartilage implants and the peripheral space surrounding the implants were elevated in a time-dependent manner. At week 16 postimplantation, airway stenosis was noticed under the thicker sheet implants, but not the thinner implants, suggesting that the thinner (1 mm thick) scaffolded cartilage sheet was an optimal candidate for tracheal reconstruction in this study. Finally, cartilage sheets could be a reconstructive therapy candidate applied to reconstruct defects in the trachea and other tissues composed of cartilage. Impact statement Tissue engineering is a promising approach to generate biological substitutes. We aimed to develop cartilage sheets as tracheal prosthesis used in tracheal reconstruction or regional repairing in the animal model. The formation of microvessels and the dynamics of reepithelialization were monitored for 16 weeks in tracheal implants of the engineered cartilage sheets. In this study, it was demonstrated that the tissue-engineered cartilage sheets are potential substitutes applied in the reconstruction of the trachea and other tissues composed of cartilage tissue. The cartilage sheets were thought of as biomaterials for personalized regenerative medicine since the dimensions, thickness, and pore sizes of cartilage sheets were tunable to fit the lesions that need to be reconstructed.

Comparing anti–aging hallmark activities of Metformin and Nano-PSO in a mouse model of genetic Creutzfeldt-Jakob Disease

Advanced age is the main risk factor for the manifestation of late onset neurodegenerative diseases. Metformin, an anti–diabetic drug, was shown to extend longevity, and to ameliorate the activity of recognized aging hallmarks. Here, we compared the clinical, pathologic and biochemical effects of Metformin to those of Nano-PSO (Granagard), a brain targeted anti–oxidant shown by us to delay disease advance in transgenic mice mimicking for genetic Creutzfeldt Jacob disease (CJD) linked to the E200KPrP mutation. We demonstrate that both Metformin and Nano-PSO reduced aging hallmarks activities such as activated AMPK, the main energy sensor of cells as well as Nrf2 and COX IV1, regulators of oxidation, and mitochondrial activity. Both compounds reduced inflammation and increased stem cells production, however did not decrease PrP accumulation. As opposed to Nano-PSO, Metformin neither delayed clinical disease advance in these mice nor reduced the accumulation of sulfated glycosaminoglycans, a pathologic feature of prion disease. We conclude that elevation of anti–aging markers may not be sufficient to delay the fatal advance of genetic CJD.

Inhibition of glucose use improves structural recovery of injured Achilles tendon in mice.

Injured tendons do not regain their native structure except at fetal or very young ages. Healing tendons often show mucoid degeneration involving accumulation of sulfated glycosaminoglycans (GAGs), but its etiology and molecular base have not been studied substantially. We hypothesized that quality and quantity of gene expression involving the synthesis of proteoglycans having sulfated GAGs are altered in injured tendons and that a reduction in synthesis of sulfated GAGs improves structural and functional recovery of injured tendons. C57BL6/j mice were subjected to Achilles tendon tenotomy surgery. The injured tendons accumulated sulfate proteoglycans as early as 1-week postsurgery and continued so by 4-week postsurgery. Transcriptome analysis revealed upregulation of a wide range of proteoglycan genes that have sulfated GAGs in the injured tendons 1 and 3 weeks postsurgery. Genes critical for enzymatic reaction of initiation and elongation of chondroitin sulfate GAG chains were also upregulated. After the surgery, mice were treated with the 2-deoxy-d-glucose (2DG) that inhibits conversion of glucose to glucose-6-phosphate, an initial step of glucose metabolism as an energy source and precursors of monosaccharides of GAGs. The 2DG treatment reduced accumulation of sulfated proteoglycans, improved collagen fiber alignment, and reduced the cross-sectional area of the injured tendons. The modulus of the 2DG-treated groups was higher than that in the vehicle group, but not of statistical significance. Our findings suggest that mucoid degeneration in injured tendons may result from the upregulated expression of genes involved the synthesis of sulfate proteoglycans and can be inhibited by reduction of glucose utilization.

The mutational spectrum of hunter syndrome reveals correlation between biochemical and clinical profiles in Tunisian patients

BackgroundMucopolysaccharidosis type II (MPS II) or Hunter syndrome is an X-linked recessive lysosomal storage disorder resulting from deficient activity of iduronate 2-sulfatase (IDS) and the progressive lysosomal accumulation of sulfated glycosaminoglycans (GAGs).MethodsA diagnosis of MPS II or Hunter syndrome was performed based on the following approach after a clinical and paraclinical suspicion. Two biochemical and molecular tests were carried out separately and according to the availability of the biological material.ResultsAll patients in this cohort presented the most common MPS II clinical features. Electrophoresis of GAGs on a cellulose acetate plate in the presence of a high concentration of heparane sulfate showed an abnormal dermatan sulfate band in the patients compared with that in a control case. Furthermore, leukocyte IDS activity ranged from 0.00 to 0.75 nmol/h/mg of leukocyte protein in patients.Five previously reported mutations were identified in this study patients: one splice site mutation, c.240 + 1G > A; two missense mutations, p.R88P and p.G94D; a large deletion of exon 1 to exon 7; and one nonsense mutation, p.Q396*. In addition, two novel alterations were identified in the MPS II patients: one frame shift mutation, p.D450Nfs*95 and one nonsense mutation, p.Q204*.Additionally, five known IDS polymorphisms were identified in the patients: c.419–16 delT, c.641C > T (p.T214M), c.438 C > T (p.T146T), c.709-87G > A, and c.1006 + 38 T > C.ConclusionsThe high level of urine GAGs and the deficiency of iduronate 2-sulfatase activity was associated with the phenotype expression of Hunter syndrome. Molecular testing was useful for the patients’ phenotypic classification and the detection of carriers.

Silk Fibroin Scaffold-Based 3D Co-Culture Model for Modulation of Chondrogenesis without Hypertrophy via Reciprocal Cross-talk and Paracrine Signaling.

In this study, the effect of cellular cross-talk on modulation of chondrogenesis and hypertrophy while minimizing the usage of articular chondrocytes (ACs) has been investigated. Herein, co-culture of ACs with adipose-derived human mesenchymal stem cells (ADhMSCs) was employed for cross-talk within silk fibroin (SF)-based three-dimensional (3D) scaffolds. The co-culture model was developed by co-culturing four different ratios of ADhMSCs to ACs: 1:0, 3:1, 1:1, and 0:1 on porous 3D SF scaffolds for 21 days. The co-culture groups were cultured in defined media without adding any exogenous growth factors except the monoculture group, ADhMSC-only controls. The co-cultured constructs indicated significantly higher cellular viability and proliferation than the control monoculture groups. The supernatants of co-culture groups indicated significantly higher levels of TGF-β1 and IL-10, which confirmed the production of the morphogens/signaling molecules by chondrocytes for induction of ADhMSCs differentiation toward the chondrogenic phenotype. Biochemical assays indicated enhanced accumulation of sulfated glycosaminoglycans, collagen, and high DNA content along with high cellularity in co-culture groups than chondrocyte-only controls. Co-culture groups revealed synergistic interactions between cells as indicated by the interaction index value ranging from 2-3. Furthermore, upregulation of putative chondrogenic markers-aggrecan, sox-9, and collagen II, and significantly reduced expression of hypertrophic genes-collagen type X and MMP-13 was revealed in co-culture constructs. Histological and immunohistochemical staining also demonstrated even distribution and deposition of ECM in co-cultured constructs. Taken together, this work presents the potential of the developed 3D co-culture model toward modulation of chondrogenesis and hypertrophy via 3D microenvironment induced by physicochemical and biological properties of SF scaffolds, synergistic interactions between cells, and paracrine signaling in the co-culture system.

Pre-transplantational Control of the Post-transplantational Fate of Human Pluripotent Stem Cell-Derived Cartilage.

SummaryCartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unmineralized chondrocytes after subcutaneous transplantation. Thus, the post-transplantational fate of chondrocytes originating from hPSC-derived chondroprogenitors can be controlled during their genesis in vitro.

Chondrocyte Differentiation of Human Endometrial Gland‐Derived MSCs in Layered Cell Sheets

Recently, regenerative medicine using engineered three-dimensional (3D) tissues has been focused. In the fields of cell therapy and regenerative medicine, mesenchymal stem cells (MSCs) are attractive autologous cell sources. While, in bioengineered tissues, a 3D environment may affect the differentiation of the stem cells, little is known regarding the effect of 3D environment on cellular differentiation. In this study, MSC differentiation in in vitro 3D tissue models was assessed by human endometrial gland-derived MSCs (hEMSCs) and cell sheet technology. hEMSC sheets were layered into cell-dense 3D tissues and were cultured on porous membranes. The tissue sections revealed that chondrocyte-like cells were found within the multilayered cell sheets even at 24 h after layering. Immunostainings of chondrospecific markers were positive within those cell sheet constructs. In addition, sulfated glycosaminoglycan accumulation within the tissues increased in proportion to the numbers of layered cell sheets. The findings suggested that a high cell density and hypoxic environment in 3D tissues by layering cell sheets might accelerate a rapid differentiation of hEMSCs into chondrocytes without the help of chondro-differentiation reagents. These tissue models using cell sheets would give new insights to stem cell differentiation in 3D environment and contribute to the future application of stem cells to cartilage regenerative therapy.

SOX9 enhanced chondrogenic differentiation potential of human umbilical cord mesenchymal stem cells through cellular aggregation

To explore the effects of transcription factor SOX9 on chondrogenic differentiation potential of human umbilical cord mesenchymal stem cells (hUC-MSCs). hUC-MSCs were harvested from human umbilical cord and their phenotypic characteristics identified by flow cytometry. To confirm their multipotency, hUC-MSCs were induced to differentiate toward adiposity and osteogenesis. After transfection with the packaging lentivirus vectors containing SOX9 in vitro, the expression of green fluorescent protein (GFP) and the efficiency of transfection were detected by fluorescence microscopy. Their cellular proliferation capacity was detected by thiazolyl blue tetrazolium bromide (MTT) assay.hUC-MSCs modified with SOX9 were seeded into monolayer and cultured for 21 days in a defined, serum-free medium supplemented with transforming growth factor (TGF)-β1. The untransduced cells or those transduced with GFP served as the controls. Morphologic changes of hUC-MSCs were observed daily and their chondrogenic differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunofluorescent staining. And the accumulation of sulfated glycosaminoglycans was detected by Alcian blue staining. Meanwhile, the expressions of collagen I, X and cell adhesion molecule N-cadherin were assayed. The hUC-MSCs isolated from human umbilical cord stromas exhibited fibroblastic morphology and they were positive for CD29 (95.9%), CD44 (96.5%), CD90 (98.9%), CD105 (94.3%) and negative for hematopoietic stem cells surface markers CD34 (3.0%) and CD45 (2.6%). At Day 21, hUC-MSCs differentiated toward adiposity and osteogenesis. Both oil red O and alkaline phosphatase stains were intensely positive and it confirmed the multilineage potential of hUC-MSCs. An intense expression of GFP was observed under flourescence microscope and the transfection efficiency of cells with Lenti-GFP-SOX9 or Lenti-GFP was more than 90% respectively. SOX9 gene was over-expressed in hUC-MSCs at 48 h post-transduction. The proliferation of hUC-MSCs had no significant effect after the transfection of lentivirus vectors (P > 0.05). In vitro high-density monolayer culture of these SOX9-transfected hUC-MSCs demonstrated that spontaneous cell aggregation appeared at Day 14 of culturing and subsequently generated large cartilage nodules. However there was no phenomenon of cell aggregation occurring in the cells transducted by Lenti-GFP or untransduced vectors. The expressions of collagen II and Aggrecan were higher in SOX9 transducted cells than those in the controls. Alcian blue staining also showed abundant accumulation of sulfated glycosaminoglycans in the SOX9-induced cartilage nodules. The expression of collagen I had no difference in all groups and collagen X was inhibited in SOX9 transduced cells. N-cadherin was strongly up-regulated by SOX9 and might result in cellular aggregation and formation of large cartilage nodules. SOX9 may enhance the chondrogenic differentiation potential of human umbilical cord mesenchymal stem cells through cellular aggregation.

Construction of osteochondral-like tissue graft combining β-tricalcium phosphate block and scaffold-free centrifuged chondrocyte cell sheet

BackgroundThe combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure. MethodsHuman and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32cm2) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1–7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined. ResultsThe use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 105 cells tended to result in a curved cell sheet. Culture involving 18.6 × 105 cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1–7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block. ConclusionCentrifugation and subsequent cultivation of chondrocytes (18.6 × 105 cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition.

Chondrogenesis of Rabbit Mesenchymal Stem Cells in Fibrin/Hyaluronan Composite ScaffoldIn Vitro

Scaffold material is expected to play a crucial role in induction of chondrogenic differentiation of mesenchymal stem cells (MSCs) for cartilage tissue engineering. Here we demonstrated the feasibility of a fibrin/hyaluronan (HA) composite hydrogel as a potent scaffold for support of chondrogenesis of rabbit MSCs (rMSCs). rMSCs were prepared in three-dimensional cultures of pellet, alginate layer, and fibrin/HA gel. Specimens in each group were cultured in chondrogenic defined media for 4 weeks in the absence or presence of transforming growth factor β1 (TGF-β1) treatment. Viability of rMSCs was somewhat reduced until 4 weeks, which was less significant in fibrin/HA gels than in the alginate layer (*p < 0.05). The fibrin/HA group showed transient size reduction by about 35% at 1 week, but showed significantly higher mechanical strength than the alginate group. In safranin-O and alcian blue stains, accumulation of sulfated glycosaminoglycans (GAGs) was observed clearly from 1 week, and homogenously in the entire area at 4 weeks in the fibrin/HA group. Of note, TGF-β1 treatment showed no additional effect on GAGs accumulation in the fibrin/HA group. The alginate and pellet groups, however, showed much lower levels of GAGs accumulation only in the presence of TGF-β1. Biochemical assays for GAGs and collagen, and expression of chondrogenic markers also showed much better results in the fibrin/HA group, even without TGF-β treatment than the other groups. These results demonstrated that fibrin/HA composite gel efficiently promoted chondrogenic differentiation of rMSCs, even without TGF-β treatment, and that it could be a useful tool for use in cartilage tissue engineering.

Ligament-Derived Matrix Stimulates a Ligamentous Phenotype in Human Adipose-Derived Stem Cells

Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p <or= 0.05). Increased seeding density increased DNA content incrementally over 28 days in culture for LDM but not COL constructs (p <or= 0.05). These findings suggest that LDM can stimulate a ligament phenotype by hASCs, and may provide a novel scaffold material for ligament engineering applications.

Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc

There is currently no suitable replacement for damaged temporomandibular joint (TMJ) discs after discectomy. In the present study, we fabricated bilayer biodegradable polylactide (PLA) discs comprising a non-woven mat of poly(L/D)lactide (P(L/D)LA) 96/4 and a P(L/DL)LA 70/30 membrane plate. The PLA disc was examined in combination with adipose stem cells (ASCs) for tissue engineering of the fibrocartilaginous TMJ disc in vitro. ASCs were cultured in parallel in control and chondrogenic medium for a maximum of six weeks. Relative expression of the genes, aggrecan, type I collagen and type II collagen present in the TMJ disc extracellular matrix increased in the ASC-seeded PLA discs in the chondrogenic medium. The hypertrophic marker, type X collagen, was moderately induced. Alcian blue staining showed accumulation of sulphated glycosaminoglycans. ASC differentiation in the PLA discs was close to that observed in pellet cultures. Comparison of the mRNA levels revealed that the degree of ASC differentiation was lower than that in TMJ disc-derived cells and tissue. The pellet format supported the phenotype of the TMJ disc-derived cells under chondrogenic conditions and also enhanced their hyalinization potential, which is considered part of the TMJ disc degeneration process. Accordingly, the combination of ASCs and PLA discs has potential for the development of a tissue-engineered TMJ disc replacement.

The influence of mechanical strain on adult rat marrow stromal cell dynamics

The combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure.Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm2) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical βTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1–7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined.The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 105 cells tended to result in a curved cell sheet. Culture involving 18.6 × 105 cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a βTCP block to the cell sheet (1–7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block.Centrifugation and subsequent cultivation of chondrocytes (18.6 × 105 cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition.

Mucopolysaccharidosis type IV: N-Acetylgalactosamine-6-sulfatase mutations in Tunisian patients

Mucopolysaccharidosis type IVA (MPS IVA; OMIM #253000) or Morquio A syndrome is an autosomal recessive inborn error resulting from the deficient activity of the lysosomal enzyme, N-acetylgalactosamine-6-sulfatase (GALNS), and the progressive lysosomal accumulation of sulfated glycosaminoglycans. Clinically, the severe form of this lysosomal storage disease is characterized by a characteristic severe bone dysplasia and normal intelligence. To date, a variety of mutations have been associated with the severe MPS IVA phenotype. Here, we report the GALNS mutations in six severe MPS IVA patients from four unrelated Tunisian families. For mutation detection, each of the 14 exons and adjacent intron–exon junctions of the GALNS gene were sequenced after PCR-amplification from genomic DNA. Two novel mutations were identified: a G to A transition in the conserved 5′ donor splice site of intron 1 (GACgt → GACat: designated IVS1 +1g→a) and a G to C transversion in codon 66 of exon 2 predicting a glycine to arginine substitution (G66R). The IVS1 +1g→a mutation was homozygous in five similarly affected patients from three presumably unrelated families, but haplotype analysis suggested a common ancestor. The affected patient in the fourth family was homozygous for the G66R mutation. These are the first GALNS mutations causing severe MPS IVA disease identified in Tunisia. These molecular findings provide genotype/phenotype correlations, and permit accurate carrier detection, prenatal diagnosis, and counseling for MPS IVA disease in Tunisia where first cousin consanguineous mating remains frequent.

Synthesis and in Vitro Evaluation of Enzymatically Cross-Linked Elastin-Like Polypeptide Gels for Cartilaginous Tissue Repair

Genetically engineered elastin-like polypeptide (ELP) hydrogels offer unique promise as scaffolds for cartilage tissue engineering because of the potential to promote chondrogenesis and to control mechanical properties. In this study, we designed and synthesized ELPs capable of undergoing enzyme-initiated gelation via tissue transglutaminase, with the ultimate goal of creating an injectable, in situ cross-linking scaffold to promote functional cartilage repair. Addition of the enzyme promoted ELP gel formation and chondrocyte encapsulation in a biocompatible process, which resulted in cartilage matrix synthesis in vitro and the potential to contribute to cartilage mechanical function in vivo. A significant increase in the accumulation of sulfated glycosaminoglycans was observed, and histological sections revealed the accumulation of a cartilaginous matrix rich in type II collagen and lacking in type I collagen, indicative of hyaline cartilage formation. These results provide evidence of chondrocytic phenotype maintenance for cells in the ELP hydrogels in vitro. In addition, the dynamic shear moduli of ELP hydrogels seeded with chondrocytes increased from 0.28 to 1.7 kPa during a 4-week culture period. This increase in the mechanical integrity of cross-linked ELP hydrogels suggests restructuring of the ELP matrix by deposition of functional cartilage extracellular matrix components.

Immature murine articular chondrocytes in primary culture: a new tool for investigating cartilage

Many genetically modified animal models are providing new keys for unlocking the pathophysiology of cartilage degradation. To produce a tool for cellular and molecular studies in genetically engineered murine models, we defined the optimal culture conditions for primary cultures of articular chondrocytes from newborn mice (C57Bl/6). To determine whether the cultured cells exhibited the typical articular chondrocyte phenotype, we examined several morphological, biochemical, and functional features. The cells had the typical chondrocyte morphology, with a rounded or polygonal shape. Immunolocalization studies showed high levels of type II collagen and aggrecan expression, together with sulfated glycosaminoglycan accumulation. Type II collagen and aggrecan expression decreased with passaging. In contrast, type I collagen expression was low in primary cultures and high after four passages, indicating a fibroblast phenotype. To evaluate the functional integrity of our cultured cells, we evaluated their ability to produce prostaglandin E2 (PGE2) and nitric oxide (NO) in response to the catabolic cytokine interleukin (IL)-1beta (10 ng/ml). Production of both PGE2 and NO increased significantly as compared to untreated controls. In addition, IL-1beta induced COX-2 expression by the cultured cells, as shown by Western blotting. Since functional and molecular parameters can be measured readily in mice, the immature murine articular chondrocyte (iMAC) model described here should prove a powerful tool for research, particularly as many transgenic and knockout mouse strains are available, even if iMACs are not optimal substitutes for human chondrocytes.

Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture.

Tissue engineering laboratories are attempting to create neocartilage that could serve as an implant material for structural support during reconstructive surgery. One approach to forming such tissue is to proliferate chondrocytes in monolayer culture and then seed the expanded cell population onto biodegradable scaffolds. However, chondrocytes are known to dedifferentiate after this type of monolayer growth and, as a result, decrease their production of cartilaginous extracellular matrix components such as sulfated glycosaminoglycans. The resultant tissue lacks the biomechanical properties characteristic of cartilage. The objective of the study was to determine whether different culture systems could induce monolayer-expanded human septal chondrocytes to redifferentiate and form extracellular matrix. Laboratory research. Chondrocytes were isolated from human nasal septal cartilage of five donor patients (age, 35.8 +/- 9.3 y). Cell populations were seeded at low density (30,000 cells/cm2) into monolayer culture and expanded for 4 to 6 days. Following trypsin release, chondrocytes were placed into three different systems for neocartilage formation: alginate beads, polyglycolic acid scaffolds, and monolayer. After 7 and 14 days of growth, neocartilage was analyzed using histological and quantitative biochemical assessment of cellularity (Hoechst 33258 assay) and sulfated glycosaminoglycan content (dimethyl methylene blue assay). Histologically, alginate beads contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage. In contrast, polyglycolic acid scaffolds and monolayer cultures contained elongated cells with scant staining for matrix sulfated glycosaminoglycans, which are features that are characteristic of dedifferentiated chondrocytes. Biochemical analysis demonstrated a lower level of cell proliferation (P <.001) in scaffolds (+52% over baseline) and alginate (+96% over baseline) than in monolayer (+366% over baseline), as well as a higher content of sulfated glycosaminoglycans per cell (P <.001), after 14 days of growth in alginate culture than in either polyglycolic acid scaffolds (19-fold difference) or monolayer (98-fold difference). Of the systems compared, monolayer-expanded human septal chondrocytes demonstrated the greatest accumulation of sulfated glycosaminoglycans per cell when grown in alginate beads. Future research on cartilage tissue engineering may use alginate culture for reverting dedifferentiated cells back to the chondrocytic phenotype.

Effects of serial expansion of septal chondrocytes on tissue-engineered neocartilage composition.

Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage. Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 [P(0)]) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P(1)) and passage 2 (P(2)). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay). Histologic sections of constructs seeded with P(0) cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P(1) and P(2) cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P(0) constructs was higher than in either P(1) (P < 0.05) or P(2) (P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages. Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation. The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs.

Preeclampsia is associated with alterations in insulin-like growth factor (IGF)-1 and IGF-binding proteins in Wharton's jelly of the umbilical cord.

Wharton's jelly is abundant in extracellular matrix, which is known as a storage site to concentrate and stabilise growth factors in the vicinity of cells. It was previously found that Wharton's jelly contains significant amounts of insulin-like growth factor (IGF)-1 and IGF-binding proteins (BPs). IGF-1 is a stimulator of biosynthetics of collagen and sulphated glycosaminoglycans. Preeclampsia (edema, proteinuria, hypertension (EPH)-gestosis) is accompanied by an accumulation of sulphated glycosaminoglycans in Wharton's jelly. IGF-1 and BPs may play an important role in such a remodelling of this tissue. It was decided to evaluate the alterations in amounts of IGF-1 and BPs in Wharton's jelly of newborns delivered by mothers with preeclampsia. Studies were performed on Wharton's jelly of 10 controls and 10 newborns delivered by mothers with preeclampsia (edema, proteinuria > 500 mg/l, arterial pressure: systolic > 140 mm Hg, diastolic > 90 mmHg). Radioimmunological techniques were employed to determine IGF-1 and IGF-BPs (BP-1 and BP-3). It was found that preeclampsia is associated with a decrease in IGF-1 and IGF-BP-1 in Wharton's jelly. A slight increase in IGF-BP-3 was found. Ligand blotting demonstrated that BP-3 (not BP-1) is a main component of Wharton's jelly, which binds IGF-1. Heparin drastically inhibited the binding of IGF-1 by BP-3. It is known from our previous studies that preeclampsia is associated with an increase in the amount of sulphated glycosaminoglycans (heparin, heparan sulphate, dermatan sulphate) in Wharton's jelly. This may be a factor, which prevents the binding of IGF-1 by BPs and facilitates the binding of IGF-1 to cells, stimulating them to produce sulphated glycosaminoglycans in Wharton's jelly.