Proteoglycan Research Articles

Effect of hydration on the ultra‐structural distribution of collagen fibrils and proteoglycans in human corneal stroma

AbstractPurpose We investigated the ultra‐structural changes in the distribution of collagen fibrils (CF) and proteoglycans (PGs) due to hydration in the human corneal stroma.Methods Six fresh human scleral corneal rings were hydrated separately in deionized water for 2hrs and 48hrs. Corneas were divided into two parts. The first part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy to assess proteoglycans. A second part of each cornea was fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer and post fixed in 1% osmium tetroxide to assess collagen fibril diameter.Results In the hydrated corneas the mean diameter of CF in the anterior stroma (23.40±2.61nm), middle stroma (22.48±3.28nm) and posterior stroma (23.58±2.46nm) was less than the CF diameter in the anterior (28.27±4.05nm), middle (28.22±3.09nm) and posterior stroma (29.73±3.72nm) of the normal cornea. Micro‐fibrils within the CF in the hydrated cornea were degenerated. In the hydrated cornea, the mean area of the PGs in the anterior stroma was reduced whereas it increased in the middle stroma compared to those in the normal cornea. The density of the PGs in the hydrated cornea was reduced in the anterior, middle and posterior stroma compared to those in the normal cornea.Conclusion The effect of hydration on the uniform organisation of CF and PGs was higher in the middle and posterior stroma than in the anterior stroma. This could be due to less penetration of water in the interlacing lamellae of the anterior stroma. The reduction in the collagen fibrils diameter could be due to degeneration of microfibrils within the collagen fibrils.

PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.

Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted proteoglycans (PGs) displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.

Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures.

The potential of hypertonic conditions at in vivo levels to promote cartilage extracellular matrix accumulation in scaffold-free primary chondrocyte cultures was investigated. Six million bovine primary chondrocytes were cultured in transwell inserts in low glucose (LG), high glucose (HG), or hypertonic high glucose (HHG) DMEM supplemented with fetal bovine serum, antibiotics, and ascorbate under 5 % or 20 % O2 tension with and without transforming growth factor (TGF)-β3 for 6 weeks. Samples were collected for histological staining of proteoglycans (PGs) and type II collagen, analysis by quantitative reverse transcription plus the polymerase chain reaction (RT-PCR) of mRNA expression of aggrecan and procollagen α1 (II) and of Sox9 and procollagen α2 (I), and quantitation of PGs and PG separation in agarose gels. Cartilage tissues produced at 20 % O2 tension were larger than those formed at 5 % O2 tension. Compared with LG, the tissues grew to larger sizes in HG or HHG medium. Histological staining showed the strongest PG and type II collagen staining in cartilage generated in HG or HHG medium at 20 % O2 tension. Quantitative RT-PCR results indicated significantly higher expression of procollagen α1 (II) mRNA in cartilage generated in HHG medium at 20 % O2 tension compared with that in the other samples. TGF-β3 supplements in the culture medium provided no advantage for cartilage formation. Thus, HHG medium used at 20 % O2 tension is the most beneficial combination of the tested culture conditions for scaffold-free cartilage production in vitro and should improve cell culture for research into cartilage repair or tissue engineering.

Raman analysis of proteoglycans simultaneously in bone and cartilage

Bone and cartilage are connective tissues with distinct organic matrix (collagen and non‐collagenous proteins) composition facilitating their biological function. Proteoglycans (PGs), a member of the non‐collagenous proteins fulfill functions that are determined by both their core protein and their glycosaminoglycan chains. The purpose of the present study was to identify Raman bands that are representative of PG concentration and may be used in both bone and cartilage tissues. To achieve this goal, we analyzed a series of reference PGs and collagens, as well as turkey leg tendon to verify the laser polarization independency of the identified bands. Additionally, the applicability of these bands in both cartilage and bone tissue simultaneously was tested in a healthy femoral head by Raman imaging and hierarchical cluster analysis to describe the distribution of PGs at the micron level from articular cartilage to subchondral bone. The results of the study show that the Raman band ~1375 cm−1 can be used as a PGs marker band in both cartilage and bone. Moreover, articular cartilage has a lower content of organic matrix (mostly type II collagen), while the middle and deep transitional zone haves a higher concentration of PGs. The calcified cartilage is characterized by a lower content of PGs and total organic matrix (estimated from the integrated area of the amide III band). Copyright © 2014 John Wiley & Sons, Ltd.

Is the T1ρ MRI profile of hyaline cartilage in the normal hip uniform?

T1ρ MRI is an imaging technique sensitive to proteoglycan (PG) content of hyaline cartilage. However, normative T1ρ values have not been established for the weightbearing cartilage of the hip, and it is not known whether it is uniform or whether there is topographic variation. Knowledge of the T1ρ profile of hyaline cartilage in the normal hip is important for establishing a baseline against which comparisons can be made to experimental and clinical arthritic subjects. In this diagnostic study, we determined (1) the T1ρ MRI values of hyaline cartilage of the normal hip; and (2) whether the T1ρ MRI profile of the normal hip hyaline cartilage is uniform. Fourteen asymptomatic volunteers (11 men, three women; mean age, 35 years) prospectively underwent 1.5-T T1ρ MRI of a single hip. The weightbearing hyaline cartilage bilayer of the acetabulum and femoral head was evaluated on sagittal images and segmented into four zones: (1) anterior; (2) anterosuperior; (3) posterosuperior; and (4) and posterior. For the full region of interest and within each zone and each sagittal slice, we calculated the mean T1ρ relaxation value, a parameter that indirectly quantifies PG content, where T1ρ is inversely related to PG concentration. There was variation in the T1ρ relaxation values depending on zone (anterior to posterior) and slice (medial to lateral). When combining the most anterior quadrants (Zones 1 and 2), the T1ρ relaxation values were lower than those in the combined posterior quadrants (Zones 3 and 4) (30.4 msec versus 32.2 msec, respectively; p = 0.002), reflecting higher PG concentration. There was a difference between the T1ρ relaxation values of the sagittal slices (p = 0.038), most pronounced anteriorly in Zone 1 (26.6 msec, p = 0.001). With a selective combination of zones and slices, there were lower mean T1ρ values in the anterolateral-most region compared with the remainder of the weightbearing portion of the hip (28.6 msec versus 32.2 msec, respectively; p = 0.001). The T1ρ profile of normal hyaline cartilage of the hip is not uniform with the topographic differences identified suggesting regional variations in PG concentration. This study, through determination of lower T1ρ relaxation values, suggests inherently greater PG concentrations in the more anterolateral region of the normal hip hyaline cartilage. Furthermore, it demonstrates that T1ρ MRI has the ability to detect even subtle, microscopic local differences in hyaline cartilage composition. This technique has the potential to facilitate basic science and clinical research by serving as a noninvasive surrogate or biomarker of cartilage health and thus may be added to the growing repertoire of advanced, biochemical MRI techniques for evaluating hyaline cartilage.

Treatment of discogenic low back pain by minimally invasive lumbar disc shunt

Objective To study the mechanism of minimaliy invasive lumbar disc shunt on treating discogenic low back pain, and analyze its short-term clinical outcome. Methods First, use nylon suture to perform a shunt study about its ability to change acid fluid＇ s PH. Second, evaluate the influence of proteoglycan＇ s content pre-and post-nylon suture implanting into lumber disc of sheep, meanwhile, observe nylon suture＇ s biocompatibility inside of lumbar disc. Third, a clinical trial is per-formed , which is named ＂disc shunt＂. Fifteen patients were diagnosed as discogenic low back pain by discography. All of them accepted ＂disc shunt＂ through pathway of discography at once, local anesthesia was used in all cases. Patients had shunts （using nylon suture） implanted into the following level of discs： one at L3-4, six at L4-5, seven at L5S1, and one at both L3-4 and L4-5. Clinical outcome evaluation is based on VAS score, ODI score and radiology at one week, three months, one year and two year after implantation. Results In virto study showed pH of lactic acid solution was gradually raised from 4.0 to 7.14 after shunt by nylon suture. Shunted discs of sheep were harvested at one, three, twelve and twenty-four months. Safranine 0 staining showed gradually increasing of proteoglycans in disc. No obvious inflammatory reaction surround nylon suture after 24 months observation. Nylon suture＇ s bioeompatibility was satisfactory inside lumbar disc. For clinical trial study, the low back pain and disability was significantly improved after operation. At the final twenty-four months follow up, evaluations showed all of the patients＇ VAS and ODI scores were reduced significantly, average rate of VAS scores improvement was 79.16%± 12.33%, and average rate of ODI was 77.42%±12.20%. Radiological examinations after implant showed no reduction of disc height index, and no aggressive disc degeneration according to Thompson scale. In addition, shunted discs were whitened under MRI, probably from disc hydration through the disc shunts. Conclusion Percutaneous disc shunt is an efficacious therapy for discogenic low back pain with multi- ple advantages such as being minimally invasive, having few risks, reducing pain and disability, and having treatment concurrent with diagnostic discography. Key words: Intervertebral disc degeneration; Low Back Pain; Intervertebral disc; Surgical procedures, minimally invasive

Bevacizumab Reduces Neurocan Content and Gene Expression in Newborn Rat Retina In Vitro

Extracellular matrix (ECM) and cellular membrane proteoglycans (PGs) play important roles in neural differentiation and cell adhesion. Vascular endothelial growth factor, an important signal protein in vascular and retinal neural cell development, is retained in the ECM due to its high affinity for PG. Bevacizumab, an anti-VEGF agent, has been extensively used for treating retinal diseases in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on neurocan, phosphacan, and syndecan-3 PG levels in newborn rat retina. Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 48 hours. Immunohistochemical staining was assessed against neurocan, phosphacan, and syndecan-3. Proteoglycan content was quantified based on the intensity of immunohistochemical labeling. Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. The results from the treatment and control groups were compared. No significant difference in the staining intensity and mRNA expression of phosphacan and syndecan-3 was observed between the groups. However, a significant decrease in neurocan content and mRNA expression was observed in bevacizumab-treated retinal explants compared with controls. Bevacizumab did not affect phosphacan and syndecan-3 levels but decreased neurocan content and gene expression. Therefore, it may interfere with early postnatal retinal cell differentiation. Although further studies are necessary to confirm our findings, we suggest anti-VEGF agents be used with caution in developing retinal tissue.

Hippocampal proteoglycans brevican and versican are linked to spatial memory of Sprague-Dawley rats in the morris water maze.

Proteoglycans (PGs) are major constituents of the extracellular matrix and have recently been proposed to contribute to synaptic plasticity. Hippocampal PGs have not yet been studied or linked to memory. The aim of the study, therefore, was to isolate and characterize rat hippocampal PGs and determine their possible role in spatial memory. PGs were extracted from rat hippocampi by anion-exchange chromatography and analyzed by nano LC-MS/MS. Twenty male Sprague-Dawley rats were tested in the morris water maze. PGs agrin, amyloid beta A4 protein, brevican, glypican-1, neurocan, phosphacan, syndecan-4, and versican were identified in the hippocampi. Brevican and versican levels in the membrane fraction were higher in the trained group, correlating with the time spent in the target quadrant. α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor GluR1 was co-precipitated with brevican and versican. Levels for a receptor complex containing GluR1 was higher in trained while GluR2 and GluR3-containing complex levels were higher in yoked rats. The findings provide information about the PGs present in the rat hippocampus, demonstrating that versican and brevican are linked to memory retrieval in the morris water maze and that PGs interact with α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor GluR1, which is linked to memory retrieval. Proteoglycans (PGs) are major constituents of the extracellular matrix of the brain and were proposed to contribute to synaptic plasticity. This report addressed PGs in rat hippocampus and suggests that PGs brevican and versican are linked to spatial memory, and form a complex with the GluR1 subunit of the AMPA receptor, a key signaling molecule in memory mechanisms.

Proteoglycans and glycosaminoglycans in misfolded proteins formation in Alzheimer's disease.

Misfolded protein amyloid-beta protein (Aβ) and tau protein are two high hallmarks of Alzheimer's disease (AD), representing significant targets in treating AD. Researches on mechanisms of the two proteins inducing neuron dysfunctions provide therapeutic strategies of AD, including inhibition of Aβ production and aggregation, acceleration of Aβ clearance as well as reduction of tau hyperphosphorylation. Proteoglycans (PGs) consist of a core protein and glycosaminoglycans (GAGs) chains, with enormous structural diversity due to variation in the core protein, the number of GAGs chains as well as extent and position of sulfation. Considerable evidences have indicated that PGs and GAGs play important roles in Aβ and tau processing. Numbers of GAGs and analogues have potential therapeutic function in AD. In this Review, we focus on the relationship of PGs and GAGs with misfolded proteins in AD and their potential therapeutic implications.

Small rigid platforms functionalization with quaternary ammonium: Targeting extracellular matrix of chondrosarcoma

This work takes place in the “cartilage targeting strategy”, consisting in using the quaternary ammonium (QA) function as a vector to proteoglycans (PGs) of extracellular matrix (ECM). The objective was to demonstrate that QA could address gadolinium based small rigid platforms (SRP) to PG-rich tumors. SRP were functionalized with QA, radiolabeled with 111Indium and evaluated for biodistribution in vivo, respectively to non functionalized SRP, in two experimental models: (i) the HEMCSS human xenograft model; (ii) the Swarm rat chondrosarcoma (SRC) orthotopic model. The contribution of cellular uptake to tumoral accumulation of nano-objects was also determined from in vitro binding. In the SRC model expressing a highly and homogeneously distributed PG content, tumor accumulation and retention of SRP@QA were increased by 40% as compared to non-functionalized SRP. When considering the radiosensitizing potential of gadolinium based SRP, these results provide hopes for the radiobiological approach of highly resistant tumor such as chondrosarcoma. From the Clinical EditorIn this study, gadolinium-based complexing DOTA-surfaced small polysiloxane nanoparticles were functionalized with quaternary ammonium derivatives that target the extracellular matrix of chondrosarcoma. The authors demonstrate in a rat model that the use of these constructs results in a 40% increase of tumor accumulation and retention compared to non-functionalized (and otherwise same) platforms. Similar approaches would be welcome additions to the clinical armamentarium addressing chondrosarcoma.

Proteoglycans affect monomer infiltration in the etch-and-rinse bonding technique

ObjectivesTo identify proteoglycans (PGs) and collagen fibrils (CF) within human dentin by means of a dual immunofluorescent labeling technique and to investigate the monomer infiltration of two etch-and-rinse adhesives to tosyl-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (TRY)-pretreated dentin. MethodsThirty-micrometer sections of middle coronal dentin were obtained and etched with 37% phosphoric acid gel for 15s. After preconditioning with or without TRY digestion, the sections were subjected to dual immunofluorescent labeling and observed with a confocal laser scanning microscope (CLSM). Demineralized dentin matrixes treated with or without TRY were observed with field emission scanning electron microscope (FE-SEM). Two etch-and-rinse adhesives, Adper Single Bond 2 (SB) and Prime & Bond NT (PBNT), were applied to the dentin surfaces that were pretreated with or without TRY. The thickness of the hybrid layers was evaluated under confocal micro-Raman spectroscopy and analyzed with a two-way ANOVA. ResultsGreen and red fluorescence was used to represent the PGs and the CF that were colocalized in the same section with different distributions. PGs were localized in the lumens of the dentin tubules and in peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TRY digestion, the red fluorescence decreased or disappeared, the organic filaments in the lumens of the dentin tubules disappeared, the tubules were enlarged, and the hybrid layer thickness for adhesives bonded to the TRY-pretreated dentin surfaces were significantly increased (p<0.001 for both SB and PBNT). SignificanceThe dual immunofluorescence labeling methodology can be used to study the human dentin matrix without decalcifying the entire dentin fragment. Proteoglycans were localized in the lumens of the dentin tubules and in peritubular dentin, which could depress the infiltration of the adhesive resin monomers. The use of TRY digestion increased the thickness of the hybrid layer created by the tested two-step etch-and-rinse adhesive.

Proteoglycan concentrations in healthy and diseased articular cartilage by Fourier transform infrared imaging and principal component regression

Fourier transform infrared imaging (FTIRI) combining with principal component regression (PCR) analysis were used to determine the reduction of proteoglycan (PG) in articular cartilage after the transection of the anterior cruciate ligament (ACL). A number of canine knee cartilage sections were harvested from the meniscus-covered and meniscus-uncovered medial tibial locations from the control joints, the ACL joints at three time points after the surgery, and their contralateral joints. The PG loss in the ACL cartilage was related positively to the durations after the surgery. The PG loss in the contralateral knees was less than that of the ACL knees. The PG loss in the meniscus-covered cartilage was less than that of the meniscus-uncovered tissue in both ACL and contralateral knees. The quantitative mapping of PG loss could monitor the disease progression and repair processes in arthritis.

June 2014: This Month in JoVE - 3D Virtual Navigation in the Human Brain, Simulation of Planet Formation, and Bioprinting Cultured Cartilage Grafts

Here's a look at what's coming up in the June 2014 issue of JoVE: The Journal of Visualized Experiments. The human brain has been called the next frontier of scientific discovery. In coming years scientists will greatly advance our understanding of how the brain works, leading to new methods of diagnosis, prevention, and intervention for many neurological disorders. This month in JoVE we feature two new techniques that can help scientists navigate through uncharted territories of the human brain. In the Clinical & Translational Medicine section, DaSilva et al. analyze changes in neurological processes that occur during migraine headaches. They scan the brain of a migraine patient using two methods: magnetic resonance imaging (MRI), which uses the magnetic properties of water molecules to image living tissues; and positron emission tomography (PET), which detects gamma rays emitted by a radionuclide tracer. The neuroimaging data are displayed in a special format, so through special shutter glasses, users can see the images in a three-dimensional, virtual-reality environment. This technique allows real-time interaction and navigation through the brains of patients with migraines, and can be applied to other neurological disorders such as neuralgia, depression, and addiction. In the Behavior section, Bathelt et al. map the sources of cognitive activities in a child's cerebral cortex. Brain activity is assessed with electroencephalography (EEG), which measures electrical changes caused by neural activity, and the sources of the EEG recordings are mapped using head models based on MRI scans. These maps show different cortical regions of activation in response to different cognitive stimuli. This technique can help scientists understand the foundations of complex cognitive skills in the developing brain. In JoVE Applied Physics we examine a very large developing system in a protocol that approximates conditions in the early Solar System so scientists can examine how planets form. Planet formation is thought to begin when microscopic dust particles accumulate to form fragile aggregates. Over a few thousand years, these aggregates can grow to around 1cm in diameter. When they collide, these aggregates may bounce off each other or break apart into fragments-but if the collision velocity is just right, the aggregates will stick to each other, gradually forming planets over millions of years. Blum et al. developed laboratory setups that generate the collision velocities thought to promote planet formation, and record these collisions with high-speed imaging. With these studies, they hope to shed light on the physical forces that created our Solar System. In the bioengineering field, 3D printing is an exciting technology with many potential biomedical applications ranging from printing microfluidic arrays for cellular assays to producing physical models of imaging data. It can also be used to engineer actual tissue structures, which Cui et al. demonstrate this month in JoVE Bioengineering. They convert a commercial inkjet printer into a bioprinter than can build tissue structures out of cells and biomaterials. They use this technology to print cultured cartilage grafts, which show excellent cell viability and production of proteoglycan, a component of connective tissue. This technique holds the potential to repair and regenerate multiple tissue types. You've just had a sneak peek of the June 2014 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

FRI0346 Activatory FC Gamma Receptor IV Plays A Crucial Role in Cartilage and Bone Erosion during the Chronic Phase of Antigen Induced Arthritis

BackgroundRheumatoid arthritis is characterized by immune complexes dependent chronic joint inflammation and severe cartilage and bone destruction. Earlier we found that in the absence of activatory FcγRI and III, joint...

SAT0443 In Vitro Effects of Canakinumab on Human Osteoarthritic Chondrocytes

BackgroundCanakinumab is a human IgGκ monoclonal antibody targeting Interleukin (IL)-1β; its action involves the neutralization of the signaling of IL-1β, the most important cytokine in the pathogenesis of Osteoartrhitis (OA)....

Impact of first trimester fasting glycemic levels on expression of proteoglycans in pregnancy.

The aim of this study was to assess the influence of glucose metabolism on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in pregnant women. Seventy-six women in the first trimester of pregnancy (10-13 weeks) attending the Gynecologic and Obstetric Clinic, University of Sassari, were enrolled and gave early morning urine samples. Groups I, II and III included women with serum glucose values of 65-89 mg/dL, 90-99 mg/dL and 100-125 mg/dL, respectively. Urine GAGs/PGs distribution was determined by electrophoresis on cellulose acetate strips. Urinary N-Acetyl-β-glucosaminidase was estimated kinetically. Analysis of urinary GAGs/PGs electrophoretic profiles showed a significant increase in heparan sulfate (HS) excretion (P = 0.017) as well as a reduced chondroitin sulfate (CS) excretion (P = 0.048) in the group II pregnant women compared with the group I, and higher values of the HS/CS ratio in groups II and III compared to group I. Furthermore, we observed a positive correlation among fasting blood glucose levels and the relative content of HS, the HS/CS and urinary trypsin inhibitor/CS ratios, and the N-Acetyl-β-glucosaminidase levels. The assessment of risk factors for gestational diabetes mellitus should also take into account fasting blood glucose values of 90-99 mg/dL, as the findings of our study indicated an alteration in the metabolism of GAGs during the early stages of pregnancy.

Understanding the chemical free enzyme based cleaner unhairing process in leather manufacturing

Abstract Higher cost of enzyme is one of the major factors for the system not being practiced through found environmentally friendly. Through understanding of enzyme based unhairing sans lime and sodium sulfide is essential to develop enzymes of low cost. Understanding of the enzyme–substrate is therefore imminent. The release of PG and carbohydrate was found to be more in the case of EBS compared to Lime–Sulfide System (LSS). The onset of complete removal of hair was found to take place after 4 h in the case of EBS. The trend of release of PG was found to be in congruence to the trend of degree of hair loosening. The micrographs of histological staining also corroborated the findings. Complete hair removal was achieved in 5 h in the case of EBS. The hair loosening in the case of EBS is primarily due to break down of PG present in the hair follicle. If protease that could break down selectively the protein core of the proteoglycans (PG), then with much lesser amount of protease hair loosening could be achieved. And the cost of EBS could therefore be brought down significantly.

Changes in the metabolism of chondroitin sulfate glycosaminoglycans in articular cartilage from patients with Kashin–Beck disease

To identify changes in the expression patterns of enzymes involved in chondroitin sulfate (CS) glycosaminoglycan (GAG) metabolism in articular cartilage proteoglycan (PG) isolated from adolescent patients with Kashin-Beck disease (KBD). Samples of articular cartilage were divided into two groups: Control samples (from five normal children), and KBD samples (from five KBD children) aged 3-12 years old. The morphology and pathology of hand joint cartilage were examined by histochemical staining. The localization and expression patterns of enzymes involved in CS GAG metabolism (i.e., PAPS synthetase 2 (PAPSS2), PAPS transporter 1 (PAPST1), Carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferases 15 (CHST15), Arylsulfatase B (ARSB) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS)) were performed using immuno-histochemical analyses. Positive immunostaining in articular cartilage was semi-quantified. Reduced aggrecan staining was observed in KBD samples compared with the control samples. The percentages of positive staining for the anabolic enzymes PAPSS2, PAPST1 and CHST15 in the upper and middle zones of KBD samples were significantly lower than that found in the Controls. In contrast, the percentages of positive staining in KBD samples for the catabolic enzymes ARSB and GALNS were significantly higher than the control samples. However, the staining for all of these GAG metabolism enzymes were hardly observed in the deep zones of KBD cartilage, suggesting that significant cell death and necrosis had occurred in this region. Our results indicate that alterations of enzymes involved in articular cartilage CS GAG metabolism on PGs in the articular cartilage play an important role in the onset and pathogenesis of KBD in adolescent children.

Effect of Short-Term Enzymatic Treatment on Cell Migration and Cartilage Regeneration:In VitroOrgan Culture of Bovine Articular Cartilage

Depending on the damage extent and adjacent tissue condition in traumatic cartilage injury, it is possible to heal the tissue by resident cells. Unlike autologous chondrocyte implantation, short-term enzymatic treatment is an effective single-step procedure without extra cell expansion. Moreover, this method has been shown to significantly increase cellularity in lesion edges, resulting in enhanced integration and interfacial strength. We hypothesize that the locally digested extracellular matrix by treatment allows effortless cell migration from the adjacent tissue. Full-thickness cartilage discs and osteochondral explants were prepared from mature bovine stifle joints. These specimens were treated with collagenase in a culture medium. Two concentrations, 0.25 and 0.5 mg/mL, were used with various treating time of 10, 30, and 180 min. The cartilages were subsequently washed and cultured with fibrin hydrogel. The effect of enzymatic treatment on cell migration was apparent in both experiments of the cartilage disc and full-thickness cartilage defect model. In the disc culture, the treatment resulted in an approximately three to four times higher number of migrated cells than nontreated control. In short-term collagenase-treated groups, the proteoglycan (PG) loss was localized in the edge of tissue with minimal cell death. The treatment also accelerated cell migration in the full-thickness cartilage defects and some cells differentiated into chondrocytes with the deposit of PG. Gene expression results could support the characteristics of migrated cells, which had migratory ability and chondrogenic differentiation potential with overexpression of collagen type I and II, respectively. Based on these results, short-term enzymatic treatment, which can accelerate cell migration into traumatically injured cartilage, has great potential for clinical application.

Modeling the role of IGF-1 on extracellular matrix biosynthesis and cellularity in intervertebral disc

The insulin-like growth factor-1 (IGF-1) is a well-known anabolic agent for intervertebral disc (IVD), promoting both proteoglycan (PG) biosynthesis and cell proliferation. Accordingly, it is believed that IGF-1 may play a central role in IVD homeostasis. Furthermore, the exogenous administration of IGF-1 has been proposed as a possible therapeutic strategy for disc degeneration. The objectives of this study were to develop a new computational framework for describing the mechanisms regulating IGF-mediated homeostasis in IVD, and to apply this numerical tool for investigating the effectiveness of exogenous administration of IGF-1 for curing disc degeneration. A diffusive–reactive model was developed for describing competitive binding of IGF-1 to its binding proteins and cell surface receptors, with the latter reaction initiating the intracellular signaling mechanism leading to PG production and cell proliferation. Because PG production increases cell metabolic rate, and cell proliferation increases nutritional demand, nutrients transport and metabolism were also included into the model, and co-regulated, together with IGF-1, IVD cellularity. The sustainability and the effectiveness of IGF-mediated anabolism were investigated for conditions of pathologically insufficient nutrient supply, and for the case of exogenous administration of IGF-1 to degenerated IVD. Results showed that pathological nutrients deprivation, by decreasing cellularity, caused a reduction of PG biosynthesis. Also, exogenous administration of IGF-1 was only beneficial in well-nourished regions of IVD, and exacerbated cell mortality in malnourished regions. These findings remark the central role of nutrition in IVD health, and suggest that adequate nutritional supply is paramount for achieving a successful IGF-based therapy for disc degeneration.