Publications by authors named "Toin H van Kuppevelt"

256 Publications

Loss of Hs3st3a1 or Hs3st3b1 enzymes alters heparan sulfate to reduce epithelial morphogenesis and adult salivary gland function.

Matrix Biol 2021 Sep 12;103-104:37-57. Epub 2021 Oct 12.

Matrix and Morphogenesis Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Heparan sulfate 3-O-sulfotransferases generate highly sulfated but rare 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes but that Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. To analyze their in vivo functions, we generated both Hs3st3a1 and Hs3st3b1 single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1 mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1 HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. Understanding how 3-O-sulfation regulates myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration.
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http://dx.doi.org/10.1016/j.matbio.2021.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8629026PMC
September 2021

Selective Binding of Heparin/Heparan Sulfate Oligosaccharides to Factor H and Factor H-Related Proteins: Therapeutic Potential for C3 Glomerulopathies.

Front Immunol 2021 18;12:676662. Epub 2021 Aug 18.

Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands.

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HS), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HS purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HS were analyzed using selectively desulfated heparins in competition with purified HS. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HS, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.
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http://dx.doi.org/10.3389/fimmu.2021.676662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416517PMC
October 2021

Polarized Secretion of APRIL by the Tonsil Epithelium Upon Toll-Like Receptor Stimulation.

Front Immunol 2021 18;12:715724. Epub 2021 Aug 18.

Translational Innovation in Medicine and Complexity, Centre National de la Recherche Scientifique UMR5525, La Tronche, France.

In mucosa such as tonsil, antibody-producing plasmocytes (PCs) lie in sub-epithelium space, which is thought to provide a suitable environment for their survival. A proliferation inducing ligand (APRIL) is one key survival factor for PCs present in this area. According to staining, apical epithelial cells produced APRIL, and the secreted product had to migrate all through the stratified surface epithelium to reach basal cells. A similar process also occurred in the less-organized crypt epithelium. Tonsil epithelial cells captured secreted APRIL, thanks to their surface expression of the APRIL coreceptor, either syndecan-1 or -4 depending on their differentiation stage. In the most basal epithelial cells, secreted APRIL accumulated inside secretory lamp-1 vesicles in a polarized manner, facing the sub-epithelium. The tonsil epithelium upregulated APRIL production by apical cells and secretion by basal cells upon Toll-like receptor stimulation. Furthermore, LPS-stimulated epithelial cells sustained PC survival in a secreted APRIL-dependent manner. Taken together, our study shows that the tonsil epithelium responds to pathogen sensing by a polarized secretion of APRIL in the sub-epithelial space, wherein PCs reside.
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http://dx.doi.org/10.3389/fimmu.2021.715724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416415PMC
August 2021

The role of MMP-14 in ovarian cancer: a systematic review.

J Ovarian Res 2021 Aug 3;14(1):101. Epub 2021 Aug 3.

Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, the Netherlands.

Aim: In order to evaluate the role of MMP-14 in ovarian cancer, a systematic review was conducted.

Methods: In March 2020, a search in Pubmed was performed with MMP-14 and ovarian cancer as search terms. After exclusion of the references not on MMP-14 or ovarian cancer or not in English, the studies found were classified into two categories: basic research and clinicopathological research.

Results: In total, 94 references were found of which 33 were excluded. Two additional articles were found in the reference lists of the included studies. Based on the full texts, another 4 were excluded. Eventually, 59 studies were included in the review, 32 on basic research and 19 on clinicopathological research. 8 studies fell in both categories. The basic research studies show that MMP-14 plays an important role in ovarian cancer in the processes of proliferation, invasion, angiogenesis and metastasis. In clinocopathological research, MMP-14 expression is found in most tumours with characteristics of poor prognosis but this immunohistochemical MMP-14 determination does not seem to be an independent predictor of prognosis.

Conclusions: From this systematic review of the literature concerning MMP-14 in ovarian cancer it becomes clear that MMP-14 plays various important roles in the pathophysiology of ovarian cancer. The exact translation of these roles in the pathophysiology to the importance of MMP-14 in clinicopathological research in ovarian cancer and possible therapeutic role of anti-MMP-14 agents needs further elucidation.
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http://dx.doi.org/10.1186/s13048-021-00852-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8336022PMC
August 2021

Regulation of fractone heparan sulfate composition in young and aged subventricular zone neurogenic niches.

Glycobiology 2021 Jul 29. Epub 2021 Jul 29.

Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Fractones, specialized extracellular matrix structures found in the subventricular zone (SVZ) neurogenic niche, can capture growth factors, such as basic fibroblast growth factor, from the extracellular milieu through a heparin-binding mechanism for neural stem cell presentation, which promotes neurogenesis. During aging, a decline in neurogenesis correlates with a change in the composition of heparan sulfate (HS) within fractones. In this study, we used antibodies that recognize specific short oligosaccharides with varying sulfation to evaluate the HS composition in fractones in young and aged brains. To further understand the conditions that regulate 6-O sulfation levels and its impact on neurogenesis, we used endosulfatase Sulf1 and Sulf2 double knock out (DKO) mice. Fractones in the SVZ of Sulf1/2 DKO mice showed immunoreactivity for the HS epitope, suggesting higher 6-O sulfation. While neurogenesis declined in the aged SVZ of both WT and Sulf1/2 DKO mice, we observed a larger number of neuroblasts in the young and aged SVZ of Sulf1/2 DKO mice. Together, these results show that the removal of 6-O-sulfation in fractones HS by endosulfatases inhibits neurogenesis in the SVZ. Our findings advance the current understanding regarding the extracellular environment that is best suited for neural stem cells to thrive, which is critical for the design of future stem cell therapies.
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http://dx.doi.org/10.1093/glycob/cwab081DOI Listing
July 2021

Low-GDP, pH-neutral solutions preserve peritoneal endothelial glycocalyx during long-term peritoneal dialysis.

Clin Exp Nephrol 2021 Sep 17;25(9):1035-1046. Epub 2021 May 17.

Department of Nephrology and Rheumatology, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan.

Background: During peritoneal dialysis (PD), solute transport and ultrafiltration are mainly achieved by the peritoneal blood vasculature. Glycocalyx lies on the surface of endothelial cells and plays a role in vascular permeability. Low-glucose degradation product (GDP), pH-neutral PD solutions reportedly offer higher biocompatibility and lead to less peritoneal injury. However, the effects on the vasculature have not been clarified.

Methods: Peritoneal tissues from 11 patients treated with conventional acidic solutions (acidic group) and 11 patients treated with low-GDP, pH-neutral solutions (neutral group) were examined. Control tissues were acquired from 5 healthy donors of kidney transplants (control group). CD31 and ratio of luminal diameter to vessel diameter (L/V ratio) were evaluated to identify endothelial cells and vasculopathy, respectively. Immunostaining for heparan sulfate (HS) domains and Ulex europaeus agglutinin-1 (UEA-1) binding was performed to assess sulfated glycosaminoglycans and the fucose-containing sugar chain of glycocalyx.

Results: Compared with the acidic group, the neutral group showed higher CD31 positivity. L/V ratio was significantly higher in the neutral group, suggesting less progression of vasculopathy. Both HS expression and UEA-1 binding were higher in the neutral group, whereas HS expression was markedly more preserved than UEA-1 binding in the acidic group. In vessels with low L/V ratio, which were found only in the acidic group, HS expression and UEA-1 binding were diminished, suggesting a loss of glycocalyx.

Conclusion: Peritoneal endothelial glycocalyx was more preserved in patients treated with low-GDP, pH-neutral solution. The use of low-GDP, pH-neutral solutions could help to protect peritoneal vascular structures and functions.
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http://dx.doi.org/10.1007/s10157-021-02078-9DOI Listing
September 2021

Growth factor mimetics for skin regeneration: In vitro profiling of primary human fibroblasts and keratinocytes.

Arch Pharm (Weinheim) 2021 Aug 8;354(8):e2100082. Epub 2021 May 8.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Small molecules have gained considerable interest in regenerative medicine, as they can effectively modulate cell fates in a spatiotemporal controllable fashion. A continuous challenge in the field represents genuine mimicry or activation of growth factor signaling with small molecules. Here, we selected and profiled three compounds for their capacity to directly or indirectly activate endogenous FGF-2, VEGF, or SHH signaling events in the context of skin regeneration. Phenotypic and functional analysis of primary skin fibroblasts and keratinocytes revealed unique, cell-specific activity profiles for the FGF-2 mimetic SUN11602 and the putative VEGF mimetic ONO-1301. Whereas SUN11602 exclusively stimulated keratinocyte differentiation, ONO-1301 mainly affected the proliferation and migration behavior of fibroblasts. In each skin cell type, both compounds selectively enhanced the expression of MMP1 and VEGFA. A combined small molecule FGF-2/VEGF mimicry may not only improve angiogenesis-related microcirculation but also reduce early fibrosis while facilitating wound remodeling at later stages. SUN11602 and ONO-1301 represent valuable tools for improving the management of difficult-to-heal wounds, particularly for the design and development of small molecule-functionalized, next-generation, engineered skin substitutes.
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http://dx.doi.org/10.1002/ardp.202100082DOI Listing
August 2021

Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage.

Mol Cell Proteomics 2021 Jun 3;20:100079. Epub 2021 Jun 3.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, Colorado, USA; Cancer Center Proteomics Core, School of Medicine, University of Colorado, Aurora, Colorado, USA. Electronic address:

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.
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http://dx.doi.org/10.1016/j.mcpro.2021.100079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188056PMC
June 2021

Abnormalities in reparative function of lung-derived mesenchymal stromal cells in emphysema.

Am J Physiol Lung Cell Mol Physiol 2021 05 3;320(5):L832-L844. Epub 2021 Mar 3.

University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Groningen, The Netherlands.

Mesenchymal stromal cells (MSCs) may provide crucial support in the regeneration of destructed alveolar tissue (emphysema) in chronic obstructive pulmonary disease (COPD). We hypothesized that lung-derived MSCs (LMSCs) from patients with emphysema are hampered in their repair capacity, either intrinsically or due to their interaction with the damaged microenvironment. LMSCs were isolated from the lung tissue of controls and patients with severe emphysema and characterized at baseline. In addition, LMSCs were seeded onto control and emphysematous decellularized lung tissue scaffolds and assessed for deposition of extracellular matrix (ECM). We observed no differences in surface markers, differentiation/proliferation potential, and expression of ECM genes between control- and COPD-derived LMSCs. Notably, COPD-derived LMSCs displayed lower expression of and messenger RNA (mRNA) and hepatocyte growth factor (HGF) and decorin protein. When seeded on control decellularized lung tissue scaffolds, control- and COPD-derived LMSCs showed no differences in engraftment, proliferation, or survival within 2  wk, with similar ability to deposit new matrix on the scaffolds. Moreover, LMSC numbers and the ability to deposit new matrix were not compromised on emphysematous scaffolds. Collectively, our data show that LMSCs from patients with COPD compared with controls show less expression of mRNA, HGF mRNA and protein, and decorin protein, whereas other features including the mRNA expression of various ECM molecules are unaffected. Furthermore, COPD-derived LMSCs are capable of engraftment, proliferation, and functioning on native lung tissue scaffolds. The damaged, emphysematous microenvironment as such does not hamper the potential of LMSCs. Thus, specific intrinsic deficiencies in growth factor production by diseased LMSCs may contribute to impaired alveolar repair in emphysema.
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http://dx.doi.org/10.1152/ajplung.00147.2020DOI Listing
May 2021

Distinct osmoregulatory responses to sodium loading in patients with altered glycosaminoglycan structure: a randomized cross-over trial.

J Transl Med 2021 01 20;19(1):38. Epub 2021 Jan 20.

Department of Internal Medicine, Section of Nephrology, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands.

Background: By binding to negatively charged polysaccharides called glycosaminoglycans, sodium can be stored in the body-particularly in the skin-without concurrent water retention. Concordantly, individuals with changed glycosaminoglycan structure (e.g. type 1 diabetes (DM1) and hereditary multiple exostosis (HME) patients) may have altered sodium and water homeostasis.

Methods: We investigated responses to acute (30-min infusion) and chronic (1-week diet) sodium loading in 8 DM1 patients and 7 HME patients in comparison to 12 healthy controls. Blood samples, urine samples, and skin biopsies were taken to investigate glycosaminoglycan sulfation patterns and both systemic and cellular osmoregulatory responses.

Results: Hypertonic sodium infusion increased plasma sodium in all groups, but more in DM1 patients than in controls. High sodium diet increased expression of nuclear factor of activated t-cells 5 (NFAT5)-a transcription factor responsive to changes in osmolarity-and moderately sulfated heparan sulfate in skin of healthy controls. In HME patients, skin dermatan sulfate, rather than heparan sulfate, increased in response to high sodium diet, while in DM1 patients, no changes were observed.

Conclusion: DM1 and HME patients show distinct osmoregulatory responses to sodium loading when comparing to controls with indications for reduced sodium storage capacity in DM1 patients, suggesting that intact glycosaminoglycan biosynthesis is important in sodium and water homeostasis. Trial registration These trials were registered with the Netherlands trial register with registration numbers: NTR4095 ( https://www.trialregister.nl/trial/3933 at 2013-07-29) and NTR4788 ( https://www.trialregister.nl/trial/4645 at 2014-09-12).
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http://dx.doi.org/10.1186/s12967-021-02700-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816310PMC
January 2021

Hypoxic Induction of Exosome Uptake through Proteoglycan-Dependent Endocytosis Fuels the Lipid Droplet Phenotype in Glioma.

Mol Cancer Res 2021 03 7;19(3):528-540. Epub 2020 Dec 7.

Department of Clinical Sciences, Section of Oncology, Lund University, Lund, Sweden.

As an adaptive response to hypoxic stress, aggressive tumors rewire their metabolic phenotype into increased malignant behavior through extracellular lipid scavenging and storage in lipid droplets (LD). However, the underlying mechanisms and potential lipid source retrieved in the hypoxic tumor microenvironment remain poorly understood. Here, we show that exosome-like extracellular vesicles (EV), known as influential messengers in the tumor microenvironment, may also serve anabolic functions by transforming hypoxic, patient-derived human glioblastoma cell lines into the LD phenotype. EVs were internalized via a hypoxia-sensitive, endocytic mechanism that fueled LD formation through direct lipid transfer, and independently of fatty acid synthase activity. EVs can enter cells through multiple and yet ill-defined pathways. On a mechanistic level, we found that hypoxia-mediated EV uptake depends on increased heparan sulfate proteoglycan (HSPG) endocytosis that preferentially followed the lipid raft pathway. The functional relevance of HSPG was evidenced by the reversal of EV-mediated LD loading by targeting of HSPG receptor function. IMPLICATIONS: Together, our data extend the multifaceted role of EVs in cancer biology by showing their LD-inducing capacity in hypoxic glioma cells. Moreover, these findings highlight a potential function for HSPG-mediated endocytosis as a salvage pathway for EV retrieval during tumor stress conditions.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0560DOI Listing
March 2021

Oncolytic Virus Therapy with HSV-1 for Hematological Malignancies.

Mol Ther 2021 02 30;29(2):762-774. Epub 2020 Sep 30.

Department of Internal Medicine, Division of Hematology, Rheumatology and Respiratory Medicine, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan. Electronic address:

Oncolytic herpes simplex virus type 1 (HSV-1) has been investigated to expand its application to various malignancies. Because hematopoietic cells are resistant to HSV-1, its application to hematological malignancies has been rare. Here, we show that the third generation oncolytic HSV-1, T-01, infected and killed 18 of 26 human cell lines and 8 of 15 primary cells derived from various lineages of hematological malignancies. T-01 replicated at low levels in the cell lines. Viral entry and the oncolytic effect were positively correlated with the expression level of nectin-1 and to a lesser extent 3-O-sulfated heparan sulfate, receptors for glycoprotein D of HSV-1, on tumor cells. Transfection of nectin-1 into nectin-1-negative tumor cells made them susceptible to T-01. The oncolytic effects did not appear to correlate with the expression or phosphorylation of antiviral molecules in the cyclic GMP-AMP (cGAS)-stimulator of interferon genes (STING) and PKR-eIF2α pathways. In an immunocompetent mouse model, intratumoral injection of T-01 into lymphoma induced regression of injected, as well as non-injected, contralateral tumors accompanied by abundant infiltration of antigen-specific CD8 T cells. These data suggest that intratumoral injection of oncolytic HSV-1 may be applicable to systemic hematological malignancies. Nectin-1 expression may be the most useful biomarker for optimal efficacy.
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http://dx.doi.org/10.1016/j.ymthe.2020.09.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854286PMC
February 2021

Construction and evaluation of an antibody phage display library targeting heparan sulfate.

Glycoconj J 2020 08 28;37(4):445-455. Epub 2020 May 28.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical center, PO Box 9101, 6500, HB, Nijmegen, the Netherlands.

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a 'single pot' phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 10 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.
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http://dx.doi.org/10.1007/s10719-020-09925-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329785PMC
August 2020

Introduction of Specific 3D Micromorphologies in Collagen Scaffolds Using Odd and Even Dicarboxylic Acids.

ACS Omega 2020 Mar 18;5(8):3908-3916. Epub 2020 Feb 18.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

The construction of scaffolds and subsequent incorporation of cells and biologics have been widely investigated to regenerate damaged tissues. Scaffolds act as a template to guide tissue formation, and their characteristics have a considerable impact on the regenerative process. Whereas many technologies exist to induce specific two-dimensional (2D) morphologies into biomaterials, the introduction of three-dimensional (3D) micromorphologies into individual pore walls of scaffolds produced from biological molecules such as collagen poses a challenge. We here report the use of dicarboxylic acids to induce specific micromorphologies in collagen scaffolds and evaluate their effect on cellular migration and differentiation. Insoluble type I collagen fibrils were suspended in monocarboxylic and dicarboxylic acids of different concentrations, and unidirectional and random pore scaffolds were constructed by freezing and lyophilization. The application of various acids and concentrations resulted in variations in 3D micromorphologies, including wall structure, wall thickness, and pore size. The use of dicarboxylic acids resulted in acid-specific micromorphologies, whereas monocarboxylic acids did not. Dicarboxylic acids with an odd or even number of C-atoms resulted in frayed/fibrillar or smooth wall structures, respectively, with varying appearances. The formation of micromorphologies was concentration-dependent. In vitro analysis indicated the cytocompatibility of scaffolds, and micromorphology-related cell behavior was indicated by enhanced myosin staining and myosin heavy chain gene expression for C2C12 myoblasts cultured on scaffolds with frayedlike micromorphologies compared to those with smooth micromorphologies. In conclusion, porous collagen scaffolds with various intrawall 3D micromorphologies can be constructed by application of dicarboxylic acids, superimposing the second level of morphology to the overall scaffold structure. Acid crystal formation is key to the specific micromorphologies observed and can be explained by the odd/even theory for dicarboxylic acids. Scaffolds with a 3D micrometer-defined topography may be used as a screening platform to select optimal substrates for the regeneration of specific tissues.
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http://dx.doi.org/10.1021/acsomega.9b03350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057318PMC
March 2020

Sustained Postnatal Skin Regeneration Upon Prenatal Application of Functionalized Collagen Scaffolds.

Tissue Eng Part A 2021 01 22;27(1-2):10-25. Epub 2020 Jan 22.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Primary closure of fetal skin in spina bifida protects the spinal cord and improves clinical outcome, but is also associated with postnatal growth malformations and spinal cord tethering. In this study, we evaluated the postnatal effects of prenatally closed full-thickness skin defects in sheep applying collagen scaffolds with and without heparin/vascular endothelial growth factor/fibroblast growth factor 2, focusing on skin regeneration and growth. At 6 months, collagen scaffold functionalized with heparin, VEGF, and FGF2 (COL-HEP/GF) resulted in a 6.9-fold increase of the surface area of the regenerated skin opposed to 1.7 × for collagen only. Epidermal thickness increased 5.7-fold at 1 month, in line with high gene expression of S100 proteins, and decreased to 2.1 at 6 months. Increased adipose tissue and reduced scaffold degradation and number of myofibroblasts were observed for COL-HEP/GF. Gene ontology terms related to extracellular matrix (ECM) organization were enriched for both scaffold treatments. In COL-HEP/GF, ECM gene expression resembled native skin. Expression of hair follicle-related genes in COL-HEP/GF was comparable to native skin, and hair follicle generation was indicated. In conclusion, closure of skin defects using functionalized collagen scaffolds resulted in long-term skin regeneration and growth. Functionalized collagen scaffolds that grow with the child may be useful for prenatal treatment of closure defects like spina bifida. Impact statement Prenatal closure of fetal skin in case of spina bifida prevents damage to the spinal cord. Closure of the defect is challenging and may result in postnatal growth malformations. In this study, the postnatal effects of a prenatally applied collagen scaffold functionalized with heparin and vascular endothelial growth factor (VEGF)/fibroblast growth factor (FGF) were investigated. An increase of the surface area of regenerated skin ("growing with the child") and generation of hair follicles was observed. Gene expression levels resembled those of native skin with respect to the extracellular matrix and hair follicles. Overall, closure of skin defects using heparin/VEGF/FGF functionalized collagen scaffolds results in long-term skin regeneration.
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http://dx.doi.org/10.1089/ten.tea.2019.0234DOI Listing
January 2021

Polymorphisms in MMP-14 and MMP-2 genes and ovarian cancer survival.

Cancer Biomark 2019 ;25(3):233-241

Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, the Netherlands.

Background: Functional polymorphisms in matrix metalloproteinases can increase or decrease the risk of cancer. This study focused on ovarian cancer and investigated how polymorphisms in the coding region of MMP-14 and the promoter region of MMP-2 are related to clinical characteristics including survival.

Methods: In 144 patients with ovarian tumours from a Caucasian population, polymorphisms of MMP-14 (+7096 and +6767) and MMP-2 (-735 and -1306) were analysed. These results were then correlated to the immunohistochemical expression of MMP-14 and MMP-2 and clinical characteristics.

Results: In these patients, the MMP-14 +7096 polymorphism showed only TT genotype, in sharp contrast to the described MAF (minimal allele frequency) C of 27%. The MMP-14 +6767 G>A polymorphism was found to have a hazard ratio of 2.09 (CI 1.00-4.35, p 0.046) for recurrence-free survival in advanced-stage patients. However, this significance disappeared after Bonferroni correction for multiple testing. No other correlations between MMP-14 and MMP-2 polymorphisms, immunohistochemistry and clinical characteristics were found, except between the MMP-2 -1306 polymorphism and differentiation grade, with a Spearman correlation coefficient of -0.19, p 0.064.

Conclusions: In ovarian cancer, the MMP-14 +6767 G>A polymorphism in the coding region seems to improve recurrence-free survival with a hazard ratio of 2.09 (CI 1.00-4.35, p 0.046). However, as this significance disappeared after correction for multiple testing, there is a need for further research on the functional effect of this change in the MMP-14 gene with larger patient sample sizes.
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http://dx.doi.org/10.3233/CBM-181826DOI Listing
January 2020

A versatile salt-based method to immobilize glycosaminoglycans and create growth factor gradients.

Glycoconj J 2019 06 4;36(3):227-236. Epub 2019 May 4.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.

Glycosaminoglycans (GAGs) are known to play pivotal roles in physiological processes and pathological conditions. To study interactions of GAGs with proteins, immobilization of GAGs is often required. Current methodologies for immobilization involve modification of GAGs and/or surfaces, which can be time-consuming and may involve specialized equipment. Here, we use an efficient and low-cost method to immobilize GAGs without any (chemical) modification using highly concentrated salt solutions. A number of salts from the Hofmeister series were probed for their capacity to immobilize heparin and chondroitin-6-sulfate on microtiter plates applying single chain antibodies against GAGs for detection (ELISA). From all salts tested, the cosmotropic salt ammonium sulfate was most efficient, especially at high concentrations (80-100% (v/v) saturation). Immobilized GAGs were bioavailable as judged by their binding of FGF2 and VEGF, and by their susceptibility towards GAG lyases (heparinase I, II and III, chondroitinase ABC). Using 80% (v/v) saturated ammonium sulfate, block and continuous gradients of heparin were established and a gradient of FGF2 was created using a heparin block gradient as a template. In conclusion, high concentrations of ammonium sulfate are effective for immobilization of GAGs and for the establishment of gradients of both GAGs and GAG-binding molecules, which enables the study to the biological roles of GAGs.
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http://dx.doi.org/10.1007/s10719-019-09872-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548755PMC
June 2019

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.

J Virol 2019 07 14;93(13). Epub 2019 Jun 14.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl. Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.
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http://dx.doi.org/10.1128/JVI.02160-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580964PMC
July 2019

Primary Ovarian Tumors With Lymphogenic and Hematogenic Metastasis Express High MMP-14, Which Colocalizes With Highly Sulfated Chondroitin Sulfate in the Stroma.

Int J Gynecol Pathol 2020 Mar;39(2):184-192

Lymphogenic and hematogenic metastases are uncommon in ovarian cancer, especially at presentation. We hypothesized that MMP-14 and MMP-2, CD44, and highly sulfated chondroitin sulfate (CS-E) may be overexpressed in tumors with these metastatic patterns. These molecules are all present in the ovarian tumor microenvironment, wherein they may interact. In an ovarian cancer cohort of 44 patients with metastases in lymph nodes, spleen, and/or liver, the presence of MMP-14, MMP-2, CD44, and CS-E in both the primary tumor and the metastases was determined with immunohistochemistry and related to clinical characteristics. Immunohistochemical expression was found for MMP-14 in all primary tumors as well as in all metastases and for MMP-2 expression in most of the samples. Most primary tumors with synchronous metastases were positive for CS-E, as well as most primary tumors with metachronous lymphogenic metastases. The expression of the MMPs and CS-E in the stroma seemed to colocalize. For CD44 immunohistochemical expression, this relationship was not found. Epithelial MMP-14 on the one hand and stromal CS-E on the other hand seem to be essential players in ovarian cancer with lymphogenic and hematogenic metastases. CD44 expression is not correlated with the other markers. More research on the interaction of these molecules and their role in the process of dissimination of disease is warranted.
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http://dx.doi.org/10.1097/PGP.0000000000000587DOI Listing
March 2020

A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships.

Nat Methods 2018 11 30;15(11):889-899. Epub 2018 Oct 30.

Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.

Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of an HS-mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell. We used this library to (1) determine that the strictly defined fine structure of HS, not its overall degree of sulfation, is more important for FGF2-FGFR1 signaling; (2) define the epitope features of commonly used anti-HS phage display antibodies; and (3) delineate the fine inter-regulation networks by which HS genes modify HS and chain length in mammalian cells at a cell-type-specific level. Our mutant-cell library will allow robust and systematic interrogation of the roles and related structures of HS in a cellular context.
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http://dx.doi.org/10.1038/s41592-018-0189-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214364PMC
November 2018

Muscle fibrosis in the soft palate: Delivery of cells, growth factors and anti-fibrotics.

Adv Drug Deliv Rev 2019 06 11;146:60-76. Epub 2018 Aug 11.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, 6500HB Nijmegen, The Netherlands. Electronic address:

The healing of skeletal muscle injuries after major trauma or surgical reconstruction is often complicated by the development of fibrosis leading to impaired function. Research in the field of muscle regeneration is mainly focused on the restoration of muscle mass while far less attention is paid to the prevention of fibrosis. In this review, we take as an example the reconstruction of the muscles in the soft palate of cleft palate patients. After surgical closure of the soft palate, muscle function during speech is often impaired by a shortage of muscle tissue as well as the development of fibrosis. We will give a short overview of the most common approaches to generate muscle mass and then focus on strategies to prevent fibrosis. These include anti-fibrotic strategies that have been developed for muscle and other organs by the delivery of small molecules, decorin and miRNAs. Anti-fibrotic compounds should be delivered in aligned constructs in order to obtain the organized architecture of muscle tissue. The available techniques for the preparation of aligned muscle constructs will be discussed. The combination of approaches to generate muscle mass with anti-fibrotic components in an aligned muscle construct may greatly improve the functional outcome of regenerative therapies for muscle injuries.
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http://dx.doi.org/10.1016/j.addr.2018.08.002DOI Listing
June 2019

Providing direction improves function: Comparison of a radial pore-orientated acellular collagen scaffold to clinical alternatives in a surgically induced rabbit diaphragmatic tissue defect model.

J Tissue Eng Regen Med 2018 11 12;12(11):2138-2150. Epub 2018 Sep 12.

Department of Development and Regeneration, Katholieke Universiteit Leuven, Leuven, Belgium.

Gore-Tex® is a widely used durable patch for repair of congenital diaphragmatic defects yet may result in complications. We compared Gore-Tex with a composite of a radial pore-orientated collagen scaffold (RP-Composite) and clinically used porcine small intestinal submucosa (SIS; Surgisis®) in a rabbit model for diaphragmatic hernia. The growing rabbit mimics the rapid rib cage growth and reherniation rates seen in children. We created and immediately repaired left hemidiaphragmatic defects in 6-week-old rabbits with Gore-Tex, SIS, and an RP-Composite scaffold. An additional group of rabbits had a sham operation. At 90 days, survivors more than doubled in weight. We observed few reherniations or eventrations in Gore-Tex (17%) and RP-Composite (22%) implanted animals. However, SIS failed in all rabbits. Maximum transdiaphragmatic pressure was lower in Gore-Tex (71%) than RP-Composite implanted animals (112%) or sham (134%). Gore-Tex repairs were less compliant than RP-Composite, which behaved as sham diaphragm (p < 0.01). RP-Composite induced less foreign body giant cell reaction than Gore-Tex (p < 0.05) with more collagen deposition (p < 0.001), although there was a tendency for the scaffold to calcify. Unlike Gore-Tex, the compliance of diaphragms reconstructed with RP-Composite scaffolds were comparable with native diaphragm, whereas reherniation rates and transdiaphragmatic pressure measurements were similar.
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http://dx.doi.org/10.1002/term.2734DOI Listing
November 2018

Self-expandable tubular collagen implants.

J Tissue Eng Regen Med 2018 06 15;12(6):1494-1498. Epub 2018 May 15.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

Collagen has been extensively used as a biomaterial, yet for tubular organ repair, synthetic polymers or metals (e.g., stents) are typically used. In this study, we report a novel type of tubular implant solely consisting of type I collagen, suitable to self-expand in case of minimal invasive implantation. Potential benefits of this collagen scaffold over conventional materials include improved endothelialization, biodegradation over time, and possibilities to add bioactive components to the scaffold, such as anticoagulants. Implants were prepared by compression of porous scaffolds consisting of fibrillar type I collagen (1.0-2.0% (w/v)). By applying carbodiimide cross-linking to the compressed scaffolds in their opened position, entropy-driven shape memory was induced. The scaffolds were subsequently crimped and dried around a guidewire. Upon exposure to water, crimped scaffolds deployed within 15-60 s (depending on the collagen concentration used), thereby returning to the original opened form. The scaffolds were cytocompatible as assessed by cell culture with human primary vascular endothelial and smooth muscle cells. Compression force required to compress the open scaffolds increased with collagen content from 16 to 32 mN for 1.0% to 2.0% (w/v) collagen scaffolds. In conclusion, we report the first self-expandable tubular implant consisting of solely type I collagen that may have potential as a biological vascular implant.
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http://dx.doi.org/10.1002/term.2685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032829PMC
June 2018

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8.

J Extracell Vesicles 2018 13;7(1):1446660. Epub 2018 Mar 13.

Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands.

Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using and stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.
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http://dx.doi.org/10.1080/20013078.2018.1446660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912193PMC
March 2018

Chemotherapeutic drug delivery by tumoral extracellular matrix targeting.

J Control Release 2018 03 31;274:1-8. Epub 2018 Jan 31.

Radboud university medical center, Radboud Institute for Molecular Life Sciences, Department of Biochemistry, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Electronic address:

Systemic chemotherapy is a primary strategy in the treatment of cancer, but comes with a number of limitations such as toxicity and unfavorable biodistribution. To overcome these issues, numerous targeting systems for specific delivery of chemotherapeutics to tumor cells have been designed and evaluated. Such strategies generally address subsets of tumor cells, still allowing the progressive growth of tumor cells not expressing the target. Moreover, tumor stem cells and tumor supportive cells, such as cancer associated fibroblasts and cancer associated macrophages, are left unaffected by this approach. In this review, we discuss an alternative targeting strategy aimed at delivery of anti-tumor drugs to the tumoral extracellular matrix with the potential to eliminate all cell types. The extracellular matrix of tumors is vastly different from that of healthy tissue and offers hooks for targeted drug delivery. It is concluded that matrix targeting is promising, but that clinical studies are required to evaluate translation.
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http://dx.doi.org/10.1016/j.jconrel.2018.01.029DOI Listing
March 2018

CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis.

Kidney Int 2018 03 21;93(3):626-642. Epub 2017 Dec 21.

Department of Nephrology, RIMLS, RIHS, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease.
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http://dx.doi.org/10.1016/j.kint.2017.09.020DOI Listing
March 2018

Unidirectional BMP2-loaded collagen scaffolds induce chondrogenic differentiation.

Biomed Mater 2017 Nov 22;13(1):015007. Epub 2017 Nov 22.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

Microfracture surgery may be improved by the implantation of unidirectional collagen scaffolds that provide a template for mesenchymal stem cells to regenerate cartilage. Incorporation of growth factors in unidirectional scaffolds may further enhance cartilage regeneration. In scaffolds, immobilization of growth factors is required to prolong in vivo activity, to limit diffusion and to reduce the amount of growth factor needed for safe clinical application. We investigated the immobilization of bone morphogenetic protein 2 (BMP2) to unidirectional collagen scaffolds and the effect on in vitro chondrogenesis. C3H10T1/2 cells were seeded on unidirectional collagen scaffolds with and without covalently attached heparin, and with and without incubation with BMP2 (1 and 10 μg), or with BMP2 present in the culture medium (10-200 ng ml). Culturing was for 2 weeks and readout parameters included histology, immunohistochemistry, biochemical analysis and molecular biological analysis. The unidirectional pores facilitated the distribution of C3H10T1/2 cells and matrix formation throughout scaffolds. The effective dose of medium supplementation with BMP2 was 100 ng ml (total exposure 1 μg BMP2), and similar production of cartilage-specific molecules chondroitin sulfate (CS) and type II collagen was found for scaffolds pre-incubated with 10 μg BMP2. Pre-incubation with 1 μg BMP2 resulted in less cartilage matrix formation. The conjugation of heparin to the scaffolds resulted in more CS and less type II collagen deposition compared to scaffolds without heparin. In conclusion, unidirectional collagen scaffolds pre-incubated with 10 μg BMP2 supported chondrogenesis in vitro and may be suitable for prolonged cartilage matrix synthesis in vivo.
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http://dx.doi.org/10.1088/1748-605X/aa8960DOI Listing
November 2017

Augmented cartilage regeneration by implantation of cellular versus acellular implants after bone marrow stimulation: a systematic review and meta-analysis of animal studies.

PeerJ 2017 27;5:e3927. Epub 2017 Oct 27.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

Bone marrow stimulation may be applied to regenerate focal cartilage defects, but generally results in transient clinical improvement and formation of fibrocartilage rather than hyaline cartilage. Tissue engineering and regenerative medicine strive to develop new solutions to regenerate hyaline cartilage tissue. This systematic review and meta-analysis provides a comprehensive overview of current literature and assesses the efficacy of articular cartilage regeneration by implantation of cell-laden versus cell-free biomaterials in the knee and ankle joint in animals after bone marrow stimulation. PubMed and EMBASE (via OvidSP) were systematically searched using tissue engineering, cartilage and animals search strategies. Included were primary studies in which cellular and acellular biomaterials were implanted after applying bone marrow stimulation in the knee or ankle joint in healthy animals. Study characteristics were tabulated and outcome data were collected for meta-analysis for studies applying semi-quantitative histology as outcome measure (117 studies). Cartilage regeneration was expressed on an absolute 0-100% scale and random effects meta-analyses were performed. Implantation of cellular biomaterials significantly improved cartilage regeneration by 18.6% compared to acellular biomaterials. No significant differences were found between biomaterials loaded with stem cells and those loaded with somatic cells. Culture conditions of cells did not affect cartilage regeneration. Cartilage formation was reduced with adipose-derived stem cells compared to other cell types, but still improved compared to acellular scaffolds. Assessment of the risk of bias was impaired due to incomplete reporting for most studies. Implantation of cellular biomaterials improves cartilage regeneration compared to acellular biomaterials.
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http://dx.doi.org/10.7717/peerj.3927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661456PMC
October 2017

Sequencing of glycosaminoglycans with potential to interrogate sequence-specific interactions.

Sci Rep 2017 11 1;7(1):14785. Epub 2017 Nov 1.

Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.

Technologies to sequence nucleic acids/proteins are widely available, but straightforward methodologies to sequence complex polysaccharides are lacking. We here put forward a strategy to sequence glycosaminoglycans, long linear polysaccharides involved in many biochemical processes. The method is based on the covalent immobilization and (immuno)chemical characterization of only those size-separated saccharides that harbor the original reducing end of the full-length chain. Using this methodology, the saccharide sequence of the chondroitin sulfate chain of the proteoglycan bikunin was determined. The method can be performed in any standard biochemical lab and opens studies to the interaction of complex saccharide sequences with other biomolecules.
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http://dx.doi.org/10.1038/s41598-017-15009-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665995PMC
November 2017

Desulfation of Heparan Sulfate by Sulf1 and Sulf2 Is Required for Corticospinal Tract Formation.

Sci Rep 2017 10 23;7(1):13847. Epub 2017 Oct 23.

Department of Molecular Neurobiology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Ibaraki, 305-8575, Japan.

Heparan sulfate (HS) has been implicated in a wide range of cell signaling. Here we report a novel mechanism in which extracellular removal of 6-O-sulfate groups from HS by the endosulfatases, Sulf1 and Sulf2, is essential for axon guidance during development. In Sulf1/2 double knockout (DKO) mice, the corticospinal tract (CST) was dorsally displaced on the midbrain surface. In utero electroporation of Sulf1/2 into radial glial cells along the third ventricle, where Sulf1/2 mRNAs are normally expressed, rescued the CST defects in the DKO mice. Proteomic analysis and functional testing identified Slit2 as the key molecule associated with the DKO phenotype. In the DKO brain, 6-O-sulfated HS was increased, leading to abnormal accumulation of Slit2 protein on the pial surface of the cerebral peduncle and hypothalamus, which caused dorsal repulsion of CST axons. Our findings indicate that postbiosynthetic desulfation of HS by Sulfs controls CST axon guidance through fine-tuning of Slit2 presentation.
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http://dx.doi.org/10.1038/s41598-017-14185-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653861PMC
October 2017
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