Mimicking the micro-environment. Construction and evaluation of complex collagen-based scaffolds for tissue engineering

Contains fulltext : 99159.pdf (publisher's version ) (Open Access)The ultimate goal in tissue engineering is to create biomaterials that mimic normal tissue or are able to encourage cells cells to generate new functional tissue. For these biomaterials, it is important to use highly purified components in order to moderate the tissue response and to control the regeneration process. Using type I collagen, elastin, glycosaminoglycans and a variety of growth factors as basic components, a wide variety of biomaterials can be prepared. The purpose of this thesis is to design, develop and evaluate a new molecularly defined skin construct that closely mimics normal tissue. A biologically active growth factor was made for that reason. Different biomaterials were developed and tested in laboratory animals to evaluate tissue reaction, calcification and vascularization. Following these results, a skin construct was made and analyzed in an animal model for acute wounds. First results are promising for future development.Radboud Universiteit Nijmegen, 19 oktober 2012Promotores : Brock, R.E., Schalkwijk, J. Co-promotores : Daamen, W.F., Kuppevelt, A.H. va

Depots of solubilised elastin promote the formation of blood vessels and elastic fibres in rat.

Contains fulltext : 49565.pdf (publisher's version ) (Open Access

Cloning, large-scale production, and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164).

Contains fulltext : 89025.pdf (publisher's version ) (Closed access)Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale production was performed by fermentation. cDNA encoding VEGF-164 was prepared from embryonic rat tissue RNA, and a recombinant pPIC9HV/rVEGF-164 plasmid, containing an AOX1 promoter, was constructed. The methylotrophic P. pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation ( approximately 124mg/L) and purified by heparin affinity chromatography. SDS-PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48kDa which was purified to near homogeneity by heparin affinity chromatography in a large quantity. A bioassay indicated a three- to fivefold increase in endothelial cell proliferation after 3days, due to the addition of the produced rrVEGF-164. The produced rrVEGF-164 showed a higher biological activity than a commercially available, mouse cell line-based, growth factor. In conclusion, using the P. pastoris expression system we were able to produce biologically active rat VEGF-164 in high quantities and this may provide a powerful tool for basic and applied life sciences.1 januari 201

Increased angiogenesis and blood vessel maturation in acellular collagen-heparin scaffolds containing both FGF2 and VEGF.

Contains fulltext : 51608schalkwijk.pdf (publisher's version ) (Closed access)An important issue in tissue engineering is the vascularisation of the implanted construct, which often takes several weeks. In vivo, the growth factors VEGF and FGF2 show a combined effect on both angiogenesis and maturation of blood vessels. Therefore, we hypothesise that the addition of these growth factors to an acellular construct increases blood vessel formation and maturation. To systematically evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, five porous scaffolds were prepared and characterised, viz.: collagen, collagen with heparin, and collagen with heparin plus one or two growth factors (rrFGF2 and rrVEGF). Scaffolds were subcutaneously implanted in 3 months old Wistar rats. Of all scaffolds tested, the one with a combination of growth factors displayed the highest density of blood vessels (type IV collagen) and most mature blood vessels (smooth muscle actin). In addition, no hypoxic cells were found in this scaffold at day 7 and 21 (hypoxia inducible factor 1-alpha). These results indicate that the addition of both FGF2 and VEGF to an acellular construct enhances an early mature vasculature. This opens prospects for (acellular) tissue-engineered constructs in conditions as ischaemic heart disease or diabetic ulcers

Increased angiogenesis in acellular scaffolds by combined release of FGF2 and VEGF.

Item does not contain fulltex

Tissue response of defined collagen-elastin scaffolds in young and adult rats with special attention to calcification.

Contains fulltext : 47698.pdf (publisher's version ) (Closed access)Collagen-elastin scaffolds may be valuable biomaterials for tissue engineering because they combine tensile strength with elasticity. In this study, the tissue response to and the calcification of these scaffolds were evaluated. In particular, the hypothesis was tested that calcification, a common phenomenon in biomaterials, may be due to microfibrils within the elastic fibre, and that these microfibrils might generate a tissue response. Four scaffolds were subcutaneously implanted, viz. collagen, collagen + pure elastin, collagen+microfibril-containing, and collagen + pulverised elastic ligament (the source for elastin). Explants were evaluated at day 3, 7 and 21. In young Sprague Dawley rats, collagen + ligament calcified substantially, whereas collagen + elastin (with and without microfibrils) calcified less, and collagen did not. Calcification started at elastic fibres. In both Sprague Dawley and Wistar adult rats, however, none of the scaffolds calcified. Mononuclear cell infiltration was prominent in young and adult Sprague Dawley rats. In adult Wistar rats, this infiltration was associated with the presence of microfibrils. Degradation of scaffolds and new matrix formation were related with cellular influx and degree of vascularisation. In conclusion, absence of microfibrils from the elastic fibre does not prevent calcification in young Sprague Dawley rats, but does reduce the tissue response in adult Wistar rats. Cellular response and calcification differs with age and strain and therefore the choice of animal model is of key importance in biomaterial evaluation

Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruch's membrane

Item does not contain fulltextPseudoxanthoma elasticum (PXE) is a heritable disorder characterized by mineralization of connective tissue, which leads to pathology in eye, skin and blood vessels. The disease is caused by mutations in ABCC6. To learn more about PXE eye pathology, we analyzed Bruch's membrane (BM) of the eye of an Abcc6 knockout mouse. With age, BM differences between Abcc6-/- and wild type mice became apparent. At two years of age, von Kossa staining indicated clear calcification of BM in Abcc6-/- mice, and not in healthy controls. Electron microscopy revealed BM changes as early as at 10 months of age: Fibrous structures with abnormal high electron-density were present in the central layers of BM of Abcc6-/- mice. EDX (Energy Dispersive X-ray) analysis demonstrated that these structures contained elevated levels of Ca, P and O. Since some of these electron-dense structures showed a banding pattern with periodicity of about 50 nm, they most likely represent calcified collagen fibers. Immunoelectron microscopy showed that the calcified structures were positive for collagen III. Remarkably, the elastic layer of BM appeared to have a normal ultrastructure, even in 2.5 year old Abcc6-/- mice. Our results suggest that Abcc6 deficiency in the mouse causes calcification of BM. While PXE is considered to affect primarily the elastic fibers, we found predominantly mineralization of collagen fibers

An overview of methods for the in vivo evaluation of tissue-engineered skin constructs

Item does not contain fulltextCutaneous wounding often leads to contraction and scarring, which may result in a range of functional, cosmetic, and psychological complications. Tissue-engineered skin substitutes are being developed to enhance restoration of the skin and improve the quality of wound healing. The aim of this review is to provide researchers in the field of tissue engineering an overview of the methods that are currently used to clinically evaluate skin wound healing, and methods that are used to evaluate tissue-engineered constructs in animal models. Clinically, the quality of wound healing is assessed by noninvasive subjective scar assessment scales and objective techniques to measure individual scar features. Alternatively, invasive technologies are used. In animal models, most tissue-engineered skin constructs studied are at least evaluated macroscopically and by using conventional histology (hematoxylin-eosin staining). Planimetry and immunohistochemistry are also often applied. An overview of antibodies used is provided. In addition, some studies used methods to assess gene expression levels and mRNA location, transillumination for blood vessel observation, in situ/in vivo imaging, electron microscopy, mechanical strength assessment, and microbiological sampling. A more systematic evaluation of tissue-engineered skin constructs in animal models is recommended to enhance the comparison of different constructs, thereby accelerating the trajectory to application in human patients. This would be further enhanced by the embracement of more clinically relevant objective evaluation methods. In addition, fundamental knowledge on construct-mediated wound healing may be increased by new developments in, for example, gene expression analysis and noninvasive imaging

High density gene expression microarrays and gene ontology analysis for identifying processes in implanted tissue engineering constructs.

Contains fulltext : 88354.pdf (publisher's version ) (Closed access)The in vivo performance of tissue-engineered constructs is often based on generally accepted read-out parameters, like (immuno)histology. In this study, high-density gene expression microarrays and gene ontology (GO) analysis were used as a read-out tool to identify the biological processes occurring after implantation of an acellular collagen-based skin construct using a rat full-thickness wound model. A freely-available program (DAVID) was used to identify up/downregulated biological processes (GO-terms) and results were compared to wound healing/regeneration without a construct. The entire process from RNA isolation to biological interpretation is explained step-by-step. Conventional (immuno)histology was used to validate the biological processes identified and indicate that microarray analysis may provide a valuable, fast and unbiased tool to evaluate the in vivo performance of tissue-engineered constructs. However, challenges remain e.g. with regards to the development of specific GO-terms and annotation of the (rat) genome.01 november 201

Familial Kleefstra syndrome due to maternal somatic mosaicism for interstitial 9q34.3 microdeletions

Item does not contain fulltextThe Kleefstra syndrome (Online Mendelian Inheritance in Man 607001) is caused by a submicroscopic 9q34.3 deletion or by intragenic euchromatin histone methyl transferase 1 (EHMT1) mutations. So far only de novo occurrence of mutations has been reported, whereas 9q34.3 deletions can be either de novo or caused by complex chromosomal rearrangements or translocations. Here we give the first descriptions of affected parent-to-child transmission of Kleefstra syndrome caused by small interstitial deletions, approximately 200 kb, involving part of the EHMT1 gene. Additional genome-wide array studies in the parents showed the presence of similar deletions in both mothers who only had mild learning difficulties and minor facial characteristics suggesting either variable clinical expression or somatic mosaicism for these deletions. Further studies showed only one of the maternal deletions resulted in significantly quantitative differences in signal intensity on the array between the mother and her child. But by investigating different tissues with additional fluorescent in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) analyses, we confirmed somatic mosaicism in both mothers. Careful clinical and cytogenetic assessments of parents of an affected proband with an (interstitial) 9q34.3 microdeletion are merited for accurate estimation of recurrence risk