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Rob Janssen,1 Emiel FM Wouters,2 Wim Janssens,3 Willeke F Daamen,4 Paul Hagedoorn,5 Hugo AJM de Wit,6 Jef Serré,3 Ghislaine Gayan-Ramirez,3 Frits ME Franssen,2 Niki L Reynaert,2 Jan H von der Thüsen,7 Henderik W Frijlink5 1Department of Pulmonary Medicine, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands; 2Department of Respiratory Medicine, Maastricht University Medical Center+, Maastricht, The Netherlands; 3Laboratory of Respiratory Diseases, Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), KU Leuven, Leuven, Belgium; 4Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands; 5Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute of Pharmacy, Groningen, University of Groningen, Groningen, The Netherlands; 6Department of Clinical Pharmacy, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands; 7Department of Pathology, Erasmus Medical Center, Rotterdam, The NetherlandsCorrespondence: Rob JanssenDepartment of Pulmonary Medicine, Canisius-Wilhelmina Hospital, Weg Door Jonkerbos 100, Nijmegen, SZ 6532, The NetherlandsEmail rob.janssen@cwz.nlAbstract: Current pharmacotherapy of chronic obstructive pulmonary disease (COPD) aims at reducing respiratory symptoms and exacerbation frequency. Effective therapies to reduce disease progression, however, are still lacking. Furthermore, COPD medications showed less favorable effects in emphysema than in other COPD phenotypes. Elastin fibers are reduced and disrupted, whereas collagen levels are increased in emphysematous lungs. Protease/antiprotease imbalance has historically been regarded as the sole cause of emphysema. However, it is nowadays appreciated that emphysema may also be provoked by perturbations in the sequential repair steps following elastolysis. Essentiality of fibulin-5 and lysyl oxidase-like 1 in the elastin restoration process is discussed, and it is argued that copper deficiency is a plausible reason for failing elastin repair in emphysema patients. Since copper-dependent lysyl oxidases crosslink elastin as well as collagen fibers, copper supplementation stimulates accumulation of both proteins in the extracellular matrix. Restoration of abnormal elastin fibers in emphysematous lungs is favorable, whereas stimulating pulmonary fibrosis formation by further increasing collagen concentrations and organization is detrimental. Heparin inhibits collagen crosslinking while stimulating elastin repair and might therefore be the ideal companion of copper for emphysema patients. Efficacy and safety considerations may lead to a preference of pulmonary administration of copper-heparin over systemic administration.Keywords: collagen, copper, desmosine, elastin, emphysema, fibulin-5, heparin, lysyl oxidase, lysyl oxidase-like protein 1, tropoelastin

Elastin, a fibrous extracellular matrix (ECM) protein, is the main component of elastic fibers that are involved in tissues' elasticity and resilience, enabling them to undergo reversible extensibility and to endure repetitive mechanical stress. After wounding, it is challenging to regenerate elastic fibers and biomaterials developed thus far have struggled to induce its biosynthesis. This review provides a comprehensive summary of elastic fibers synthesis at the cellular level and its implications for biomaterial formulation, with a particular focus on dermal substitutes. The review delves into the intricate process of elastogenesis by cells and investigates potential triggers for elastogenesis encompassing elastin-related compounds, ECM components, and other molecules for their potential role in inducing elastin formation. Understanding of the elastogenic processes is essential for developing biomaterials that trigger not only the synthesis of the elastin protein, but also the formation of a functional and branched elastic fiber network., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Background: The developmental abnormality spina bifida is hallmarked by missing tissues (e.g. skin) and exposure of the spinal cord to the amniotic fluid, which can negatively impact neurological development. Surgical closure of the skin in utero limits neurological damage, but in large defects this results in scarring and contractures. Stimulating skin regeneration in utero would greatly benefit treatment outcome. Previously, we demonstrated that a porous type I collagen (COL) scaffold, functionalized with heparin (HEP), fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) (COL-HEP/GF) improved pre- and postnatal skin regeneration in a fetal sheep full thickness wound model. In this study we uncover the early events associated with enhanced skin regeneration., Methods: We investigated the gene expression profiles of healing fetal skin wounds two weeks after implantation of the COL(-HEP/GF) scaffolds. Using laser dissection and microarrays, differentially expressed genes (DEG) were identified in the epidermis and dermis between untreated wounds, COL-treated wounds and wounds treated with COL-HEP/GF. Biological processes were identified using gene enrichment analysis and DEG were clustered using protein-protein-interaction networks., Results: COL-HEP/GF influences various interesting biological processes involved in wound healing. Although the changes were modest, using protein-protein-interaction networks we identified a variety of clustered genes that indicate COL-HEP/GF induces a tight but subtle control over cell signaling and extracellular matrix organization., Conclusion: These data offer a novel perspective on the key processes involved in (fetal) wound healing, where a targeted and early interference during wound healing can result in long-term enhanced effects on skin regeneration., (© 2024. The Author(s).)

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs., (© The Author(s) 2024. Published by Oxford University Press.)

Achieving regeneration in humans has been a long-standing goal of many researchers. Whereas amphibians like the axolotl ( Ambystoma mexicanum ) are capable of regenerating whole organs and even limbs, most mammals heal their wounds via fibrotic scarring. Recently, the African spiny mouse ( Acomys sp.) has been shown to be injury resistant and capable of regenerating several tissue types. A major focal point of research with Acomys has been the identification of drivers of regeneration. In this search, the matrisome components related to the extracellular matrix (ECM) are often overlooked. In this review, we compare Acomys and axolotl skin wound healing and blastema-mediated regeneration by examining their wound healing responses and comparing the expression pattern of matrisome genes, including glycosaminoglycan (GAG) related genes. The goal of this review is to identify matrisome genes that are upregulated during regeneration and could be potential candidates for inclusion in pro-regenerative biomaterials. Research papers describing transcriptomic or proteomic coverage of either skin regeneration or blastema formation in Acomys and axolotl were selected. Matrisome and GAG related genes were extracted from each dataset and the resulting lists of genes were compared. In our analysis, we found several genes that were consistently upregulated, suggesting possible involvement in regenerative processes. Most of the components have been implicated in regulation of cell behavior, extracellular matrix remodeling and wound healing. Incorporation of such pro-regenerative factors into biomaterials may help to shift pro-fibrotic processes to regenerative responses in treated wounds.

Objectives: Fetal endoscopic tracheal occlusion (FETO) improves neonatal survival of fetuses with congenital diaphragmatic hernia (CDH). However, FETO also increases the risk of preterm prelabor rupture of membranes (PPROM) and preterm delivery (PTD), as fetal membrane defects after fetoscopy do not heal. To solve this issue, an advanced sealing plug for closing the membrane defect is being developed. Using early-stage health economic modeling, we aimed to estimate the potential value of this innovative plug in terms of costs and effects, and to determine the properties required for it to become cost-effective., Methods: Early-stage health economic modeling was applied to the case of performing FETO in women with a singleton pregnancy whose fetus is diagnosed prenatally with CDH. We simulated a cohort of patients using a state-transition model over a 45-year time horizon. In our best-case-scenario analysis, we compared the current-care strategy with the perfect-plug strategy, which reduces the risk of PPROM and PTD by 100%, to determine the maximum quality-adjusted life years (QALYs) gained and costs saved. Using threshold analysis, we determined the minimum percentage reduction in the risk of PPROM and PTD required for the plug to be considered cost-effective. The impact of model parameters on outcome was investigated using a sensitivity analysis., Results: Our model indicated that a perfect-plug strategy would yield on average an additional 1.94 QALYs at a cost decrease of €2554 per patient. These values were influenced strongly by the percentage of cases with early PTD (27-34 weeks). Threshold analysis showed that, for €500 per plug, the plug strategy needs a minimum percentage reduction of 1.83% in the risk of PPROM and PTD (i.e. reduction in the risk from 47.50% to 46.63% for PPROM and from 71.50% to 70.19% for PTD) to be cost-effective., Conclusions: Our model-based approach showed clear potential of the plug strategy when applied in the context of FETO for CDH fetuses, as only a minor reduction in the risk of PPROM and PTD is needed for the plug to be cost-effective. Its value is expected to be even higher when used in conditions associated with a higher rate of early PTD. Continued investment in research and development of the plug strategy appears to provide value for money. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology., (© 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.)

The process of wound healing is a tightly controlled cascade of events, where severe skin wounds are resolved via scar tissue. This fibrotic response may be diminished by applying anti-fibrotic factors to the wound, thereby stimulating regeneration over scarring. The development of tunable biomaterials that enable spatiotemporal control over the release of anti-fibrotics would greatly benefit wound healing. Herein, harnessing the power of click-to-release chemistry for regenerative medicine, we demonstrate the feasibility of such an approach. For this purpose, one side of a bis-N-hydroxysuccinimide-trans-cyclooctene (TCO) linker was functionalized with human epidermal growth factor (hEGF), an important regulator during wound healing, whereas on the other side a carrier protein was conjugated-either type I collagen scaffolds or bovine serum albumin (BSA). Mass spectrometry demonstrated the coupling of hEGF-TCO and indicated a release following exposure to dimethyl-tetrazine. Type I collagen scaffolds could be functionalized with the hEGF-TCO complex as demonstrated by immunofluorescence staining and Western blotting. The hEGF-TCO complex was also successfully ligated to BSA and the partial release of hEGF upon dimethyl-tetrazine exposure was observed through Western blotting. This work establishes the potential of click-to-release chemistry for the development of pro-regenerative biomaterials.

In our aging society, the number of patients suffering from poorly healing bone defects increases. Bone morphogenetic proteins (BMPs) are used in the clinic to promote bone regeneration. However, poor control of BMP delivery and thus activity necessitates high doses, resulting in adverse effects and increased costs. It has been demonstrated that messenger RNA (mRNA) provides a superior alternative to protein delivery due to local uptake and prolonged expression restricted to the site of action. Here, we present the development of porous collagen scaffolds incorporating peptide-mRNA nanoparticles (NPs). Nanoparticles were generated by simply mixing aqueous solutions of the cationic cell-penetrating peptide PepFect14 (PF14) and mRNA. Peptide-mRNA complexes were uniformly distributed throughout the scaffolds, and matrices fully preserved cell attachment and viability. There was a clear dependence of protein expression on the incorporated amount of mRNA. Importantly, after lyophilization, the mRNA formulation in the collagen scaffolds retained activity also at 4 °C over two weeks. Overall, our results demonstrate that collagen scaffolds incorporating peptide-mRNA complexes hold promise as off-the-shelf functional biomaterials for applications in regenerative medicine and constitute a viable alternative to lipid-based mRNA formulations.

Iatrogenic preterm premature rupture of fetal membranes (iPPROM) after fetal surgery remains a strong trigger for premature birth. As fetal membrane defects do not heal spontaneously and amniotic fluid leakage and chorioamniotic membrane separation may occur, we developed a biocompatible, fetoscopically-applicable collagen plug with shape memory to prevent leakage. This plug expands directly upon employment and seals fetal membranes, hence preventing amniotic fluid leakage and potentially iPPROM. Lyophilized type I collagen plugs were given shape memory and crimped to fit through a fetoscopic cannula (Ø 3 mm). Expansion of the plug was examined in phosphate buffered saline (PBS). Its sealing capacity was studied ex vivo using human fetal membranes, and in situ in a porcine bladder model. The crimped plug with shape memory expanded and tripled in diameter within 1 min when placed into PBS, whereas a crimped plug without shape memory did not. In both human fetal membranes and porcine bladder, the plug expanded in the defect, secured itself and sealed the defect without membrane rupture. In conclusion, collagen plugs with shape memory are promising as medical device for rapid sealing of fetoscopic defects in fetal membranes at the endoscopic entry point., Competing Interests: WD, RM, and TvK are mentioned as inventors on related patent applications. Other authors: no conflicts of interest., (© 2022 The Authors.)

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 in situ 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 in vitro 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sturm, Quinterot, Guyot, Righini, Daamen, van Kuppevelt and Huard.)

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., (© 2021 The Authors. Archiv der Pharmazie Published by Wiley-VCH Verlag GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

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 FGF10 and HGF 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 FGF10 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.

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 de novo hair follicle generation was indicated. In conclusion, in utero 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, in utero closure of skin defects using heparin/VEGF/FGF functionalized collagen scaffolds results in long-term skin regeneration.

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., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

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 6 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.

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., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Type I collagen scaffolds for tissue reconstruction often have impaired mechanical characteristics such as limited stiffness and lack of strength. In this study, a new technique is presented to fine-tune stiffness and biodegradability of collagen scaffolds by treatment with concentrated salt solutions. Collagen scaffolds were prepared by a casting, freezing and lyophilization process. Scaffolds were treated with 90% saturated salt solutions, the salts taken from the Hofmeister series, followed by chemical crosslinking. Treatment with salts consisting of a divalent cation in combination with a monovalent anion, e.g. CaCl 2 , resulted in fast shrinkage of the scaffolds up to approximately 10% of the original surface area. Effective salts were mostly at the chaotropic end of the Hofmeister series. Shrunken scaffolds were more than 10 times stiffer than non-shrunken control scaffolds, and displayed reduced pore sizes and swollen, less organized collagen fibrils. The effect could be pinpointed to the level of individual collagen molecules and indicates the shrinking effect to be driven by disruption of stabilizing hydrogen bonds within the triple helix. No calcium deposits remained in CaCl 2 treated scaffolds. Subcutaneous implantation in rats showed similar biocompatibility compared to H 2 O and NaCl treated scaffolds, but reduced cellular influx and increased structural integrity without signs of major degradation after 3 months. In conclusion, high concentrations of chaotropic salts can be used to adjust the mechanical characteristics of collagen scaffolds without affecting biocompatibility. This technique may be used in regenerative medicine to stiffen collagen scaffolds to better comply with the surrounding tissues, but may also be applied for e.g. slow release drug delivery systems., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

For many years elastin was considered as the matrix component structurally required to provide tissue elasticity. However, the expanded knowledge on the regulation of connective tissue homeostasis has revealed that elastic fibers also represent a source of elastokines and are the target of a number of signaling pathways mainly involving the TGF-β/BMP axis. A better understanding of these complex regulatory networks may pave the way for targeted therapeutic strategies in a number of genetic as well as acquired diseases and for the development of new functionalized biomaterials., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Iron-containing metallic implants are shown herein to mediate hydrolysis of glycosidic linkages. Using glucuronide prodrugs for broad-spectrum fluoroquinolone antibacterial agents, we capitalize on this behaviour and perform localized synthesis of antimicrobials which affords a significant zone of inhibition of bacterial growth around the metallic material.

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., (© 2018 John Wiley & Sons, Ltd.)

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.

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., (© 2018 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.)

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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Tissue engineering may become an alternative to current bladder augmentation techniques. Large scaffolds are needed for clinically significant augmentation, but can result in fibrosis and graft shrinkage. The purpose of this study was to investigate the use of multiple scaffolds instead of one large scaffold, to enhance bladder tissue regeneration and bladder capacity. Second, acellular collagen, collagen-heparin, and collagen-heparin scaffolds with growth factors (GFs) were used and the biological activity of the different scaffolds was compared in a large animal model., Materials and Methods: Scaffolds were made of bovine type I collagen with or without heparin (Ø = 3.2 cm). Collagen-heparin scaffolds were loaded with GFs, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and heparin-binding epidermal growth factor (HB-EGF). Three identical scaffolds prepared from collagen (COL-group), collagen with heparin (COLHEP-group), or collagen-heparin with growth factors (COLHEPGF-group) were implanted in one porcine bladder. The outcome was compared with sham-operated animals (Sham-group), in which no scaffold was used. Urodynamic evaluation was performed before surgery and 3 months after bladder reconstruction, together with histological evaluation., Results: Survival rate was 92%, 12 animals completed the study, 3 of every group, 1 animal developed peritonitis due to urine leakage and was sacrificed. The regenerated area was largest in the COLHEP-group, and least in the COL-group (p = 0.002). Histological evaluation revealed a normal urothelial layer and good angiogenesis in all groups, and comparable ingrowth of smooth muscle cells. Urodynamics showed no statistically significant differences in bladder capacity and compliance between groups. Bladder capacity and compliance was very high in this animal model, which made it impossible to study the increase due to augmentation., Conclusions: Implantation of multiple collagen-heparin scaffolds in one bladder is feasible in a porcine model, resulting in tissue almost indistinguishable from native tissue involving all cell layers of the bladder. Collagen scaffolds with heparin incorporated resulted in a larger area of regenerated tissue. To reach clinically significant augmentation, multiple larger collagen-heparin scaffolds, with or without GFs, need to be tested to study the largest possible diameter of scaffold and number of used scaffolds still resulting in well-vascularized tissue.

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 -1 ). 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 -1 (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.

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.

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., Competing Interests: The authors declare there are no competing interests.

Context: Urethra repair by tissue engineering has been extensively studied in laboratory animals and patients, but is not routinely used in clinical practice., Objective: To systematically investigate preclinical and clinical evidence of the efficacy of tissue engineering for urethra repair in order to stimulate translation of preclinical studies to the clinic., Evidence Acquisition: A systematic search strategy was applied in PubMed and EMBASE. Studies were independently screened for relevance by two reviewers, resulting in 80 preclinical and 23 clinical studies of which 63 and 13 were selected for meta-analysis to assess side effects, functionality, and study completion. Analyses for preclinical and clinical studies were performed separately. Full circumferential and inlay procedures were assessed independently. Evaluated parameters included seeding of cells and type of biomaterial., Evidence Synthesis: Meta-analysis revealed that cell seeding significantly reduced the probability of encountering side effects in preclinical studies. Remarkably though, cells were only sparsely used in the clinic (4/23 studies) and showed no significant reduction of side effects. ln 21 out of 23 clinical studies, decellularized templates were used, while in preclinical studies other biomaterials showed promising outcomes as well. No direct comparison to current clinical practice could be made due to the limited number of randomized controlled studies., Conclusions: Due to a lack of controlled (pre)clinical studies, the efficacy of tissue engineering for urethra repair could not be determined. Meta-analysis outcome measures were similar to current treatment options described in literature. Surprisingly, it appeared that favorable preclinical results, that is inclusion of cells, were not translated to the clinic. Improved (pre)clinical study designs may enhance clinical translation., Patient Summary: We reviewed all available literature on urethral tissue engineering to assess the efficacy in preclinical and clinical studies. We show that improvements to (pre)clinical study design is required to improve clinical translation of tissue engineering technologies., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

The use of bowel tissue for urinary diversion can be associated with severe complications, and regenerative medicine may circumvent this by providing an engineered conduit. In this study, a novel tubular construct was identified for this purpose. Three constructs (diameter 15 mm) were prepared from type I collagen and either (a) a semi-biodegradable Vypro II polymer (COL-Vypro), (b) a rapidly biodegradable Vicryl polymer (COL-Vicryl) or (c) an additional collagenous layer (COL-DUAL). After freezing, lyophilization and crosslinking, all constructs showed a porous structure with a two-fold higher strength for the polymer-containing constructs. These constructs were connected to full bladder defects of 11 female pigs and evaluated after 1 (n = 4) or 3 months (n = 5). With respect to surgical handling, the polymer-containing constructs were superior. All pigs voided normally without leakage and the survival rate was 82%. For the implanted COL-Vypro constructs (8/9), stone formation was observed. COL-DUAL and COL-Vicryl showed better biocompatibility and only small remnants were found 1 month post-implantation. Histological and immunohistochemical analysis showed the best regeneration for COL-Vicryl with respect to urothelium; muscle pedicles and elastin formation were best developed in the COL-Vicryl constructs. In this study, COL-Vicryl constructs were superior in both biocompatibility and bladder tissue regeneration and have high potential for artificial urinary diversions. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Purpose: Implantation of pre-endothelialized stents could enhance cellular recovery of a damaged vessel wall provided attached cells remain viable, functional and are present in sufficient numbers after deployment. The purpose of this study was to evaluate the feasibility of grooved stainless steel (SS) stents as a primary endothelial cell (EC) carrier with potentially enhanced EC protection upon stent deployment., Materials and Methods: Attachment and behavior of enzymatically harvested human adult venous ECs seeded onto gelatin-coated vascular stents were evaluated in an in vitro setting. Smooth and grooved SS stents and smooth nitinol stents were studied., Results: All cells expressed EC markers vWF and CD31. Using rotational seeding for a period of 16-24 h, ECs attached firmly to the stents with sufficient coverage to form a confluent EC monolayer. The grooved SS wire design was found to enable attachment of three times the number of cells compared to smooth wires. This also resulted in an increased number of cells remaining on the stent after deployment and after pulsatile flow of 180 ml/min for 24 h, which did not result in additional EC detachment., Conclusions: The grooved stent provides a potential percutaneous means to deliver sufficient numbers of viable and functional cells to a vessel segment during vascular intervention. The grooves were found to offer a favorable surface for EC attachment and protection during stent deployment in an in vitro setting.

Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed., (Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.)

Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy. Here, we report on the functionalization of drug-loaded lyophilisomes (albumin-based biocapsules) to specifically target the stroma of ovarian carcinomas with the potential to eliminate cancer cells. To achieve specific targeting, we conjugated single chain antibodies reactive with CS-E to lyophilisomes using a two-step approach comprising sortase-mediated ligation and bioorthogonal click chemistry. Antibody-functionalized lyophilisomes specifically targeted the ovarian cancer stroma through CS-E. In a CS-E rich micro-environment in vitro lyophilisomes induced cell death by extracellular release of doxorubicin which localized to the nucleus. Immunohistochemistry identified CS-E rich stroma in a variety of solid tumors other than ovarian cancer, including breast, lung and colon cancer indicating the potential versatility of matrix therapy and the use of highly sulfated chondroitin sulfates in cancer stroma as a micro-environmental hook for targeted drug delivery., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Tubular collagen scaffolds have been used for the repair of damaged hollow organs in regenerative medicine, but they generally lack the ability to reversibly expand in radial direction, a physiological characteristic seen in many native tubular organs. In this study, tubular collagen scaffolds were prepared that display a shape recovery effect and therefore exhibit radial elasticity. Scaffolds were constructed by compression of fibrillar collagen around a star-shaped mandrel, mimicking folds in a lumen, a typical characteristic of empty tubular hollow organs, such as ureter or urethra. Shape recovery effect was introduced by in situ fixation using a star-shaped mandrel, 3D-printed clamps and cytocompatible carbodiimide crosslinking. Prepared scaffolds expanded upon increase of luminal pressure and closed to the star-shaped conformation after removal of pressure. In this study, we applied this method to construct a scaffold mimicking the dynamics of human urethra. Radial expansion and closure of the scaffold could be iteratively performed for at least 1000 cycles, burst pressure being 132±22mmHg. Scaffolds were seeded with human epithelial cells and cultured in a bioreactor under dynamic conditions mimicking urination (pulse flow of 21s every 2h). Cells adhered and formed a closed luminal layer that resisted flow conditions. In conclusion, a new type of a tubular collagen scaffold has been constructed with radial elastic-like characteristics based on the shape of the scaffold, and enabling the scaffold to reversibly expand upon increase in luminal pressure. These scaffolds may be useful for regenerative medicine of tubular organs., Statement of Significance: In this paper, a new type I collagen-based tubular scaffold is presented that possesses intrinsic radial elasticity. This characteristic is key to the functioning of a number of tubular organs including blood vessels and organs of the gastrointestinal and urogenital tract. The scaffold was given a star-shaped lumen by physical compression and chemical crosslinking, mimicking the folding pattern observed in many tubular organs. In rest, the lumen is closed but it opens upon increase of luminal pressure, e.g. when fluids pass. Human epithelial cells seeded on the luminal side adhered well and were compatible with voiding dynamics in a bioreactor. Collagen scaffolds with radial elasticity may be useful in the regeneration of dynamic tubular organs., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Type I collagen is widely applied as a biomaterial for tissue regeneration. In the extracellular matrix, collagen provides strength but not elasticity under large deformations, a characteristic crucial for dynamic organs and generally imparted by elastic fibers. In this study, a methodology is described to induce elastic-like characteristics in a scaffold consisting of solely type I collagen. Tubular scaffolds are prepared from collagen fibrils by a casting, molding, freezing and lyophilization process. The lyophilized constructs are compressed, corrugated and subsequently chemically crosslinked with carbodiimide in the corrugated position. This procedure induces elastic-like properties in the scaffolds that could be repeatedly stretched five times their original length for at least 1000 cycles. The induced elasticity is entropy driven and can be explained by the introduction of hydrophobic patches that are disrupted upon stretching thus increasing the hydrophobic-hydrophilic interface. The scaffolds are cytocompatible as demonstrated by fibroblast cell culture. In conclusion, a new straightforward technique is described to endow unique elastic characteristics to scaffolds prepared from type I collagen alone. Scaffolds may be useful for engineering of dynamic tissues such as blood vessels, ligaments, and lung., Statement of Significance: In this research report, a methodology is presented to introduce elasticity to biomaterials consisting of only type I collagen fibrils. The method comprises physical compression and corrugation in combination with chemical crosslinking. By introducing elasticity to collagen biomaterials, their application in regenerative medicine may be expanded to dynamic organs such as blood vessels, ligaments and lung. The combination of strength and elasticity in one single natural biomaterial may also "simplify" the design of new scaffolds., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: The field of regenerative medicine has developed promising techniques to improve current neobladder strategies used for radical cystectomies or congenital anomalies. Scaffolds made from molecularly defined biomaterials are instrumental in the regeneration of tissues, but are generally confined to small flat patches and do not comprise the whole organ. We have developed a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold, mimicking the shape of the whole bladder, and with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized, with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. Human and porcine bladder urothelial and smooth muscle cells were able to attach to the scaffold and maintained their phenotype in vitro. The closed luminal side and the porous outside of the scaffold facilitated the formation of an urothelial lining and infiltration of smooth muscle cells, respectively. The cells aligned according to the provided scaffold template. The technology used is highly adjustable (shape, size, materials) and may be used as a starting point for research to an off-the-shelf medical device suitable for neobladders., Statement of Significance: In this study, we describe the development of a simple, one-step casting method to produce a seamless large hollow collagen-based scaffold mimicking the shape of the whole bladder with integrated anastomotic sites for ureters and urethra. The hollow bladder scaffold is highly standardized with uniform wall thickness and a unidirectional pore structure to facilitate cell infiltration in vivo. The closed luminal surface and the porous exterior of the scaffold facilitated the formation of a urothelial lining and infiltration of smooth muscle cells, respectively. The applied technology is highly adjustable (shape, size, materials) and can be the starting point for research to an off-the-shelf medical device suitable for neobladders., (Copyright © 2016. Published by Elsevier Ltd.)

Microfracture surgery may be applied to treat cartilage defects. During the procedure the subchondral bone is penetrated, allowing bone marrow-derived mesenchymal stem cells to migrate towards the defect site and form new cartilage tissue. Microfracture surgery generally results in the formation of mechanically inferior fibrocartilage. As a result, this technique offers only temporary clinical improvement. Tissue engineering and regenerative medicine may improve the outcome of microfracture surgery. Filling the subchondral defect with a biomaterial may provide a template for the formation of new hyaline cartilage tissue. In this study, a systematic review and meta-analysis were performed to assess the current evidence for the efficacy of cartilage regeneration in preclinical models using acellular biomaterials implanted after marrow stimulating techniques (microfracturing and subchondral drilling) compared to the natural healing response of defects. The review aims to provide new insights into the most effective biomaterials, to provide an overview of currently existing knowledge, and to identify potential lacunae in current studies to direct future research. A comprehensive search was systematically performed in PubMed and EMBASE (via OvidSP) using search terms related to tissue engineering, cartilage and animals. Primary studies in which acellular biomaterials were implanted in osteochondral defects in the knee or ankle joint in healthy animals were included and study characteristics tabulated (283 studies out of 6,688 studies found). For studies comparing non-treated empty defects to defects containing implanted biomaterials and using semi-quantitative histology as outcome measure, the risk of bias (135 studies) was assessed and outcome data were collected for meta-analysis (151 studies). Random-effects meta-analyses were performed, using cartilage regeneration as outcome measure on an absolute 0-100% scale. Implantation of acellular biomaterials significantly improved cartilage regeneration by 15.6% compared to non-treated empty defect controls. The addition of biologics to biomaterials significantly improved cartilage regeneration by 7.6% compared to control biomaterials. No significant differences were found between biomaterials from natural or synthetic origin or between scaffolds, hydrogels and blends. No noticeable differences were found in outcome between animal models. The risk of bias assessment indicated poor reporting for the majority of studies, impeding an assessment of the actual risk of bias. In conclusion, implantation of biomaterials in osteochondral defects improves cartilage regeneration compared to natural healing, which is further improved by the incorporation of biologics., Competing Interests: The authors declare there are no competing interests.

Identifying collagen produced de novo by cells in a background of purified collagenous biomaterials poses a major problem in for example the evaluation of tissue-engineered constructs and cell biological studies to tumor dissemination. We have developed a universal strategy to detect and localize newly deposited collagen based on its inherent association with dermatan sulfate. The method is applicable irrespective of host species and collagen source.

In skeletal muscle, the stem cell niche is important for controlling the quiescent, proliferation and differentiation states of satellite cells, which are key for skeletal muscle regeneration after wounding. It has been shown that type I collagen, often used as 3D-scaffolds for regenerative medicine purposes, impairs myoblast differentiation. This is most likely due to the absence of specific extracellular matrix proteins providing attachment sites for myoblasts and/or myotubes. In this study we investigated the differentiation capacity of primary murine myoblasts on type I collagen films either untreated or modified with elastin, laminin, type IV collagen, laminin/entactin complex, combinations thereof, and Matrigel as a positive control. Additionally, increased reactive oxygen species (ROS) and ROCK signaling might also be involved. To measure ROS levels with live-cell microscopy, fibronectin-coated glass coverslips were additionally coated with type I collagen and Matrigel onto which myoblasts were differentiated. On type I collagen-coated coverslips, myotube formation was impaired while ROS levels were increased. However, anti-oxidant treatment did not enhance myotube formation. ROCK inhibition, which generally improve cellular attachment to uncoated surfaces or type I collagen, enhanced myoblast attachment to type I collagen-coated coverslips and -films, but slightly enhanced myotube formation. Only modification of type I collagen films by Matrigel and a combination of laminin/entactin significantly improved myotube formation. Our results indicate that type I collagen scaffolds can be modified by satellite cell niche factors of which specifically laminin and entactin enhanced myotube formation. This offers a promising approach for regenerative medicine purposes to heal skeletal muscle wounds., Statement of Significance: In this manuscript we show for the first time that impaired myotube formation on type I collagen scaffolds can be completely restored by modification with laminin and entactin, two extracellular proteins from the satellite cell niche. This offers a promising approach for regenerative medicine approaches to heal skeletal muscle wounds., (Copyright © 2015. Published by Elsevier Ltd.)

Tissue engineering may become an alternative to current bladder augmentation techniques. Large scaffolds are needed for clinically significant augmentation, but can result in fibrosis and graft shrinkage. The purpose of this study was to investigate whether smart acellular collagen-heparin scaffolds with growth factors (GFs) VEGF, FGF2, and HB-EGF enhance bladder tissue regeneration and bladder capacity in a large animal model of diseased bladder. Scaffolds of bovine type I collagen with heparin and VEGF, FGF2, and HB-EGF measuring 3.2 cm in diameter were prepared. In 23 fetal sheep, a bladder exstrophy was surgically created at 79 days of gestation. One week after birth (at full term), the bladder was reconstructed by primary closure (PC group) or using a collagen-heparin scaffold with GFs (COLGF group) and compared to a historical group reconstructed with a collagen scaffold without GFs (COL group). Functional (video urodynamics) and histological evaluation was performed 1 and 6 months after bladder repair. The overall survival rate was 57%. Cystograms were normal in all animals, except for low-grade reflux in all groups. Urodynamics showed no statistically significant differences in bladder capacity and compliance between groups. Histological evaluation at 1 month revealed increased urothelium formation, improved angiogenesis, and enhanced ingrowth of smooth muscle cells (SMCs) in the COLGF group compared to the COL group. At 6 months, improved SMC ingrowth was found in the COLGF group compared to the COL group; both scaffold groups showed normal urothelial lining and standard extracellular matrix development. Bladder regeneration using a collagen-heparin scaffold with VEGF, FGF2, and HB-EGF improved bladder tissue regeneration in a large animal model of diseased bladder. Larger GF-loaded constructs need to be tested to reach clinically significant augmentation.

Autologous skin grafts are the gold standard for the treatment of burn wounds. In a number of cases, treatment with autologous tissue is not possible and skin substitutes are used. The outcome, however, is not optimal and improvements are needed. Inspired by scarless healing in early embryonic development, we here set out a strategy for the design and construction of embryonic-like scaffolds for skin tissue engineering. This strategy may serve as a general approach in the construction of embryonic-like scaffolds for other tissues/organ. As a first step, key effector molecules upregulated during embryonic and neonatal skin formation were identified using a comparative gene expressing analysis. A set of 20 effector molecules was identified, from which insulin-like growth factor 2 (IGF2) and sonic hedgehog (SHH) were selected for incorporation into a type I collagen-heparin scaffold. Porous scaffolds were constructed using purified collagen fibrils and 6% covalently bound heparin (to bind and protect the growth factors), and IGF2 and SHH were incorporated either individually (~0.7 and 0.4 µg/mg scaffolds) or in combination (combined ~1.5 µg/mg scaffolds). In addition, scaffolds containing hyaluronan (up to 20 µg/mg scaffold) were prepared, based on the up- or downregulation of genes involved in hyaluronan synthesis/degradation and its suggested role in scarless healing. In conclusion, based on a comprehensive gene expression analysis, a set of effector molecules and matrix molecules was identified and incorporated into porous scaffolds. The scaffolds thus prepared may create an 'embryonic-like' environment for cells to recapitulate embryonic events and for new tissues/organs., (Copyright © 2013 John Wiley & Sons, Ltd.)

Current ovarian cancer treatment involves chemotherapy that has serious limitations, such as rapid clearance, unfavorable biodistribution and severe side effects. To overcome these limitations, drug delivery systems (DDS) have been developed to encapsulate chemotherapeutics for delivery to tumor cells. However, no systematic assessment of the efficacy of chemotherapy by DDS compared to free chemotherapy (not in a DDS) has been performed for animal studies. Here, we assess the efficacy of chemotherapy in DDS on survival and tumor growth inhibition in animal studies. We searched PubMed and EMBASE (via OvidSP) to systematically identify studies evaluating chemotherapeutics encapsulated in DDS for ovarian cancer treatment in animal studies. Studies were assessed for quality and risk of bias. Study characteristics were collected and outcome data (survival/hazard ratio or tumor growth inhibition) were extracted and used for meta-analyses. Meta-analysis was performed to identify and explore which characteristics of DDS influenced treatment efficacy. A total of 44 studies were included after thorough literature screening (2,735 studies found after initial search). The risk of bias was difficult to assess, mainly because of incomplete reporting. A total of 17 studies (377 animals) and 16 studies (259 animals) could be included in the meta-analysis for survival and tumor growth inhibition, respectively. In the majority of the included studies chemotherapeutics entrapped in a DDS significantly improved efficacy over free chemotherapeutics regarding both survival and tumor growth inhibition. Subgroup analyses, however, revealed that cisplatin entrapped in a DDS did not result in additional tumor growth inhibition compared to free cisplatin, although it did result in improved survival. Micelles did not show a significant tumor growth inhibition compared to free chemotherapeutics, which indicates that micelles may not be a suitable DDS for ovarian cancer treatment. Other subgroup analyses, such as targeted versus non-targeted DDS or IV versus IP administration route, did not identify specific characteristics of DDS that affected treatment efficacy. This systematic review shows the potential, but also the limitations of chemotherapy by drug delivery systems for ovarian cancer treatment. For future animal research, we emphasize that data need to be reported with ample attention to detailed reporting.

Lyophilisomes are a novel class of proteinaceous biodegradable nano/microparticle capsules developed for tumor drug delivery. The in vivo characteristics of lyophilisomes are unknown and, therefore, the time course of biodistribution of sized albumin-based lyophilisomes in CD1 mice after intravenous administration was studied. Lyophilisomes, prepared from Dylight680-labeled albumin, were sized using a sucrose gradient centrifugation methodology and four fractions with a mean size of approximately 200nm, 400nm, 550nm, and 650nm were pooled for in/ex vivo localization, (immuno)histochemistry and biochemical analysis. Lyophilisomes were rapidly taken out of the circulation by the liver and spleen. Immunohistochemistry revealed that lyophilisomes were taken up in the liver by F4/80 positive macrophages, and in the spleen by Sign-R1 positive macrophages specifically located in the marginal zones. Lyophilisomes were most likely degraded by the liver and spleen and subsequently excreted via the urine, as high levels of degraded Dylight680-labeled albumin were detected in the urine. This was corroborated by electron microscopy of the spleen, which showed intact lyophilisomes in the marginal zone 5 and 30min after injection, but not after 2h. In conclusion, IV injected lyophilisomes are rapidly entrapped by liver and splenic macrophages, biodegraded, and excreted in the urine., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aligned unidirectional collagen scaffolds may aid regeneration of those tissues where alignment of cells and extracellular matrix is essential, as for instance in cartilage, nerve bundles, and skeletal muscle. Pores can be introduced by ice crystal formation followed by freeze-drying, the pore architecture reflecting the ice crystal morphology. In this study we developed a wedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directional freezing. Insoluble type I collagen suspensions were frozen using a custom-made wedge system, facilitating the formation of a horizontal as well as a vertical temperature gradient and providing a controlled solidification area for ice dendrites. The system permitted the growth of aligned unidirectional ice crystals over a large distance (>2.5 cm), an insulator prolonging the freezing process and facilitating the construction of crack-free scaffolds. Unidirectional collagen scaffolds with tunable pore sizes and pore morphologies were constructed by varying freezing rates and suspension media. The versatility of the system was indicated by the construction of unidirectional scaffolds from albumin, poly(vinyl alcohol) (a synthetic polymer), and collagen-polymer blends producing hybrid scaffolds. Macroscopic observations, temperature measurements, and scanning electron microscopy indicated that directed horizontal ice dendrite formation, vertical ice crystal nucleation, and evolutionary selection were the basis of the aligned unidirectional ice crystal growth and, hence, the aligned unidirectional pore structure. In conclusion, a simple, highly adjustable freezing system has been developed allowing the construction of large (hybrid) bioscaffolds with tunable unidirectional pore architecture.

A persistent clinical demand exists for a suitable arterial prosthesis. In this study, a vascular conduit mimicking the native 3-layered artery, and constructed from the extracellular matrix proteins type I collagen and elastin, was evaluated for its performance as a blood vessel equivalent. A tubular 3-layered graft (elastin-collagen-collagen) was prepared using highly purified type I collagen fibrils and elastin fibers, resembling the 3-layered native blood vessel architecture. The vascular graft was crosslinked and heparinised (37 ± 4 μg heparin/mg graft), and evaluated as a vascular graft using a porcine bilateral iliac artery model. An intra-animal comparison with clinically-used heparinised ePTFE (Propaten®) was made. Analyses included biochemical characterization, duplex scanning, (immuno)histochemistry and scanning electron microscopy. The tubular graft was easy to handle with adequate suturability. Implantation resulted in pulsating grafts without leakage. One week after implantation, both ePTFE and the natural acellular graft had 100% patencies on duplex scanning. Grafts were partially endothelialised (Von Willebrand-positive endothelium with a laminin-positive basal membrane layer). After one month, layered thrombi were found in the natural (4/4) and ePTFE graft (1/4), resulting in occlusion which in case of the natural graft is likely due to the porosity of the inner elastin layer. In vivo application of a molecularly-defined tubular graft, based on nature's matrix proteins, for vascular surgery is feasible.

The bio-inspired engineering of tissue equivalents should take into account anisotropic morphology and the mechanical properties of the extracellular matrix. This especially applies to collagen fibrils, which have various, but highly defined, orientations throughout tissues and organs. There are several methods available to control the alignment of soluble collagen monomers, but the options to direct native insoluble collagen fibers are limited. Here we apply a controlled counter-rotating cone extrusion technology to engineer tubular collagen constructs with defined anisotropy. Driven by diverging inner and outer cone rotation speeds, collagen fibrils from bovine skin were extruded and precipitated onto mandrels as tubes with oriented fibers and bundles, as examined by second harmonic generation microscopy and quantitative image analysis. A clear correlation was found whereby the direction and extent of collagen fiber alignment during extrusion were a function of the shear forces caused by a combination of the cone rotation and flow direction. A gradual change in the fiber direction, spanning +50 to -40°, was observed throughout the sections of the sample, with an average decrease ranging from 2.3 to 2.6° every 10μm. By varying the cone speeds, the collagen constructs showed differences in elasticity and toughness, spanning 900-2000kPa and 19-35mJ, respectively. Rotational extrusion presents an enabling technology to create and control the (an)isotropic architecture of collagen constructs for application in tissue engineering and regenerative medicine., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

Objective: To compare the regenerative capacity of diseased bladder in a large animal model of bladder exstrophy with regeneration in healthy bladder using a highly porous collagen scaffold., Materials and Methods: Highly porous bovine type I collagen scaffolds with a diameter of 32 mm were prepared. In 12 fetal sheep a bladder exstrophy was surgically created at 79 days' gestation. Lambs were born at full term (140 days' gestation). After 1 week the bladder lesion was reconstructed and augmented with a collagen scaffold (group 1). In nine normal newborn lambs the bladder was augmented with a collagen scaffold 1 week after birth (group 2). Functional (video-urodynamics) and histological evaluation was performed at 1 and 6 months after surgery., Results: The survival rate was 58% in group 1 and 100% in group 2. Cystograms were normal in all lambs, besides low-grade reflux in both groups. Urodynamics showed comparable capacity between both groups and a trend to lower compliance in group 1. Histological evaluation at 1 month revealed a non-confluent urothelial layer, an immature submucosa, and initial ingrowth of smooth muscle cells. At 6 months both groups showed normal urothelial lining, standard extracellular matrix development, and smooth muscle cell ingrowth., Conclusions: Bladder tissue regeneration with a collagen scaffold in a diseased bladder model and in healthy bladder resulted in comparable functional and histological outcome, with a good quality of regenerated tissue involving all tissue layers. Improvements may still be needed for larger augmentations or more severely diseased bladders.

Current methods for closure of congenital diaphragmatic hernia using patches are unsatisfactory, and novel collagen-based scaffolds have been developed, and successfully applied in a rat model. However, for translation to the human situation constructs must be evaluated in larger animal models. We developed collagen scaffolds enforced with Vicryl, loaded either with or without the muscle stimulatory growth factor insulin-like growth factor 1 (IGF1). We describe our steps to a surgical method to implant these scaffolds into a diaphragmatic defect in 1.5–3 week old lambs, and evaluate the scaffolds 6 months after implantation. Omentum was attached to the scaffold. At sacrifice, eventration of the implantation site was observed in all animals with a thin layer of tissue separating the abdomen from the thorax. Histologically, no scaffold remnants could be observed. Fatty tissue surrounded by fibrous tissue was seen, resembling encapsulated omentum, with collagen-rich tissue present between this tissue and the original diaphragmatic muscle. Outcomes were not different for scaffolds with or without IGF1. In conclusion, the scaffolds integrated well into the surrounding tissue, but slower degrading materials are needed to prevent eventrations.