Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials

The access to defined protein-based material systems is a major challenge in bionanotechnology and regenerative medicine. Exact control over sequence composition and modification is an important requirement for the intentional design of structure and function. Herein structural- and matrix proteins provide a great potential, but their large repetitive sequences pose a major challenge in their assembly. Here we introduce an integrative “one-vector-toolbox-platform” (OVTP) approach which is fast, efficient and reliable. The OVTP allows for the assembly, multimerization, intentional arrangement and direct translation of defined molecular DNA-tecton libraries, in combination with the selective functionalization of the yielded protein-tecton libraries. The diversity of the generated tectons ranges from elastine-, resilin, silk- to epitope sequence elements. OVTP comprises the expandability of modular biohybrid-materials via the assembly of defined multi-block domain genes and genetically encoded unnatural amino acids (UAA) for site-selective chemical modification. Thus, allowing for the modular combination of the protein-tecton library components and their functional expansion with chemical libraries via UAA functional groups with bioorthogonal reactivity. OVTP enables access to multitudes of defined protein-based biohybrid-materials for self-assembled superstructures such as nanoreactors and nanobiomaterials, e.g. for approaches in biotechnology and individualized regenerative medicine.     

Influence of the stiffness of three-dimensional alginate/collagen-I interpenetrating networks on fibroblast biology

Wound dressing biomaterials are increasingly being designed to incorporate bioactive molecules to promote healing, but the impact of matrix mechanical properties on the biology of resident cells orchestrating skin repair and regeneration remains to be fully understood. This study investigated whether tuning the stiffness of a model wound dressing biomaterial could control the behavior of dermal fibroblasts. Fully interpenetrating networks (IPNs) of collagen-I and alginate were fabricated to enable gel stiffness to be tuned independently of gel architecture, polymer concentration or adhesion ligand density. Three-dimensional cultures of dermal fibroblasts encapsulated within matrices of different stiffness were shown to promote dramatically different cell morphologies, and enhanced stiffness resulted in upregulation of key-mediators of inflammation such as IL-10 and COX-2. These findings suggest that simply modulating the matrix mechanical properties of a given wound dressing biomaterial deposited at the wound site could regulate the progression of wound healing.     

New advances in the pathophysiologic and radiologic basis of the exstrophy spectrum

The exstrophy–epispadias complex is a rare spectrum of anomalies affecting the genitourinary system, anterior abdominal wall, and pelvis. Recent advances in the repair of classic bladder exstrophy (CBE) and cloacal exstrophy (CE) have resulted in significant changes in outcomes of surgical management (including higher continence rate, fewer surgical complications, and better cosmesis) and health-related quality of life in these patients. These noteworthy changes resulted from advances in the pathophysiological and genetic backgrounds of this disease and better radiologic assessment of the three-dimensional anatomy of the bony pelvis and its musculature. A PubMed search was performed with the keyword exstrophy. The resulting literature pertaining to genetics, stem cells, imaging, tissue engineering, epidemiology, and endocrinology was reviewed. The following represents an overview of the advances in basic science understanding and imaging of the exstrophy–epispadias spectrum and discusses their possible and future effects on the management of CBE and CE.     

Application of hiPSCs in tooth regeneration via cellular modulation

BackgroundInduced pluripotent stem cell (iPSC)-based technology provides limitless resources for customized development of organs without any ethical concerns. In theory, iPSCs generated from terminally differentiated cells can be induced to further differentiate into all types of organs that are derived from the embryonic germ layers. Since iPSC reprogramming technology is relatively new, extensive efforts by the researchers have been put together to optimize the protocols to establish in vitro differentiation of human iPSCs (hiPSCs) into various desirable cell types/organs.HighlightsIn the present study, we review the potential application of iPSCs as an efficient alternative to primary cells for modulating signal molecules. Furthermore, an efficient culture system that promotes the differentiation of cell lineages and tissue formation has been reviewed. We also summarize the recent studies wherein tissue engineering of the three germ layers has been explored. Particularly, we focus on the current research strategies for iPSC-based tooth regeneration via molecular modulation.ConclusionIn recent decades, robust knowledge regarding the hiPSC-based regenerative therapy has been accumulated, especially focusing on cellular modulation. This review provides the optimization of the procedures designed to regenerate specific organs.     

Cloning of hamster osteopontin and expression distribution in normal tissues and experimentally induced oral squamous-cell carcinoma

Osteopontin (OPN) is a non-collagenous extracellular matrix (ECM) protein expressed and secreted by several human cancers. This study investigated the expression pattern of OPN during development of oral squamous-cell carcinoma by using 7,12-dimethylbenz[a]anthracene (DMBA)-induced squamous-cell carcinomas in buccal pouch of syrian golden hamsters. We first identified the hamster OPN cDNA sequence by screening of a hamster calvariae cDNA library with a rat OPN cDNA probe. The resulting 1,449 bp of hamster OPN cDNA led to a deduced protein sequence of 305 amino acids containing several putative binding sites to integrins, CD44 receptors, calcium ions and hydroxyapatite, as well as multiple sites for phosphorylation, glycosylation and sulphation. Hamster OPN cDNA was then used as a probe to analyze the expression of OPN mRNA by Northern blot and in situ hybridization analyses of normal and malignant tissues. OPN mRNA was detected in several non-mineralized tissues as well as in mineralized tissues, but was not present in normal hamster buccal epithelium. DMBA-treated hamster buccal pouches expressed OPN mRNA as early as 4 weeks and displayed the highest level of expression at 15 weeks. The specimens treated with DMBA for 15 weeks exhibited histological features of squamous-cell carcinoma, presented microcrystalline deposits and showed OPN expression associated with malignant epithelium and tumor-associated macrophages. To summarize, our results suggest that buccal-pouch carcinogenesis of Syrian golden hamster may constitute an excellent experimental model to study the mechanisms by which OPN is associated with oral cancer pathogenesis, and to validate OPN-based therapeutic approaches to ameliorate oral cancer progression and metastasis.     

In vitro assessment of motility and proliferation of human osteogenic cells on different isolated extracellular matrix components compared with enamel matrix derivative by continuous single-cell observation

OBJECTIVES: The composition of the extracellular matrix (ECM) plays a substantial role in bone remodelling, fracture healing and osseointegration of dental implants by regulating proliferation, migration and finally differentiation of osteogenic cell populations. Emdogain, a composition of an enamel matrix derivative (EMD), has been introduced as a potential candidate to promote tissue regeneration. We investigated whether EMD could serve as a potential promoter of cell proliferation and motility as a dynamic cell response and compared the results with the ubiquitous single ECM components type I collagen and laminin. MATERIAL AND METHODS: In the investigation presented, we used a continuous observation method for the analysis of migratory and proliferative patterns of individual cells. We analyzed the response of four osteoblastic cell lines to specific extracellular ligands (type I collagen, laminin and EMD) over a period of 24 h compared with untreated glass surface and bovine serum albumin (BSA) as control groups. RESULTS: Type I collagen and laminin promoted cell motility significantly compared with the control groups and, in part, compared with EMD as well. The analysis of all 451 investigated cells revealed the following mean values for cell motiliy: untreated glass (n=99): 5.46+/-2.74 microm/h, BSA (n=89): 6.35+/-2.43 microm/h, type I collagen (n=108): 8.77+/-3.42 microm/h, laminin (n=74): 9.89+/-5.10 microm/h and EMD (n=81): 7.92+/-3.35 microm/h. Proliferation rates on the different surfaces were heterogenous for all investigated cell lines and varied from 0% to 50% within 24 h without a correlation to cell motility. CONCLUSION: In our study, EMD promotes cell motility better than the control groups. The two investigated single ECM components type I collagen and laminin promoted cell motility superior to EMD. This supports the hypothesis that EMD promotes a less mobile but more differentiated osteogenic phenotype.     

材料生物力学2021年研究进展

机体组织具有复杂的三维动态结构,且受到多种形式的作用力。细胞从细胞外基质（extracellular matrix, ECM）中感受力学刺激,ECM构建的力学微环境调控细胞不同生物学功能。制备可模拟机体组织ECM力学微环境的生物材料是生物力学领域研究的热点和难点之一。生物材料的不同理化性质赋予材料特定的力学性能,进而影响细胞行为和功能。本文结合2021年材料生物力学领域的最新文献,特别关注新型材料生物力学对细胞生物学行为的调控和在组织工程中的应用,并探讨材料生物力学研究领域的未来发展方向。     

Role of cell–matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels

Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen‐1 (P15). By day 14, VICs became significantly more elongated in RGDS‐containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS‐functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α‐smooth muscle actin (αSMA) at day 14. The percentage of αSMA⁺ VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG‐functionalized gels had a significantly higher collagen‐X:collagen‐1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide‐functionalized PEG hydrogels are a useful system for culturing VICs three‐dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential gene expression identifies subgroups of renal cell carcinoma

Clear cell carcinoma of the kidney, the most common subtype of renal cell cancer, displays different biological behavior in different patients. This heterogeneity cannot be recognized by light microscopy. In this study, gene expression in 16 clear cell renal cell carcinoma samples and 17 non-malignant tissue types comprising 539 samples was determined using oligonucleotide microarrays representing approximately 40,000 known genes and ESTs. Differences in gene expression were quantified as the fold change in gene expression between the various sets of samples. A set of genes was identified that was overexpressed in the renal cell carcinoma samples compared with the normal kidney samples. Principle component analysis of the set of renal cell carcinomas using this set of genes overexpressed in renal cell cancer revealed the existence of 2 major subgroups among the renal carcinomas. A series of principle component analyses of the set of renal cell carcinomas using different gene sets composed of genes involved in different metabolic pathways also revealed the same 2 major subgroups of the renal cell cancers. Eisen clustering using the same genes also revealed the same 2 major renal cell cancer subsets. Review of the pathology suggested that these 2 subgroups differed in pathologic grade. Genes differentially expressed between the 2 renal cell cancer subsets were identified. Examination of gene expression in each renal cell cancer subset and the pool of renal cell carcinoma samples compared with that in 17 different normal tissues revealed genes specifically overexpressed in renal cell cancer compared with these normal tissues. The authors conclude that gene expression patterns may be useful in helping to further classify subtypes of renal cell carcinoma that may have clinical significance. In addition, the genes identified as overexpressed in each set of clear cell renal cell carcinomas compared with normal tissues may represent useful targets for therapy.