A novel, cell-permeable, collagen-based membrane promotes fibroblast migration.

BACKGROUND AND OBJECTIVE: Growth factors are frequently incorporated into scaffolds to promote periodontal regeneration but many currently used scaffolds do not encourage cell migration towards the dentogingival junction. We examined the proliferation and migration of human gingival fibroblasts in a novel, physically robust, collagen-Vicryl™ membrane loaded with fibronectin (FN) and/or insulin-like growth factor (IGF-I). Biocompatibility of the membranes was evaluated in rat dorsal skin. MATERIAL AND METHODS: Chemotaxis was examined in Boyden chambers and cell migration by confocal imaging of membranes, which were fabricated from rat tail type I collagen with embedded Vicryl knitted mesh, IGF-I (50, 100 ng/mL) and FN (10 µg/mL). Membranes (Vicryl alone, collagen+Vicryl, collagen+Vicryl+IGF-I, collagen+Vicryl+FN') were implanted subcutaneously in 8 rats and were evaluated by histomorphometry after 7 and 14 days. RESULTS: IGF-I (50 or 100 ng/mL) promoted chemotaxis compared with vehicle controls (P = .02, P = .001, respectively). IGF-I did not affect cell proliferation. Incorporation of FN retarded time-dependent release of IGF-I from collagen gels. Three dimensional confocal microscopy imaging of cell migration through collagen+Vicryl membranes showed enhanced migration in the IGF+FN group compared to all other groups at 8, 10 and 14 days (P < .05). In a rat skin model, implanted membranes were surrounded by thin collagen capsules and mild inflammatory infiltrates. CONCLUSION: Incorporation of FN into IGF-I-loaded collagen+Vicryl membranes reduced IGF release from collagen and increased the migration of human gingival fibroblasts. The new membrane may promote healing and reformation of the dentogingival junction.

The spatial patterning of RGD and BMP-2 mimetic peptides at the subcellular scale modulates human mesenchymal stem cells osteogenesis.

Engineering artificial extracellular matrices, based on the biomimicry of the spatial distribution of proteins and growth factors within their native microenvironment, is of great importance for understanding mechanisms of bone tissue regeneration. Herein, photolithography is used to decorate glass surfaces with subcellular patterns of RGD and BMP-2 ligands; two mimetic peptides recognized to be involved in stem cells osteogenesis. The biological relevance of well-defined RGD and BMP-2 patterned surfaces is evaluated by investigating the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts, in the absence of induction media. The extent of hMSCs differentiation is revealed to be dependent on both the pattern shape and the ligand type. Indeed, the spatial patterning of BMP-2, but not RGD peptide, significantly enhances the extent of hMSCs differentiation, suggesting that geometric cues guide stem cells specification into specialized cells in a ligand type dependent manner. Such cell culture models provide an interesting tool to investigate how stem cells perceive and respond to their microenvironment and may contribute to the development of next-generation biomaterials capable of producing clinically relevant volume of bone tissue. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 959-970, 2018.

RGD and BMP-2 mimetic peptide crosstalk enhances osteogenic commitment of human bone marrow stem cells.

UNLABELLED: Human bone marrow mesenchymal stem cells (hBMSCs) commitment and differentiation are dictated by bioactive molecules sequestered within their Extra Cellular Matrix (ECM). One common approach to mimic the physiological environment is to functionalize biomaterial surfaces with ECM-derived peptides able to recruit stem cells and trigger their linage-specific differentiation. The objective of this work was to investigate the effect of RGD and BMP-2 ligands crosstalk and density on the extent of hBMSCs osteogenic commitment, without recourse to differentiation medium. RGD peptide promotes cell adhesion via cell transmembrane integrin receptors, while BMP-2 peptide, corresponding to residues 73-92 of Bone Morphogenetic Protein-2, was shown to induce hBMSCs osteoblast differentiation. The immobilization of peptides on aminated glass was ascertained by X-ray Photoelectron Spectroscopy (XPS), the density of grafted peptides was quantified by fluorescence microscopy and the surface roughness was evaluated using Atomic Force Microscopy (AFM). The osteogenic commitment of hBMSCs cultured on RGD and/or BMP-2 surfaces was characterized by immunohistochemistry using STRO-1 as specific stem cells marker and Runx-2 as an earlier osteogenic marker. Biological results showed that the osteogenic commitment of hBMSCs was enhanced on bifunctionalized surfaces as compared to surfaces containing BMP-2, while on RGD surfaces cells mainly preserved their stemness character. These results demonstrated that RGD and BMP-2 mimetic peptides act synergistically to enhance hBMSCs osteogenesis without supplementing the media with osteogenic factors. These findings contribute to the development of biomimetic materials, allowing a deeper understanding of signaling pathways that govern the transition of stem cells towards the osteoblastic lineage. STATEMENT OF SIGNIFICANCE: For a long time, scientists thought that the differentiation of Mesenchymal Stem Cells (MSCs) into bone cells was dictated by growth factors. This manuscript shed light on other ligands that play a crucial role in regulating MSCs fate. In concrete terms, it was demonstrated that the osteoinductive effect of BMP-2 peptide is 2 folds improved in the presence of adhesive RGD peptide. Compared to previous works highlighting this synergistic cooperation between RGD and BMP-2 peptides, the main strength of this work lies to the use of primitive human cells (hMSCs) and well-defined biomimetic material surfaces (controlled surface roughness and peptide densities). This work provides valuable insights to develop custom-designed in vitro cell culture models, capable of targeting the desired cell response.

Mineralization defects in cementum and craniofacial bone from loss of bone sialoprotein.

Bone sialoprotein (BSP) is a multifunctional extracellular matrix protein found in mineralized tissues, including bone, cartilage, tooth root cementum (both acellular and cellular types), and dentin. In order to define the role BSP plays in the process of biomineralization of these tissues, we analyzed cementogenesis, dentinogenesis, and osteogenesis (intramembranous and endochondral) in craniofacial bone in Bsp null mice and wild-type (WT) controls over a developmental period (1-60 days post natal; dpn) by histology, immunohistochemistry, undecalcified histochemistry, microcomputed tomography (microCT), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative PCR (qPCR). Regions of intramembranous ossification in the alveolus, mandible, and calvaria presented delayed mineralization and osteoid accumulation, assessed by von Kossa and Goldner's trichrome stains at 1 and 14 dpn. Moreover, Bsp(-/-) mice featured increased cranial suture size at the early time point, 1 dpn. Immunostaining and PCR demonstrated that osteoblast markers, osterix, alkaline phosphatase, and osteopontin were unchanged in Bsp null mandibles compared to WT. Bsp(-/-) mouse molars featured a lack of functional acellular cementum formation by histology, SEM, and TEM, and subsequent loss of Sharpey's collagen fiber insertion into the tooth root structure. Bsp(-/-) mouse alveolar and mandibular bone featured equivalent or fewer osteoclasts at early ages (1 and 14 dpn), however, increased RANKL immunostaining and mRNA, and significantly increased number of osteoclast-like cells (2-5 fold) were found at later ages (26 and 60 dpn), corresponding to periodontal breakdown and severe alveolar bone resorption observed following molar teeth entering occlusion. Dentin formation was unperturbed in Bsp(-/-) mouse molars, with no delay in mineralization, no alteration in dentin dimensions, and no differences in odontoblast markers analyzed. No defects were identified in endochondral ossification in the cranial base, and craniofacial morphology was unaffected in Bsp(-/-) mice. These analyses confirm a critical role for BSP in processes of cementogenesis and intramembranous ossification of craniofacial bone, whereas endochondral ossification in the cranial base was minimally affected and dentinogenesis was normal in Bsp(-/-) molar teeth. Dissimilar effects of loss of BSP on mineralization of dental and craniofacial tissues suggest local differences in the role of BSP and/or yet to be defined interactions with site-specific factors.

Epidermolysis bullosa simplex associated with pyloric atresia is a novel clinical subtype caused by mutations in the plectin gene (PLEC1).

Epidermolysis bullosa (EB) is an inherited mechano-bullous disorder of the skin, and is divided into three major categories: EB simplex (EBS), dystrophic EB, and junctional EB (JEB). Mutations in the plectin gene (PLEC1) cause EBS associated with muscular dystrophy, whereas JEB associated with pyloric atresia (PA) results from mutations in the alpha6 and beta4 integrin genes. In this study, we examined three EB patients associated with PA from two distinct families. Electron microscopy detected blister formation within the basal keratinocytes leading to the diagnosis of EBS. Surprisingly, immunohistochemical studies using monoclonal antibodies to a range of basement membrane proteins showed that the expression of plectin was absent or markedly attenuated. Sequence analysis demonstrated four novel PLEC1 mutations. One proband was a compound heterozygote for a nonsense mutation of Q305X and a splice-site mutation of 1344G-->A. An exon-trapping experiment suggested that the splice-site mutation induced aberrant splicing of the gene. The second proband harbored a heterozygous maternal nonsense mutation, Q2538X and homozygous nonsense mutations R1189X. Analysis of the intragenic polymorphisms of PLEC1 suggested that R1189X mutations were due to paternal segmental uniparental isodisomy. These results indicate that PLEC1 is a possible causative gene in this clinical subtype, EBS associated with PA. Furthermore, two patients out of our three cases died in infancy. In terms of clinical prognosis, this novel subtype is the lethal variant in the EBS category.

[Selenium deficiency and electrocardiography changes in patients with severe motor and intellectual disabilities on long-term tube feeding].

In order to investigate the effect of selenium (Se) on electrocardiograms (ECG), we studied the serum Se levels and ECG in 25 patients with severe motor and intellectual disabilities. Serum Se levels in patients receiving tube feeding with enteral formulae were lower than those in orally fed patients. ECG abnormalities including an inverted T wave and depression of the ST segment were more common in patients under tube feeding. During tube feeding with enteral formulae, it is necessary to examine serum Se levels and ECG. Serum Se levels should be kept at 5 to 6 micrograms/dl to prevent cardiac dysfunction. To prevent selenium deficiency, either addition of ordinary foods, replacement by selenium-rich formula, or Se supplementation is recommended.

Improving perception of letters and visual structure of language.

Information about letters and the physical structure of language printed in Roman characters was given to children beginning to read. Experimental investigations coupled three alternative graphic modes of printing upper- and lower-case letters with an instructional intervention termed "Alpha-Beta" which provides practice in letter sorting, matching of letters, associative matching, and memory matching. In respect to graphics, Mode A letters were in standard alphabet form. Mode B provided standard letters with each backed by a unique half-tone (Visually Stippled Alphabet); Mode C provided standard letters with each backed by a unique visual texture (Visually Patterned Alphabet). Pre-posttest change in reading readiness was measured using the Metropolitan Readiness Test. In the first study 224 English-speaking 5- to 6-yr.-old children were tested. In the second there were 158 Spanish-speaking girls and boys 6 to 7 years old. It was predicted that Alpha-Beta intervention involving visually patterned alphabet would lead to the greatest increases in readiness scores. This is confirmed in both studies for children low in reading readiness preexperiment. Children high in reading readiness are less affected. The second experiment involved Spanish-speaking children and investigated intervention by Alpha-Beta against a no-intervention control. This confirms the value of Alpha-Beta per se. Possible explanations for the improvements are identified.