Synthesis and biological evaluation of modified laminin peptide (N2S2-KDP) with enhanced affinity for neuronal growth and targeted molecular imaging (SPECT).

An analog of γ1 laminin (RDIAEIIKDI) decapeptide has been used to augment neuronal survival and regeneration after injuries, during aging and other CNS disorder. As a prime synthetic peptide, KDI, is responsible for the neurite outgrowth of human embryonic neurons. In this study, we have designed, modified a KDI derivative and synthesized by replacing isoleucine (I) with Pro (P) amino acid at C-terminal to enhance its potency towards neurite growth. -Cys-Gly-Cys (-CGC) N2S2 motif was also incorporated in the present design for peptide radiolabeling. The modified peptide showed a better binding with the desired 3T1M receptor for neurite growth. The peptide was synthesized using solid phase peptide synthesis and Fmoc-strategy with more than 80% yield. The receptor binding studies of 99mTc-N2S2-KDP in Neuro2A cell lines showed Kd value in 31 nM range and the complex showed appreciable brain uptake in mice. The results on human SH-SY5Y indicate that the unlabeled N2S2-KDP may perhaps be useful for neurite growth in neurodegenerative disorder.

SEMA3A suspended in matrigel improves titanium implant fixation in ovariectomized rats.

The aim of this study was to evaluate the effect of SEMA3A released from matrigel on implant fixation in ovariectomized (OVX) rats. Sixty female rats were subjected to bilateral ovariectomy. Twelve weeks later, rats were randomly divided into three groups according to implants they accepted: (1) Control, implants with distilled water; (2) Matrigel, implants with matrigel coating; (3) Matrigel + SEMA3A, implants with coating of SEMA3A suspended in matrigel. Implants were inserted in metaphysis of proximal tibiae in all animas bilaterally. In vitro release of SEMA3A was tested using enzyme linked immunosorbent assay. In vitro release of SEMA3A was detectable during the first 10 days, and a burst release of was observed during the first 3 days. No significant difference was observed between Control and Matrigel group. The protective effects of SEMA3A in matrigel on peri-implant bone, implant osseointegration and fixation was confirmed. Compared to matrigel alone, SEMA3A suspended in matrigel increased percent bone volume by 88.7% and 83.3% (p < 0.01), bone-to-implant contact ratio by 148.9% (p < 0.01), and 24.8% (p < 0.05), the maximal push-out force by 149.3% and 209.2% (p < 0.01) at 4 and 8 weeks after implant insertion, respectively. Surface modification with SEMA3A suspended in matrigel improved implant osseointegration and fixation in the proximal tibiae of OVX rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2060-2065, 2017.

Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF.

Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury.

Controllable production of transplantable adult human high-passage dermal papilla spheroids using 3D matrigel culture.

We have succeeded in culturing human dermal papilla (DP) cell spheroids and developed a three-dimensional (3D) Matrigel (basement membrane matrix) culture technique that can enhance and restore DP cells unique characteristics in vitro. When 1 × 10(4) DP cells were cultured on the 96-well plates precoated with Matrigel for 5 days, both passage 2 and passage 8 DP cells formed spheroidal microtissues with a diameter of 150-250 µm in an aggregative and proliferative manner. We transferred and recultured these DP spheroids onto commercial plates. Cells within DP spheres could disaggregate and migrate out, which was similar to primary DP. Moreover, we examined the expression of several genes and proteins associated with hair follicle inductivity of DP cells, such as NCAM, Versican, and α-smooth muscle actin, and confirmed that their expression level was elevated in the spheres compared with the dissociated DP cells. To examine the hair-inducing ability of DP spheres, hair germinal matrix cells (HGMCs) and DP spheres were mixed and cultured on Matrigel. Unlike the dissociated DP cells and HGMCs cocultured in two dimensions, HGMCs can differentiate into hair-like fibers under the induction of the DP spheres made from the high-passage cells (passage 8) in vitro. We are the first to show that passage 3 human HGMCs differentiate into hair-like fibers in the presence of human DP spheroids. These results suggest that the 3D Matrigel culture technique is an ideal culture model for forming DP spheroids and that sphere formation partially models the intact DP, resulting in hair induction, even by high-passage DP cells.

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics.

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.

Rapid repair and regeneration of damaged rabbit sciatic nerves by tissue-engineered scaffold made from nano-silver and collagen type I.

A tissue-engineered scaffold with nano-silver and collagen type I was constructed and investigated for its ability to adsorb laminin and the usefulness in the repair and regeneration of damaged peripheral nerves in animals. The nano-silver scaffold displayed ideal microtubule structure under electronic microscope; even distribution of the nano-silver particles was also seen with energy spectrometry. After immersion in a laminin solution, the laminin-attached scaffolds were implanted into rabbits to repair a 10-mm injury of the sciatic nerve. At 30 days post-implantation, regeneration of the damaged nerve was evaluated by transmission electron microscopy, electrophysiological examination and fluoro-gold (FG) retrograde labelling. Compared with the control collagen-scaffold without nano-silver, the nano-silver-containing scaffold showed a higher rate of laminin adsorption, regenerated a nerve with a thicker myelin sheath and improved the nerve conduction velocity and nerve potential amplitude. FG retrograde labelled the newly grown axons in the spinal cord cortex anterior horn and the dorsal root ganglion. These results demonstrate the superior functionality of the nano-silver-collagen scaffold in the adsorption to laminin and subsequent regeneration of damaged peripheral nerves.

Cultivo in vitro con colágeno y fibroblastos humanos de un equivalente de mucosa oral de espesor total / In vitro culture with collagen and human fibroblasts of a full-thickness oral mucosa equivalent

Objetivos. El presente trabajo tiene por objetivo obtener, mediantecultivo in vitro, láminas de tejido oral en las que se pueda identificar lasestructuras de una mucosa oral completa. La aplicación clínica del presenteestudio permitiría, en determinados casos, la sustitución del empleo de injertoslibres de piel o autólogos de mucosa oral por esta técnica. Material y Método.A partir de pequeñas biopsias de mucosa oral se hicieron cultivos primariosde queratinocitos. A partir de estos cultivos primarios se realizaron cultivossecundarios sobre una submucosa artificial constituida por colágeno y fibroblastoshumanos. Se analizaron histológicamente sus características in vitro, yulteriormente se procedió a la realización de injertos en ratones atímicospara conocer su comportamiento in vivo. Resultados. Los cultivos primariosfueron confluentes en un plazo mínimo de 10 días y máximo de 12 días, periodosimilar al observado para la confluencia de los cultivos secundarios. El tiempotranscurrido desde la toma de la muestra hasta la obtención de una mucosaartificial completa osciló entre los 20 y los 22 días, mostrando las característicashistológicas de una mucosa normal. Tras 17 días de injerto en ratonesinmunoincompetentes, sin ningún tipo de contingencia clínica, la caracterizaciónhistológica e inmunohistoquímica (citoqueratinas 13 y 19, colágenoIV y laminina) confirmó la similitud de la mucosa in vitro con la mucosa oralsana. Conclusión. Es posible mediante técnicas de cultivo in vitro la obtenciónde un equivalente de mucosa oral completa con colágeno y fibroblastos. Sibien esta mucosa muestra un importante grado de retracción, su manejo clínicoes muy favorable(AU)

Objectives. The objective of this study was to obtain,by in vitro culture, sheets of oral tissue in which complete oral mucosastructures can be identified. Clinical application of the findings ofthis study will allow the replacement of free skin grafts or autologousoral mucosa grafts by this technique in certain cases.Material and Method. Primary keratinocyte cultures were preparedfrom small biopsy samples of oral mucosa. Secondary cultures wereprepared from these primary cultures on an artificial submucosaconstituted by collagen and human fibroblasts. The cell cultureswere analyzed histologically in vitro and then used for graft implantsin athymic mice to study their behavior in vivo.Results. The primary cultures were confluent within a minimumperiod of 10 days and maximum of 12 days, which is similar to theperiod that the secondary cultures required to reach confluence.The time from sampling to achieving a complete artificial mucosaranged from 20 to 22 days. The artificial mucosa showed histologiccharacteristics of a normal mucosa. After 17 days of graftimplantation in immunoincompetent mice without any clinicalcontingency, histologic and immunohistochemical characterization(cytokeratins 19 and 13, collagen IV, and laminin) confirmed thesimilarity of the mucosa in vitro to healthy oral mucosa.Conclusion. A complete oral mucosa equivalent can be preparedwith collagen and fibroblasts using in vitro culture techniques.Although this mucosa shows considerable retraction, its clinicalhandling is very favorable(AU)

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations.

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.

Matrigel: a useful tool to study endothelial differentiation.

Matrigel is a matrix of a mouse basement membrane neoplasm. It represents a complex mixture of basement membrane proteins including laminin, type IV collagen, entactin/nitrogen and proteoheparan sulfate, but it also contains growth factors. Matrigel induces endothelial cells to differentiate as evidenced by both the morphologic changes and by the reduction in proliferation and, therefore, offers a convenient model to study biochemical and molecular events associated with angiogenesis. Further, Matrigel permits to study the roles of the extracellular matrix in angiogenesis.

Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma.

The influence of laminin on cell cultures derived from unilateral acoustic nerve schwannomas was investigated. Cell cultures were initiated from 12 schwannomas, removed via the enlarged middle cranial fossa approach. Tumor tissue was dispersed by collagenase treatment and cells seeded in uncoated or laminin-coated culture dishes. Confluent cultures were immunocytochemically characterized with antibodies against S-100, CD 68, factor VIII-related antigen and type IV collagen. Cell adhesion in response to different doses of laminin was evaluated with an electronic cell counter. The effect of laminin on cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA. Cells cultured on laminin as substrate appeared more differentiated with long, fusiform, cytoplasmic processes. Cultured cells stained positive for S-100, not for factor VIII-related antigen or CD 68. Only cells cultured on laminin deposited a dense extracellular network of type IV collagen. When laminin was added to the culture medium, cell attachment and proliferation was stimulated in a dose dependent manner. Maximal stimulation of both was observed with a laminin concentration of 50 micrograms/ml, which induced a nearly 2-fold increase in cell attachment and an approximately 66% increase in DNA content. Since laminin is a major component of the extracellular matrix in schwannomas, the possibility exists that laminin is also mitogenic for human neoplastic Schwann cells in situ.

Synthetic cell-adhesive laminin peptide YIGSR conjugated with polyethylene glycol has improved antimetastatic activity due to a longer half-life in blood.

This study was conducted to determine the mechanisms for the enhanced inhibitory effect of cell-adhesive peptides conjugated to polyethylene glycol (PEG) on tumor metastasis. Tyr-Ile-Gly-Ser-Arg (YIGSR), a laminin-derived peptide, conjugated with amino-PEG (YIGSR-aPEG) inhibited lung metastasis of B16-BL6 melanoma cells more effectively than unconjugated YIGSR peptide. [125I]-YIGSR-aPEG and native [125I]-YIGSR showed similar biphasic elimination and profiles after intravenous injection into C57BL/6 mice. Both [125I]-YIGSR and [125I]-YIGSR-aPEG expressed similar plasma half-lives and organ distributions. The radioactivity of both compounds was transported rapidly from the blood to the kidneys, and immediately excreted into the urine. [125I]-YIGSR was almost completely degraded in the urine, but [125I]-YIGSR-aPEG was not. In an in vitro stability assay, [125I]-YIGSR was degraded immediately upon incubation with mouse serum, whereas [125I]-YIGSR-aPEG was not degraded after 180 min incubation in mouse serum. These findings indicate that the enhanced inhibitory effect of YIGSR-aPEG on lung metastasis might be due to its increased stability in the blood.

Biological distribution of 99mTc-labeled YIGSR and IKVAV laminin peptides in rodents: 99mTc-IKVAV peptide localizes to the lung.

Two laminin-derived peptides containing either YIGSR or IKVAV (single amino acid code) sequences were radiolabeled with 99mTc and their biological distribution evaluated in rodents. Both 99mTc-peptides cleared rapidly from the circulation though the kidney, and to a lesser extent, through the liver. 99mTc-YIGSR peptide did not accumulate in any organ examined in normal, tumored, and emphysemic mice. The 99mTc-IKVAV peptide localized within 10 min to the lung of normal animals, resulting in lung-to-blood ratios of approximately 23:1. The 99mTc-IKVAV peptide localized to lung after submicron filtration and after intraperitoneal injection, suggesting that particulates do not major role in localization. Pre-incubation of 99mTc-IKVAV peptide in whole blood decreased lung localization, suggesting that margination of radiolabeled cells does not play a major role in the lung localization. When 99mTc-IKVAV was injected into mice with tumored lungs (melanoma), the lung uptake was markedly increased (up to 20% injected dose higher than control lungs) at all time points examined (10, 30, and 120 min). When 99mTc-IKVAV was injected into mice with genetic emphysema, the lung uptake was markedly decreased at all time points. The localization of the 99mTc-IKVAV-containing peptide to the lung is consistent with a receptor-based mechanism.

Cell-adhesive immunoglobulin M in human plasma.

Human plasma contains a cell-adhesive protein that has a structure related to immunoglobulins. This protein was purified by affinity chromatography on an elastin-Sepharose column and by Mono Q anion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing and reducing conditions revealed that this protein is a kind of immunoglobulin M (IgM). Antibodies against the mu chain and against the Fc region of IgM inhibited the adhesion of cells to this protein. Addition of the peptide GRGDS into media inhibited the adhesion, too. These results suggest that this protein is a special subset of IgM having a cell-binding sequence in the Fc region. We propose the name "cell-adhesive immunoglobulin M (CA-IgM)" for this protein. CA-IgM binds to alpha-elastin and laminin suggesting that it may play a role in the interaction between cells and the extracellular matrix.

Uptake and degradation in vivo and in vitro of laminin and nidogen by rat liver cells.

Laminin antigens are known to be present in the blood of normal individuals. In the present study we have investigated the fate of laminin-related molecules in the circulation. After intravenous injection in rats, the native laminin-nidogen complex, as well as the separated proteins, were rapidly eliminated from the blood (half-lives 2-10 min) by the liver. The large laminin fragments E1 and E8 (Mr 400,000 and 280,000 respectively), which contain the major cell-adhesion-promoting activities of laminin, were also cleared from the blood mainly by the liver, but the rate of uptake was an order of magnitude lower for these fragments than for laminin. This indicates that the uptake of laminin did not occur via cell-adhesion receptors. The endothelial cells of liver were the most important cell type in the uptake of laminin-nidogen complex, nidogen, laminin and fragment E1, whereas the parenchymal cells were responsible for more than 50% of the uptake of E8 in the liver. Studies in vitro with cultured liver endothelial cells and parenchymal cells demonstrated that the ligands were endocytosed and degraded independently of plasma factors. The results reveal that the level of laminin antigens in blood is a very complex parameter. It is not only dependent on the turnover of basement membranes, but also on the degree of degradation of the material released into the blood and on the functional state of the liver, particularly the liver endothelial cells.

Differences in the solubility and susceptibility to proteolytic degradation of basement-membrane components in adult and embryonic mouse tissues.

We have studied susceptibility of basement membranes in a variety of tissues to solubility in guanidine hydrochloride and to proteolytic degradation by trypsin and thermolysin. Unfixed sections from embryonic and adult mouse tissues and the EHS tumor were subjected to solvent buffers or digested with enzymes. The retention or disappearance of the basement-membrane components nidogen, laminin, collagen IV, and heparan sulfate proteoglycan was subsequently assayed by immunofluorescence. Our data showed that in all tissues nidogen was the most readily solubilized component and the most susceptible to proteolytic degradation. With few exceptions, nidogen in embryonic tissues was more susceptible to degradation than that in adult tissues, and this correlated well with the susceptibility of the other basement-membrane components to be degraded. We conclude that basement membranes differ quite markedly in their solubility and their susceptibility to proteolytic degradation and that these properties reflect differences in their molecular structure.

Preferential adhesion of brain astrocytes to laminin and central neurites to astrocytes.

Astrocytes from either fetal or newborn rat brain adhered preferentially to surfaces coated with active laminin. Neurites from septal neurons did not show a preference for active over inactive laminin. However, when given a choice between laminin and an astrocytic surface, septal cells preferred to extend neurites over astroglial processes. Hence, laminin paths can guide astrocyte migration, which, in turn, can guide neurite elongation.