Adipogenic cell sheets to recreate in vitro adipose tissue microenvironments

Cell sheet (CS) engineering, taking advantage of cellular self-matrix organized as in native tissue, has been largely explored, including by us, for different purposes [1â 3]. Herein we propose for the ï¬ rst time, the use of human adipose stem cells (hASCs)-derived CS to create adipose tissue analogues with different levels of maturation. hASCs were cultured on UpCellTM thermo-responsive dishes for 1, 3 and 5 days under basal conditions previously established by us [3]. The inï¬ uence of pre-differentiation time and respective cell number, over CS stability and differentiation was assessed. Mechanically robust CS were only obtained with 5 days pre-differentiation period. Adipogenesis was followed along the culture assessing the variation of expression of mesenchymal (CD73, CD105 but not CD90) and adipogenic (PPARg, FABP4 and LPL) markers by ï¬ ow cytometry, immunocytochemistry and RT-PCR. Increased ratio of differentiated cells was achieved for longer pre-differentiation periods, while maturation degree was modulated by the maintenance medium. Independently of the overall CS differentiation/maturation level, 3D constructs were fabricated by stacking and further culturing 3 CS. Thus, by varying the culture conditions, different 3D adipose tissue-like microenvironments were recreated, enabling future development of new tissue engineering strategies, as well as further study of adipose tissue role in the regeneration of different tissues.

Extracellular matrix components in peripheral nerve repair: how to affect neural cellular response and nerve regeneration?

Peripheral nerve injury is a serious problem affecting significantly patients' life. Autografts are the "gold standard" used to repair the injury gap, however, only 50% of patients fully recover from the trauma. Artificial conduits are a valid alternative to repairing peripheral nerve. They aim at confining the nerve environment throughout the regeneration process, and providing guidance to axon outgrowth. Biocompatible materials have been carefully designed to reduce inflammation and scar tissue formation, but modifications of the inner lumen are still required in order to optimise the scaffolds. Biomicking the native neural tissue with extracellular matrix fillers or coatings showed great promises in repairing longer gaps and extending cell survival. In addition, extracellular matrix molecules provide a platform to further bind growth factors that can be released in the system over time. Alternatively, conduit fillers can be used for cell transplantation at the injury site, reducing the lag time required for endogenous Schwann cells to proliferate and take part in the regeneration process. This review provides an overview on the importance of extracellular matrix molecules in peripheral nerve repair.

A cell-based model of extracellular-matrix-guided endothelial cell migration during angiogenesis.

Angiogenesis, the formation of new blood vessels sprouting from existing ones, occurs in several situations like wound healing, tissue remodeling, and near growing tumors. Under hypoxic conditions, tumor cells secrete growth factors, including VEGF. VEGF activates endothelial cells (ECs) in nearby vessels, leading to the migration of ECs out of the vessel and the formation of growing sprouts. A key process in angiogenesis is cellular self-organization, and previous modeling studies have identified mechanisms for producing networks and sprouts. Most theoretical studies of cellular self-organization during angiogenesis have ignored the interactions of ECs with the extra-cellular matrix (ECM), the jelly or hard materials that cells live in. Apart from providing structural support to cells, the ECM may play a key role in the coordination of cellular motility during angiogenesis. For example, by modifying the ECM, ECs can affect the motility of other ECs, long after they have left. Here, we present an explorative study of the cellular self-organization resulting from such ECM-coordinated cell migration. We show that a set of biologically-motivated, cell behavioral rules, including chemotaxis, haptotaxis, haptokinesis, and ECM-guided proliferation suffice for forming sprouts and branching vascular trees.

Perfusion-CT guided intravenous thrombolysis in patients with unknown-onset stroke: a randomized, double-blind, placebo-controlled, pilot feasibility trial.

INTRODUCTION: Patients with unknown stroke onset are generally excluded from acute recanalisation treatments. We designed a pilot study to assess feasibility of a trial of perfusion computed tomography (PCT)-guided thrombolysis in patients with ischemic tissue at risk of infarction and unknown stroke onset. METHODS: Patients with a supratentorial stroke of unknown onset in the middle cerebral artery territory and significant volume of at-risk tissue on PCT were randomized to intravenous thrombolysis with alteplase (0.9 mg/kg) or placebo. Feasibility endpoints were randomization and blinded treatment of patients within 2 h after hospital arrival, and the correct application (estimation) of the perfusion imaging criteria. RESULTS: At baseline, there was a trend towards older age [69.5 (57-78) vs. 49 (44-78) years] in the thrombolysis group (n = 6) compared to placebo (n = 6). Regarding feasibility, hospital arrival to treatment delay was above the allowed 2 h in three patients (25%). There were two protocol violations (17%) regarding PCT, both underestimating the predicted infarct in patients randomized in the placebo group. No symptomatic hemorrhage or death occurred during the first 7 days. Three of the four (75%) and one of the five (20%) patients were recanalized in the thrombolysis and placebo group respectively. The volume of non-infarcted at-risk tissue was 84 (44-206) cm(3) in the treatment arm and 29 (8-105) cm(3) in the placebo arm. CONCLUSIONS: This pilot study shows that a randomized PCT-guided thrombolysis trial in patients with stroke of unknown onset may be feasible if issues such as treatment delays and reliable identification of tissue at risk of infarction tissue are resolved. Safety and efficiency of such an approach need to be established.

Bone tissue engineering using foetal cell therapy.

Different cell sources for bone tissue engineering are reviewed. In particular, adult cell source strategies have been based on the implantation of unfractionated fresh bone marrow; purified, culture expanded mesenchymal stem cells, differentiated osteoblasts, or cells that have been modified genetically to express rhBMP. Several limiting factors are mentioned for these strategies such as low number of available cells or possible immunological reaction of the host. Foetal bone cells are presented as an alternative solution and review of actual treatments using these cells is presented. Finally, foetal cells used specifically for bone tissue engineering are characterised and potentially interesting therapeutic options are proposed.

Wound repair and regeneration.

The repair of wounds is one of the most complex biological processes that occur during human life. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In human adults, the wound repair process commonly leads to a non-functioning mass of fibrotic tissue known as a scar. By contrast, early in gestation, injured fetal tissues can be completely recreated, without fibrosis, in a process resembling regeneration. Some organisms, however, retain the ability to regenerate tissue throughout adult life. Knowledge gained from studying such organisms might help to unlock latent regenerative pathways in humans, which would change medical practice as much as the introduction of antibiotics did in the twentieth century.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas-mediated cell death in mice.

Tissue transglutaminase (TG2) is a protein cross-linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha-1b-adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti-Fas antibodies, which is sublethal for wild-type mice, kills all the TG2 knock-out mice within 20 hours. Although TG2-/- thymocytes exposed to anti-Fas antibodies die at the same rate as wild-type mice, TG2-/- hepatocytes show increased sensitivity toward anti-Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIP(L) (FLICE-inhibitory protein), or the rate of I-kappaBalpha degradation, but a decrease in the Bcl-xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl-xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand-induced hepatocyte proliferation with a simultaneous inhibition of the Fas-death pathway plays a determinant role.

Lack of Smad3 signaling leads to impaired skeletal muscle regeneration.

Smad3 is a key intracellular signaling mediator for both transforming growth factor-β and myostatin, two major regulators of skeletal muscle growth. Previous published work has revealed pronounced muscle atrophy together with impaired satellite cell functionality in Smad3-null muscles. In the present study, we have further validated a role for Smad3 signaling in skeletal muscle regeneration. Here, we show that Smad3-null mice had incomplete recovery of muscle weight and myofiber size after muscle injury. Histological/immunohistochemical analysis suggested impaired inflammatory response and reduced number of activated myoblasts during the early stages of muscle regeneration in the tibialis anterior muscle of Smad3-null mice. Nascent myofibers formed after muscle injury were also reduced in number. Moreover, Smad3-null regenerated muscle had decreased oxidative enzyme activity and impaired mitochondrial biogenesis, evident by the downregulation of the gene encoding mitochondrial transcription factor A, a master regulator of mitochondrial biogenesis. Consistent with known Smad3 function, reduced fibrotic tissue formation was also seen in regenerated Smad3-null muscle. In conclusion, Smad3 deficiency leads to impaired muscle regeneration, which underscores an essential role of Smad3 in postnatal myogenesis. Given the negative role of myostatin during muscle regeneration, the increased expression of myostatin observed in Smad3-null muscle may contribute to the regeneration defects.

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Neural differentiation and therapeutic potential of adipose tissue derived stem cells.

Neural tissue has historically been regarded as having poor regenerative capacity but recent advances in the growing fields of tissue engineering and regenerative medicine have opened new hopes for the treatment of nerve injuries and neurodegenerative disorders. Adipose tissue has been shown to contain a large quantity of adult stem cells (ASC). These cells can be easily harvested with low associated morbidity and because of their potential to differentiate into multiple cell types, their use has been suggested for a wide variety of therapeutic applications. In this review we examine the evidence indicating that ASC can stimulate nerve regeneration by both undergoing neural differentiation and through the release of a range of growth factors. We also discuss some of the issues that need to be addressed before ASC can be developed as an effective cellular therapy for the treatment of neural tissue disorders.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

Delayed priming promotes CNS regeneration post-rhizotomy in Neurocan and Brevican-deficient mice.

A wealth of literature has provided evidence that reactive tissue at the site of CNS injury is rich in chondroitin sulfate proteoglycans which may contribute to the non-permissive nature of the CNS. We have recently demonstrated using a murine model of human brachial plexus injury that the chondroitin sulfate proteoglycans Neurocan and Brevican are differentially expressed by two subsets of astrocytes in the spinal cord dorsal root entry zone (DREZ) following dorsal root lesion (Beggah et al., Neuroscience 133: 749-762, 2005). However, direct evidence for a growth-inhibitory role of these proteoglycans in vivo is still lacking. We therefore performed dorsal root lesion (rhizotomy) in mice deficient in both Neurocan and Brevican. Rhizotomy in these animals resulted in no significant increase in the number of sensory fibres regenerating through the DREZ compared to genetically matched controls. Likewise, a conditioning peripheral nerve lesion prior to rhizotomy, which increases the intrinsic growth capacity of sensory neurons, enhanced growth to the same extent in transgenic and control mice, indicating that absence of these proteoglycans alone is not sufficient to further promote entry into the spinal cord. In contrast, when priming of the median nerve was performed at a clinically relevant time, i.e. 7 weeks post-rhizotomy, the growth of a subpopulation of sensory axons across the DREZ was facilitated in Neurocan/Brevican-deficient, but not in control animals. This demonstrates for the first time that (i) Neurocan and/or Brevican contribute to the non-permissive environment of the DREZ several weeks after lesion and that (ii) delayed stimulation of the growth program of sensory neurons can facilitate regeneration across the DREZ provided its growth-inhibitory properties are attenuated. Post-injury enhancement of the intrinsic growth capacity of sensory neurons combined with removal of inhibitory chondroitin sulfate proteoglycans may therefore help to restore sensory function and thus attenuate the chronic pain resulting from human brachial plexus injury.

Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

In-vivo performance of high-density collagen gel tubes for urethral regeneration in a rabbit model.

Congenital malformations or injuries of the urethra can be treated using existing autologous tissue, but these procedures are sometimes associated with severe complications. Therefore, tissue engineering may be advantageous for generating urethral grafts. We evaluated engineered high-density collagen gel tubes as urethral grafts in 16 male New Zealand white rabbits. The constructs were either acellular or seeded with autologous smooth muscle cells, isolated from an open bladder biopsy. After the formation of a urethral defect by excision, the tissue-engineered grafts were interposed between the remaining urethral ends. No catheter was placed postoperatively. The animals were evaluated at 1 or 3 months by contrast urethrography and histological examination. Comparing the graft caliber to the control urethra at 3 months, a larger caliber was found in the cell-seeded grafts (96.6% of the normal caliber) than in the acellular grafts (42.3%). Histology of acellular and cell-seeded grafts did not show any sign of inflammation, and spontaneous regrowth of urothelium could be demonstrated in all grafts. Urethral fistulae, sometimes associated with stenosis, were observed, which might be prevented by urethral catheter application. High-density collagen gel tubes may be clinically useful as an effective treatment of congenital and acquired urethral pathologies.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-β1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.