Self-assembled human osseous cell sheets as living biopapers for the laser-assisted bioprinting of human endothelial cells.

A major challenge during the engineering of voluminous bone tissues is to maintain cell viability in the central regions of the construct. In vitro prevascularization of bone substitutes relying on endothelial cell bioprinting has the potential to resolve this issue and to replicate the native bone microvasculature. Laser-assisted bioprinting (LAB) commonly uses biological layers of hydrogel, called 'biopapers', to support patterns of printed cells and constitute the basic units of the construct. The self-assembly approach of tissue engineering allows the production of biomimetic cell-derived bone extracellular matrix including living cells. We hypothesized that self-assembled osseous sheets can serve as living biopapers to support the LAB of human endothelial cells and thus guide tubule-like structure formation. Human umbilical vein endothelial cells were bioprinted on the surface of the biopapers following a predefined pattern of lines. The osseous biopapers showed relevant matrix mineralization and pro-angiogenic hallmarks. Our results revealed that formation of tubule-like structures was favored when the cellular orientation within the biopaper was parallel to the printed lines. Altogether, we validated that human osseous cell sheets can be used as biopapers for LAB, allowing the production of human prevascularized cell-based osseous constructs that can be relevant for autologous bone repair applications.

Adipose-tissue engineering: taking advantage of the properties of human adipose-derived stem/stromal cells.

Adipose tissue is now recognized as an important source of postnatal mesenchymal stem cells for regenerative medicine applications. For example, adipose-tissue engineering is an emerging approach that enables the development of autologous substitutes that could be used as an alternative to fat transplantation methods currently yielding variable outcomes for the long-term repair of soft-tissue defects. Here, we describe the production of unique tissue-engineered adipose tissues devoid of exogenous biomaterials produced from human adipose-derived stem/stromal cells. Our strategy is based on the dual self-assembly of extracellular components secreted and organized by the adipose-derived stromal cells after ascorbic acid stimulation, as well as their concomitant differentiation into adipocytes after adipogenic induction. When compared to stromal cells isolated from resected fat, lipoaspirated fat-derived cells featured an increased adipogenic potential and the enhanced ability to recreate three-dimensional adipose substitutes in vitro. These substitutes were histologically similar to native adipose tissue. They featured lipid-filled adipocytes embedded into an extracellular matrix rich in fibronectin as well as collagens I and V. On a functional level, the reconstructed adipose tissues expressed adipocyte-related transcripts and secreted adipokines typical of adipose tissue, such as leptin. Finally, the successful in vitro production of human adipose substitutes featuring an increased surface area (>30cm2) is described, reinforcing the notion that customized autologous reconstructed adipose tissues could be produced in the future to repair a wide range of soft-tissue defects.

[Porous matrix and primary-cell culture: a shared concept for skin and cornea tissue engineering]. / Matrice poreuse et culture de cellules primaires : un même concept pour la reconstruction cutanée et cornéenne.

Skin and cornea both feature an epithelium firmly anchored to its underlying connective compartment: dermis for skin and stroma for cornea. A breakthrough in tissue engineering occurred in 1975 when skin stem cells were successfully amplified in culture by Rheinwald and Green. Since 1981, they are used in the clinical arena as cultured epidermal autografts for the treatment of patients with extensive burns. A similar technique has been later adapted to the amplification of limbal-epithelial cells. The basal layer of the limbal epithelium is located in a transitional zone between the cornea and the conjunctiva and contains the stem cell population of the corneal epithelium called limbal-stem cells (LSC). These cells maintain the proper renewal of the corneal epithelium by generating transit-amplifying cells that migrate from the basal layer of the limbus towards the basal layer of the cornea. Tissue-engineering protocols enable the reconstruction of three-dimensional (3D) complex tissues comprising both an epithelium and its underlying connective tissue. Our in vitro reconstruction model is based on the combined use of cells and of a natural collagen-based biodegradable polymer to produce the connective-tissue compartment. This porous substrate acts as a scaffold for fibroblasts, thereby, producing a living dermal/stromal equivalent, which once epithelialized results into a reconstructed skin/hemicornea. This paper presents the reconstruction of surface epithelia for the treatment of pathological conditions of skin and cornea and the development of 3D tissue-engineered substitutes based on a collagen-GAG-chitosan matrix for the regeneration of skin and cornea.

HSV vector-mediated transduction and GDNF secretion from adipose cells.

The accessibility of adipose tissue and its ability to secrete various bioactive molecules suggest that adipose cells may be attractive targets for gene therapy applications. Here, we report the use of highly defective herpes simplex virus (HSV) vectors as suitable gene transfer agents for adipose cells in culture and fat tissue in animals. Using an in vitro model of human adipose differentiation, we first demonstrated that mature adipocytes and their precursor cells express the two principal HSV viral entry receptors HveA and HveC (nectin-1) and are efficiently transduced at a low multiplicity of infection by HSV-lacZ reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene vectors. Extended expression of beta-galactosidase and secretion of GDNF occurred in transduced fat tissue explants from rabbits. In vivo gene transfer to rabbit subcutaneous adipose tissue resulted in local GDNF expression for at least 2 months. These experiments establish the efficient transduction of adipose cells by HSV vectors and suggest that fat tissue may represent a useful site for HSV-mediated gene delivery with potential for therapeutic applications.

Herpesvirus-mediated systemic delivery of nerve growth factor.

Sustained systemic dissemination of therapeutic proteins from peripheral sites is an attractive prospect for gene therapy applications. Replication-defective genomic herpes simplex virus type 1 (HSV-1) vectors were evaluated for their ability to express nerve growth factor (NGF) as a model gene product both locally and systemically. Intra-articular inoculation of NGF expression vectors in rabbits resulted in significant increases in joint lavage and blood plasma NGF that persisted for 1 year. A rhesus macaque injected intra-articularly displayed a comparable increase in plasma NGF for at least 6 months, at which time the serum NGF levels of this animal were sufficient to cause differentiation of PC12 cells in culture, but not to increase footpad epidermis innervation. Long-term reporter transgene expression was observed primarily in ligaments, a finding confirmed by direct inoculation of patellar ligament. Patellar ligament inoculation with a NGF vector resulted in elevated levels of circulating NGF similar to those observed following intra-articular vector delivery. These results represent the first demonstration of sustained systemic release of a transgene product using HSV vectors, raising the prospect of new applications for HSV-1 vectors in the treatment of systemic disease.

The mixed lineage kinase leucine-zipper protein kinase exhibits a differentiation-associated localization in normal human skin and induces keratinocyte differentiation upon overexpression.

Leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase is a recently described protein serine/threonine kinase which belongs to the mixed lineage kinase family. The overall pattern of expression of the leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase gene in embryonic and adult mouse tissues suggested that this kinase could be involved in the regulation of epithelial cell proliferation and differentiation. In order to get more insights into the potential role of leucine-zipper protein kinase in these cellular processes, we characterized its expression in normal human skin, both at the mRNA and protein levels. In situ hybridization, western blotting, and indirect immunofluorescence studies were conducted to localize leucine-zipper protein kinase on various human skin tissues. This is one of the first reports that leucine-zipper protein kinase has a very precise pattern of expression in human skin epithelia, as both mRNA and protein are restricted to the granular layer of the epidermis and inner root sheath of hair follicles. Detection of leucine-zipper protein kinase protein on skin from various body sites, donors of different ages as well as on reconstructed human skin always reveals that leucine-zipper protein kinase is present only in the very differentiated keratinocytes of epidermis and hair follicles. To determine directly whether leucine-zipper protein kinase exhibits any effect on cell growth and differentiation, keratinocytes were transfected with an expression vector harboring the leucine-zipper protein kinase cDNA. The presence of this construct in keratinocytes results in growth arrest together with a concomitant increase in filaggrin expression. Collectively, our results indicate that leucine-zipper protein kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes.

Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius.

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.

The type I keratin 19 possesses distinct and context-dependent assembly properties.

Keratins (K), the cytoplasmic intermediate filament (IF) proteins of epithelial cells, are encoded by a multigene family and expressed in a tissue- and differentiation-specific manner. In human skin, keratinocytes of the basal layer of epidermis and the outer root sheath of hair follicles express K5 and K14 as their main keratins. A small subpopulation of basal cells exhibiting stem-cell like characteristics express, in addition, K19. At 40 kDa, this keratin is the smallest IF protein due to an exceptionally short carboxyl-terminal domain. We examined the assembly properties of K19 and contrasted them to K14 in vitro and in vivo. Relative to K5-K14, we find that K5-K19 form less stable tetramers that polymerize into shorter and narrower IFs in vitro. When transiently co-expressed in cultured baby hamster kidney cells, the K5 and K19 combination fails to form a filamentous array, whereas the K5-K14 and K8-K19 ones readily do so. Transient expression of K19 in the epithelial cell lines T51B-Ni and A431 results in its integration into the endogenous keratin network with minimal if any perturbation. Collectively, these results indicate that K19 possesses assembly properties that are distinct from those of K14 and suggest that it may impart unique properties to the basal cells expressing it in skin epithelia.

Localization of Merkel cells at hairless and hairy human skin sites using keratin 18.

Merkel cells are neurosecretory cells of the skin with epithelial features such as desmosomes and expression of keratins 8, 18, 19, and 20. Merkel cells are scarcely distributed in adult human skin. Although they are present in hair follicles, their density is higher at hairless anatomic sites such as palms and soles. These cells are often innervated by sensory nerve fibers and are thought to be specialized mechanosensory skin receptor cells. However, their precise origin and function are not clearly established. The aim of this study was to localize Merkel cells in human hairless and hairy skin by immunohistochemistry with antibodies Ks18.174 and Ks19.1 directed against keratins 18 and 19, respectively. In glabrous skin of palm and sole, Merkel cells have been localized at the bottom of the rete ridges, in the epidermal basal layer. To study Merkel cell distribution at hairy anatomic sites, we have chosen breast skin, a tissue containing small hair follicles typical of those covering most of the body's surface. Merkel cells were present in the interfollicular epidermis. In hair follicles, they have been identified in the isthmus region.

Amino-terminal methionine processing of the protein HPr in Streptococcus salivarius grown in continuous culture.

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Streptococci possess two forms of HPr which differ by the presence or the absence of the N-terminal methionine (Met). These forms are called HPr-1 (without Met) and HPr-2 (with Met). In order to determine whether the ratio of these two forms varies with growth conditions, we measured the amount of HPr-1 and HPr-2 present in Streptococcus salivarius grown in continuous culture at pH 7.5. The results indicated that the HPr-1/HPr-2 ratio: 1) was not related to the cellular amount of total HPr; 2) was highest (10.2 +/- 3.5) under glucose (a PTS sugar) limitation (10 mM) and low dilution rate (D = 0.1 h-1; g = 6.9 h); 3) was decreased 2.4- to 5.7-fold when the amount of glucose and/or D was increased; 4) was not influenced by D when cells were cultured on galactose (a non-PTS sugar) but was two-fold higher under conditions of galactose excess (200 mM). We suggest that the cleavage of the N-terminal HPr Met is not a stochastic phenomenon but is dictated by growth conditions.