Bioengineering Human Upper Respiratory Mucosa: A Systematic Review of the State of the Art of Cell Culture Techniques.

Background: The upper respiratory mucosa plays a crucial role in both the physical integrity and immunological function of the respiratory tract. However, in certain situations such as infections, trauma, or surgery, it might sustain damage. Tissue engineering, a field of regenerative medicine, has found applications in various medical fields including but not limited to plastic surgery, ophthalmology, and urology.

Sensory neurons increase keratinocyte proliferation through CGRP release in a tissue engineered in vitro model of innervation in psoriasis.

Skin denervation has been shown to cause remission of psoriatic lesions in patients, which can reappear if reinnervation occurs. This effect can be induced by the activation of dendritic cells through sensory innervation. However, a direct effect of nerves on the proliferation of keratinocytes involved in the formation of psoriatic plaques has not been investigated.

In Vitro Characterization of Motor Neurons and Purkinje Cells Differentiated from Induced Pluripotent Stem Cells Generated from Patients with Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease mainly characterized by spasticity in the lower limbs and poor muscle control. The disease is caused by mutations in the gene leading in most cases to a loss of function of the sacsin protein, which is highly expressed in motor neurons and Purkinje cells. To investigate the impact of the mutated sacsin protein in these cells , induced pluripotent stem cell- (iPSC-) derived motor neurons and iPSC-derived Purkinje cells were generated from three ARSACS patients.

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers.

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation.

Differentiation of Human Induced Pluripotent Stem Cells into Mature and Myelinating Schwann Cells.

In the peripheral nervous system, Schwann cells (SCs) play a crucial role in axonal growth, metabolic support of neurons, and the production of myelin sheaths. Expansion of SCs after extraction from human or animal nerves is a long and often low-yielding process. We established a rapid cell culture method using a defined serum-free medium to differentiate human induced pluripotent stem cells (iPSCs) into SCs in only 21 days.

In Vitro 3D Modeling of Neurodegenerative Diseases.

The study of neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis) is very complex due to the difficulty in investigating the cellular dynamics within nervous tissue. Despite numerous advances in the in vivo study of these diseases, the use of in vitro analyses is proving to be a valuable tool to better understand the mechanisms implicated in these diseases. Although neural cells remain difficult to obtain from patient tissues, access to induced multipotent stem cell production now makes it possible to generate virtually all neural cells involved in these diseases (from neurons to glial cells).

Tridimensional cell culture of dermal fibroblasts promotes exosome-mediated secretion of extracellular matrix proteins.

Extracellular matrix (ECM) secretion, deposition and assembly are part of a whole complex biological process influencing the microenvironment and other cellular behaviors. Emerging evidence is attributing a significant role to extracellular vesicles (EVs) and exosomes in a plethora of ECM-associated functions, but the role of dermal fibroblast-derived EVs in paracrine signalling is yet unclear. Herein, we investigated the effect of exosomes isolated from stimulated human dermal fibroblasts.

Tissue-engineered in vitro modeling of the impact of Schwann cells in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons (MNs). To investigate whether Schwann cells could be involved in the disease pathogenesis, we developed a tissue-engineered three-dimensional (3D) in vitro model that combined MNs cocultured with astrocytes and microglia seeded on top of a collagen sponge populated with epineurium fibroblasts to enable 3D axonal migration. C2C12 myoblasts were seeded underneath the sponge in the presence or absence of Schwann cells.

Moyamoya Disease Susceptibility Gene Regulates Endothelial Barrier Function.

Background: Variants in the ring finger protein 213 () gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease.

Repair of peripheral nerve injuries using a prevascularized cell-based tissue-engineered nerve conduit.

One of the major challenges in the development of a larger and longer nerve conduit for peripheral nerve repair is the limitation in oxygen and nutrient diffusion within the tissue after transplantation preventing Schwann cell and axonal migration. This restriction is due to the slow neovascularization process of the graft starting from both nerve endings. To overcome this limitation, we propose the design of a living tissue-engineered nerve conduit made of an internal tube with a three-dimensional structure supporting axonal migration, which is inserted inside a hollow external tube that plays the role of an epineurium and is strong enough to be stitched to the severed nerve stumps.

[Tridimensional in vitro models of nervous and immune systems in the skin].

The immune system and the sensory nervous system are responsible for perceiving danger under distinct yet complementary forms. In the last few years, neuroimmune interactions have become an important topic of dermatological research for conditions including wound healing, atopic dermatitis and psoriasis. We present here a selection of tridimensional in vitro models that reproduce skin structure and integrate an immune or a sensory function.

Prevascularized Tissue-Engineered Human Vaginal Mucosa: Optimization and Validation.

Tissue engineering offers novel therapies for vaginal reconstruction in patients with congenital vaginal agenesis such as Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. This study aims to reconstruct a prevascularized tissue-engineered model of human vaginal mucosa (HVM) using the self-assembly approach, free of exogenous materials. In this study, a new cell culture method was used to enhance microcapillary network formation while maintaining sufficient biomechanical properties for surgical manipulation.

Lifting the veil on the keratinocyte contribution to cutaneous nociception.

Cutaneous nociception is essential to prevent individuals from sustaining injuries. According to the conventional point of view, the responses to noxious stimuli are thought to be exclusively initiated by sensory neurons, whose activity would be at most modulated by keratinocytes. However recent studies have demonstrated that epidermal keratinocytes can also act as primary nociceptive transducers as a supplement to sensory neurons.

Cutaneous nociception: Role of keratinocytes.

Recent years have brought an enhanced understanding of keratinocyte contribution to cutaneous nociception. While intra-epidermal nerve endings were classically considered as the exclusive transducers of cutaneous noxious stimuli, it has now been demonstrated that epidermal keratinocytes can initiate nociceptive responses, like Merkel cells do for the innocuous mechanotransduction. In the light of recent in vivo findings, this article outlines this paradigm shift that points to a not yet considered population of sensory epidermal cells.

Development of an innervated tissue-engineered skin with human sensory neurons and Schwann cells differentiated from iPS cells.

Cutaneous innervation is increasingly recognized as a major element of skin physiopathology through the neurogenic inflammation driven by neuropeptides that are sensed by endothelial cells and the immune system. To investigate this process in vitro, models of innervated tissue-engineered skin (TES) were developed, yet exclusively with murine sensory neurons extracted from dorsal root ganglions. In order to build a fully human model of innervated TES, we used induced pluripotent stem cells (iPSC) generated from human skin fibroblasts.

Biotechnological Management of Skin Burn Injuries: Challenges and Perspectives in Wound Healing and Sensory Recovery.

Many wound management protocols have been developed to improve wound healing after burn with the primordial aim to restore the barrier function of the skin and also provide a better esthetic outcome. Autologous skin grafts remain the gold standard in the treatment of skin burn, but this treatment has its limitation especially for patients presenting limited donor sites due to extensive burn areas. Deep burn injuries also alter the integrity of skin-sensitive innervation and have an impact on patient's quality of life by compromising perceptions of touch, temperature, and pain.

High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord.

Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice.

In vitro glycation of an endothelialized and innervated tissue-engineered skin to screen anti-AGE molecules.

Glycation is one of the major processes responsible for skin aging through induction of the detrimental formation of advanced glycation end-products (AGEs). We developed an innovative tissue-engineered skin combining both a capillary-like and a nerve networks and designed a protocol to induce continuous AGEs formation by a treatment with glyoxal. We determined the optimal concentration of glyoxal to induce AGEs formation identified by carboxymethyl-lysin expression while keeping their toxic effects low.

Early detection of structural abnormalities and cytoplasmic accumulation of TDP-43 in tissue-engineered skins derived from ALS patients.

Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the selective degeneration of motor neurons in the brain and spinal cord progressively leading to paralysis and death. Current diagnosis of ALS is based on clinical assessment of related symptoms. The clinical manifestations observed in ALS appear relatively late in the disease course after degeneration of a significant number of motor neurons.

A Longitudinal Low Dose μCT Analysis of Bone Healing in Mice: A Pilot Study.

Low dose microcomputed tomography (μCT) is a recently matured technique that enables the study of longitudinal bone healing and the testing of experimental treatments for bone repair. This imaging technique has been used for studying craniofacial repair in mice but not in an orthopedic context. This is mainly due to the size of the defects (approximately 1.

Sensory neurons accelerate skin reepithelialization via substance P in an innervated tissue-engineered wound healing model.

Keratinocytes are responsible for reepithelialization and restoration of the epidermal barrier during wound healing. The influence of sensory neurons on this mechanism is not fully understood. We tested whether sensory neurons influence wound closure via the secretion of the neuropeptide substance P (SP) with a new tissue-engineered wound healing model made of an upper-perforated epidermal compartment reconstructed with human keratinocytes expressing green fluorescent protein, stacked over a dermal compartment, innervated or not with sensory neurons.

Concise review: tissue-engineered skin and nerve regeneration in burn treatment.

Burns not only destroy the barrier function of the skin but also alter the perceptions of pain, temperature, and touch. Different strategies have been developed over the years to cover deep and extensive burns with the ultimate goal of regenerating the barrier function of the epidermis while recovering an acceptable aesthetic aspect. However, patients often complain about a loss of skin sensation and even cutaneous chronic pain.

Nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and glial-derived neurotrophic factor enhance angiogenesis in a tissue-engineered in vitro model.

Skin is a major source of secretion of the neurotrophic factors nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial-derived neurotrophic factor (GDNF) controlling cutaneous sensory innervation. Beside their neuronal contribution, we hypothesized that neurotrophic factors also modulate the cutaneous microvascular network. First, we showed that NGF, BDNF, NT-3, and GDNF were all expressed in the epidermis, while only NGF and NT-3 were expressed by cultured fibroblasts, and BDNF by human endothelial cells.

Spontaneous fibroblast-derived pericyte recruitment in a human tissue-engineered angiogenesis model in vitro.

Cooperation between endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. We developed a unique in vitro tissue-engineered model to study angiogenesis. The human endothelialized reconstructed connective tissue model promotes the formation of a three-dimensional branching network of capillary-like tubes (CLT) with closed lumens.

Comparative study of bovine, porcine and avian collagens for the production of a tissue engineered dermis.

Combining bovine collagen with chitosan followed by freeze-drying has been shown to produce porous scaffolds suitable for skin and connective tissue engineering applications. In this study collagen extracted from porcine and avian skin was compared with bovine collagen for the production of tissue engineered scaffolds. A similar purity of the collagen extracts was shown by electrophoresis, confirming the reliability of the extraction process.