François Berthod

Collagen-Based Biomaterials for Tissue Engineering Applications

Collagen is the most widely distributed class of proteins in the human body. The use of collagen-based biomaterials in the field of tissue engineering applications has been intensively growing over the past decades. Multiple cross-linking methods were investigated and different combinations with other biopolymers were explored in order to improve tissue function. Collagen possesses a major advantage in being biodegradable, biocompatible, easily available and highly versatile. However, since collagen is a protein, it remains difficult to sterilize without alterations to its structure. This review presents a comprehensive overview of the various applications of collagen-based biomaterials developed for tissue engineering, aimed at providing a functional material for use in regenerative medicine from the laboratory bench to the patient bedside.

In vitro reconstruction of a human capillary-like network in a tissue-engineered skin equivalent

For patients with extensive burns, wound coverage with an autologous in vitro reconstructed skin made of both dermis and epidermis should be the best alternative to split‐thickness graft. Unfortunately, various obstacles have delayed the widespread use of composite skin substitutes. Insufficient vascularization has been proposed as the most likely reason for their unreliable survival. Our purpose was to develop a vascular‐like network inside tissue‐engineered skin in order to improve graft vascularization. To reach this aim, we fabricated a collagen biopolymer in which three human cell types—keratinocytes, dermal fibroblasts, and umbilical vein endothelial cells—were cocultured. We demonstrated that the endothelialized skin equivalent (ESE) promoted spontaneous formation of capillary‐like structures in a highly differentiated extracellular matrix. Immunohistochemical analysis and transmission electron microscopy of the ESE showed characteristics associated with the microvasculature in vivo (von Willebrand factor, Weibel‐Palade bodies, basement membrane material, and intercellular junctions). We have developed the first endothelialized human tissue‐engineered skin in which a network of capillary‐like tubes is formed. The transplantation of this ESE on human should accelerate graft revascularization by inosculation of its preexisting capillary‐like network with the patients own blood vessels, as it is observed with autografts. In addition, the ESE turns out to be a promising in vitro angiogenesis model.—Black, A. F., Berthod, F., L’Heureux, N., Germain, L., Auger, F. A. In vitro reconstruction of a human capillary‐like network in a tissue‐engineered skin equivalent. FASEB J. 12, 1331–1340 (1998)

Inosculation of Tissue-Engineered Capillaries with the Host's Vasculature in a Reconstructed Skin Transplanted on Mice

The major limitation for the application of an autologous in vitro tissue‐engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary‐like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14‐day period was necessary to achieve a similar result with the non‐endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the hosts capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue‐engineered organs requiring a rapid vascularization.

Vasculature Guides Migrating Neuronal Precursors in the Adult Mammalian Forebrain via Brain-Derived Neurotrophic Factor Signaling

Adult neuronal precursors retain the remarkable capacity to migrate long distances from the posterior (subventricular zone) to the most anterior [olfactory bulb (OB)] parts of the brain. The knowledge about the mechanisms that keep neuronal precursors in the migratory stream and organize this long-distance migration is incomplete. Here we show that blood vessels precisely outline the migratory stream for new neurons in the adult mammalian forebrain. Real-time video imaging of cell migration in the acute slices demonstrate that neuronal precursors are retained in the migratory stream and guided into the OB by blood vessels that serve as a physical substrate for migrating neuroblasts. Our data suggest that endothelial cells of blood vessels synthesize brain-derived neurotrophic factor (BDNF) that fosters neuronal migration via p75NTR expressed on neuroblasts. Interestingly, GABA released from neuroblasts induces Ca2+-dependent insertion of high-affinity TrkB receptors on the plasma membrane of astrocytes that trap extracellular BDNF. We hypothesize that this renders BDNF unavailable for p75NTR-expressing migrating cells and leads to their entrance into the stationary period. Our findings provide new insights into the functional organization of substrates that facilitate the long-distance journey of adult neuronal precursors.

Tissue-engineered skin substitutes: from in vitro constructs to in vivo applications.

The field of skin tissue engineering is a paradigm for the various efforts towards the reconstruction of other tissues and organ substitutes. As skin replacement, this biotechnological approach has evolved from simple cultured autologous epidermal sheets to more complex bilayered cutaneous substitutes. The various types of such substitutes are herein presented with their intended use. However, two integrative characteristics are analysed more specifically because of their critical role: neovascularization and re‐innervation. Furthermore, the in vitro use of these various skin substitutes has shed light on various physiological and pathological phenomena. Thus, not only the in vivo application of these skin substitutes as grafts, but also their in vitro value as skin models, are presented.

Tissue-engineered human skin substitutes developed from collagen- populated hydrated gels: clinical and fundamental applications

The field of tissue engineering has opened several avenues in biomedical sciences, through ongoing progress. Skin substitutes are currently optimised for clinical as well as fundamental applications. The paper reviews the development of collagen-populated hydrated gels for their eventual use as a therapeutic option for the treatment of burn patients or chronic wounds: tools for pharmacological and toxicological studies, and cutaneous models for in vitro studies. These skin substitutes are produced by culturing keratinocytes on a matured dermal equivalent composed of fibroblasts included in a collagen gel. New biotechnological approaches have been developed to prevent contraction (anchoring devices) and promote epithelial cell differentiation. The impact of dermo-epidermal interactions on the differentiation and organisation of bio-engineered skin tissues has been demonstrated with human skin cells. Human skin substitutes have been adapted for percutaneous absorption studies and toxicity assessment. The evolution of these human skin substitutes has been monitored in vivo in preclinical studies showing promising results. These substitutes could also serve as in vitro models for better understanding of the immunological response and healing mechanism in human skin. Thus, such human skin substitutes present various advantages and are leading to the development of other bio-engineered tissues, such as blood vessels, ligaments and bronchi.

Collagen synthesis by fibroblasts cultured within a collagen sponge

We prepared a collagen sponge made of type I and III bovine collagen, glycosaminoglycans (GAG) and chitosan. Fibroblasts grown within the collagen sponge express a sixfold increase of their collagen synthesis, compared with fibroblasts embedded in a collagen gel. Moreover, collagen synthesis is twice as high in the collagen sponge than in a monolayer culture. The collagen sponge culture system promotes a dynamic model for us to perform studies on the regulations of collagen synthesis. Increased collagen production within the collagen sponge leads fibroblasts to reconstitute their own extracellular matrix, which should be more physiological than a bovine collagen gel.

A tissue-engineered endothelialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary-like tube formation in vitro.

Summary Background Because angiogenesis is a major feature of different physiological and pathological situations, the identification of factors that stimulate or inhibit this process and the elucidation of their mechanisms of action are most certainly of clinical relevance. We have produced a new model of endothelialized reconstructed dermis that promotes the spontaneous formation of a human capillary‐like network and its stabilization in vitro for a period longer than 1 month.

Extracellular Matrix Deposition by Fibroblasts Is Necessary to Promote Capillary-Like Tube Formation In Vitro

The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized‐reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary‐like tubes (CLT) formed by endothelial cells was reduced by up to 10‐fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP‐2 and MMP‐9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25–70% of the capillary‐like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF‐blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast‐conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary‐like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three‐dimensional cell organization. J. Cell. Physiol. 207: 491–498, 2006.

Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice

Wound healing of deep and extensive burns can induce hypertrophic scar formation, which is a detrimental outcome for skin functionality. These scars are characterized by an impaired collagen fibril organization with fibril bundles oriented parallel to each other, in contrast with a basket weave pattern arrangement in normal skin. We prepared a reconstructed skin made of a collagen sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 20 days. We transplanted it on the back of nude mice to assess whether this reconstructed skin could prevent scar formation. After transplantation, murine blood vessels had revascularized one-third of the sponge thickness on the fifth day and were observed underneath the epidermis at day 15. The reconstructed skin extracellular matrix was mostly made of human collagen I, organized in loosely packed fibrils 5 days after transplantation, with a mean diameter of 45 nm. After 40-90 days, fibril bundles were arranged in a basket weave pattern while their mean diameter increased to 56 nm, therefore exactly matching mouse skin papillary dermis organization. Interestingly, we showed that an elastic system remodeling was started off in this model. Indeed, human elastin deposits were organized in thin fibrils oriented perpendicular to epidermis at day 90 whereas elastic system usually took years to be re-established in human scars. Our reconstructed skin model promoted in only 90 days the remodeling of an extracellular matrix nearly similar to normal dermis (i.e. collagen fibril diameter and arrangement, and the partial reconstruction of the elastic system).