As is well documented, the presence of a healthy derma is essential for the success of an epidermal graft. Consequently, a derivative of hyaluronic acid (the HYAFF 11) has been processed into a tridimensional scaffold and then seeded with fibroblasts. The resulting device has been largely tested in vitro, in vivo and in clinical pilot studies. It appeared that, thanks to its mechano-biological and histological performances, the new derma equivalent has all the characteristics required for an optimal dermal bed for a successful keratinocytes graft. To our knowledge, this is the first autologous derma equivalent device prototype ever engineered. Several properties make this device a highly innovative and effective prototype: - The scaffold is made of a natural, non-immunogenic, biocompatible, and bioactive material (hyaluronic acid). This greatly decreases the risk of an immunitary rejection of the scaffold, as for example is usually seen with collagen-based scaffolds, and guarantees the feasibility of multiple successive grafts of the same device. At the same time, as hyaluronic acid and benzyl alcohol are the only degradation products of the HYAFF 11, no toxic molecules are released by the scaffold. Moreover, the long multimeric chain of hyaluronic acid is naturally cleaved in short oligomers by the ialuronidases. These small molecules of ialuronic acid have a proven angiogenic effect. Consequently, the HYAFF 11 scaffold degradation promotes the revascularisation of the wound bed, which is an essential step for a successful kerationocytes growth. - The scaffold degrades slowly and progressively, while the seeded cells multiply and deposit new ECM. Therefore, on one side, the scaffold remains in place while the newly forming derma still requires a tridimensional support, while on the other side the seeded fibroblasts deposit the new ECM necessary for repair of the damaged derma and for the physical support of the keratinocytes layer. - The number of cells seeded is relatively low (105/cm2). The advantage is double. On the one hand, only a small biopsy (1-2 cm2) of healthy skin is required as source of cells for the autologous derma and epidermis production, thus reducing the patient discomfort and enhancing the chances of finding a non-burned donor area. On the other hand, cells need to proliferate in vitro only for 2-3 weeks before the device is ready for the grafting, thus allowing a short delay before graft, and consequently, a better protection and repair of the burned area. - The scaffold is seeded with autologous cells, thus eliminating those risks of immunitary rejection of the graft and contraction of diseases associated instead with both the allologous and heterologous derma grafts.
A unique off the shelf prototype device for burn care providing immediate protection and simultaneous regenerative effect
A composite device, made of a support of hyaluronic acid derivative (HYAFF 11) overlaid by a layer of silicone was engineered. This prototype has the following novelties: - The silicone layer prevents fluid loss maintaining a moisture vapour transmission rate similar to the healthy epidermis. As the silicone is a semi-permeable material, while it protects the body against dehydration, it also permits an exchange of oxygen and carbon dioxide, thus allowing the wound to "breathe". - The silicone membrane creates a physical barrier to bacterial infection. - When in contact with the wound, a thick hyaluronic acid-rich interface is created providing a micro environment that enables the formation of a granulation tissue. HYAFF 11 has highly hydrophilic proprieties and absorbs immediately the wound essudate creating a gel-like matrix. This matrix adheres perfectly to the wound and because of the high concentration of hyaluronic acid recreates an environment similar to the fetal extracellular matrix, thus promoting the regeneration of the burned tissue. Additionally, the angiogenetic proprieties of the small molecules of ialuronic acid enhance the neoformation of vases which largely contribute to nourishing the newly forming derma and the eventual successive keratinocytes graft. Burn wounds are a significant cause of mortality and/or long-term health care. The uniqueness of this device prototype is based on the fact that it assembles the impermeability to liquids of the silicone layer with the bioactivity of hyaluronic acid, thus providing an immediate protection to the burned area and, at the same time, a long-term effect for the healing of the wound. From a commercial perspective, even the number of burns in developed markets being relatively small, the clinical need for an effective device is huge, with only one similar product already on the market (Integra Dermal Regeneration Template from Integra LifeSciences). As a consequence, we expect this prototype to become a highly competitive and commercially aggressive product. Therefore, independently from the Consortium, and based only in its own resources, FAB has recently supported a few pilot clinical evaluation tests to assess the efficacy of this prototype and expects to launch a new product at the beginning of 2002.
All skin substitute volume moves through professional channels. This kind of product is surgically applied, and thus is appropriate only to a surgical setting, including hospitals, surgicentres, and burn centres. Consequently, we aimed at converting the experimental manufacture of the derma equivalent prototype into a pilot production setting. We focused our standardisation process on five main areas of the production: the biopsy harvesting, biopsy arrival and processing, cells extraction, cells expansion phase and seeding of biomaterials, and finally, packaging and shipping. We established check points all along the production line to guarantee precision, quality, and safety for both the patient and the operator. As a result, we were able to reduce altogether the dramatic experimental variability observed when working with cells from different patients and, at the same time, we assured that each patient biopsy was treated individually and with extreme care but without losing the production line process. The setting of this highly controlled production was quite a challenging task, due to all the variables involved, which could explain why such a kind of production has yet to be realised even by the US-based companies. Before the beginning of the EC funded project, FAB had launched a product (Laserskin) which is composed of a hyaluronic-based membrane and autologous keratinocytes. This product is commercialised as an epidermis substitute. Nevertheless, it is well documented that a good dermis is essential for a successful keratinocytes graft. Thereafter, independently from EC funding, but in close scientific collaboration with researchers in the consortium, FAB has completed the scaling up of its two-stage dermo-epidermal autologous grafting procedure for the treatment of chronic wounds. FAB has set up an 800 m2 facility for cell culture, developed CE-accredited SOP's for cell culture and graft delivery, organised the logistics of autologous skin grafting, and developed and trained a group of product specialists currently promoting the product in Italy. As a consequence, FAB is currently the first and only European organisation with CE-approved products, and one of the few companies worldwide generating sales with tissue-engineered products.
During the execution of this project the following technologies have been developed: - The technologies to process into specific tri-dimensional forms a biocompatible and biodegradable semi-synthetic class of polymers. - The development of technologies to cultivate and grow cells to be used for tissue engineering purposes. In particular, we applied FAB's know-how for the modification of a highly biocompatible but otherwise not mechanically workable material, such as the gel-like hyaluronic acid, to obtain a solid and modifiable material, which maintains all the biological characteristics of the native molecule and, additionally, becomes conveniently modifiable in pre-determined physical structures. First, we developed the knowledge and the tools to transform hyaluronic acid-based fibres (HYAFF 11) into tridimensional devices of variable thickness (HYAFF 11 non woven meshes). We then coupled the technology for the production of these devices with newly developed technologies to either silicon-coat or cell-seed these devices. In the first case, we acquired the ability to manufacture a composite prototype which marries the biocompatibility and bioactivity of hyaluronic acid with the impermeability and mechanic resistance of silicon. In the second case we specifically adapted standard cell culture methods to the production of human derma. During the scaling up process of the newly acquired technologies and devices, specific efforts were made to engineer and realise a new piece of equipment: - Prototype carding machine. Although the carding technology can be transferred from the textile industry, we needed to build a new carding machine, especially projected to produce non woven fleece based on hyaluronic acid derivatives. The carding machine developed by the Consortium, based on HDB's experience in textile machine engineering, is designed to process hyaluronic acid-based fibres, characterised by a very low tensile strength, and to produce only a few kilograms of fleece per day, in order to minimise the expenses of the starting material. Very important for the manufacture of material intended for clinical use, this prototype machine does not need the use of lubricating materials such as oil, enzyme or heat. It could be realistically envisaged to utilise these newly developed technologies and the carding machine, separately or together, for new applications. For instance: - During this project we acquired the knowledge needed to overcome some relevant obstacles to tissue engineering technology such as the realisation of tailor-made scaffolds, the seeding and proliferation of primary cells, their culture in a tridimensional structure rather than a flat surface, the organisation of a tissue-like structure in vitro. This knowledge is exploited independently by several partners in new projects, some of them EC funded, aiming to develop new human engineered tissues or organs. - The use of the carding machine, so far limited to the production of scaffolds for fibroblasts tridimensional expansion, could be extended to the manufacture of other fabric-like materials intended for medical use and thus requiring high standard quality and special production care. For example, within the frame of some of the EC funded projects in which it is presently participating, FAB is already testing the production feasibility of new hyaluronic acid-based scaffolds with the carding machine. - An immediate application of all the above detailed technologies and of the carding machine prototype is finalised at the realisation of the one-step skin graft. Indeed, FAB is currently completing the pre-clinical and clinical testing of a single device, composed of a hyaluronic-based scaffold able to support the proliferation of fibroblasts in its core and of keratinocytes on top. The aim is to obtain an autologous cell-seeded device to be used as complete skin substitute for acute or chronic wounds of small size. This new product, the first of its kind, is expected to be launched in the next 12 months. Based on its past experience and on the niche market already occupied with its own partial skin substitute devices, FAB is confident that this unique complete skin substitute device will quickly gain a large portion of the European and possibly American market.