Project nº:
BE 96-3524
Development
of Biodegradable Scaffold
for Dermo-Epidermal Skin Grafts
Questions and answer for an
article on
skin reconstruction
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Can you explain, briefly, the principal challenges when we have to reconstruct skin ? (problems of biocompatibility, of immunity, of biodegradation, ……) ?
The general objective of this project was that to develop and clinically test new biomaterials, able to support the proliferation of dermal and epidermal cells, for use as composite scaffolds for the production of artificial skin.
For a better understanding of the objective it should be kept in mind that when considering tissue engineering in wound repair, the possible approaches generally fall into the following three categories:
· Epidermal replacements -consisting of keratinocytes grown either alone (on the surface of a tissue culture flask), or in close association with a carrier vehicle such as a polymeric film or bioresorbable matrix.
· Dermal replacements -consisting of a support structure able to support the infiltration, adherence, proliferation and neo-matrix production by fibroblasts (and in some cases endothelial cells).
· Skin substitutes -are a combination of the above, able to support both dermal and epidermal components.
Moreover, formulation of a suitable scaffold, able to facilitate the several biological processes needed for skin reconstruction is a major issue for groups developing dermal and epidermal substitutes. Suitability of the materials forming the scaffold is dependent upon several factors, these include the ability to:
·
support
cell ingrowth
·
provide a
suitable substrate for adherence
·
facilitate
cell proliferation and production of extracellular matrix
·
resorb
from the wound site in a controlled fashion.
·
further
requirements include minimal toxicity, low immunogenicity and tensile properties
similar to uninjured tissue.
The other important issue in tissue engineering is the cellular component which could fall (when clearly needed) into one of three categories:
· Autologous cells -the obvious benefit of this approach is that there is little potential for rejection and the risk of disease transfer is minimal.
·
Allogeneic
cells -this approach increase the
potential for graft rejection and disease transfer, however using this technique
'off the shelf' product can be developed that are available for immediate
application to a wound.
·
Xenogeneic
cells: such cell sources have found
little favors in tissue engineered approaches to wound repair, although
xenografted tissues from porcine sources are being investigated for use in renal
and hepatic disease.
Finally, composition and performance of skin substitutes must be evaluated before initiation of clinical studies. Similar to other experimental therapies, cultured skin substitutes may be validated for function by evaluation in vitro and in animals. Factors to be evaluated include: regulation of cellular proliferation and attachment, morphogenesis of cells into skin analogues, biochemical and biophysical assessment and histogenesis of skin analogues into functional skin.
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What has been your “philosophy” in this project ? what is your originality ?
Based on the above considerations, the philosophy of the project was to develop materials and tissue engineering technologies to produce a complete skin equivalent consisting of both a dermal and an epidermal layer, an entirely autologous product, designed for the treatment of acute wounds (consequent to burn, trauma and surgery) and chronic wounds (diabetic foot ulcers, venous leg ulcers).
The
challenge was to develop tissue engineered materials which could be reproducibly
developed at an industrial scale and could easily fit in the clinical practice
of the end users.
The idea is to combine fibroblasts (dermal cells) and keratinocytes (epidermal cells) in a scaffold made from a hyaluronic acid, a molecule naturally present in skin. The device would consist of a thin top membrane (for the keratinocytes) and an underlying sponge-like layer (for the fibroblasts). Designed to favour growth and joining of the two skin layers, the scaffold would be biodegraded in time with the healing process
Specific aims of the project were:
· To develop new biocompatible materials based on biodegradable polymers which can be processed into tri-dimensional scaffolds.
· To design appropriate configurations of the architecture of the scaffolds in order to enable effective cellular infiltration and proliferation
· To determine the relevant mechanical, chemical, surface and biological properties of such materials in order to fulfill the essential requirements described in the EN 93/42 directive.
· To test in vivo take rate and healing properties of the artificial skin grafts on animal models.
· To carry out preliminary controlled clinical trials on burns and on scar/tattoo revision.
· To scale-up the technologies developed.
· To establish a more widespread culture on the clinical use of skin grafts in the treatment not only of burn wounds and other acute wounds but also in the case of chronic ulcers.
Concerning the originality of the project, important aspects are the choice to use a naturally derived material (hyaluronic acid) for building the scaffold and to use autologous cells.
The material
for the scaffold:
To date, the vast majority of biomaterials are designed on the basis that implantable medical devices should be constructed of inert materials and their functional performance was restricted to simple mechanical or physical parameter. Recently, the conceptual approach to medical devices is changing and bio-interactivity is now the major requirement, rather than inertness.
FAB
has dedicated much attention to the chemical derivatization of Hyaluronic acid
(HA, Hyaluronan), with the aim of developing biopolymers which retain the
biocompatibility of the parent molecule but have different physical properties
(i.e. elasticity, viscosity and plasticity).
Hyaluronan
is a naturally occurring glycosaminoglycan, consisting of a repeating dimer of
glucuronic acid and N-acetyl-glucosamine. HA is a widely distributed and highly
conserved polysaccharide, which plays an important role in a variety of
vertebrate soft tissues, due to its peculiar physico-chemical and biological
properties. By nature of its propensity to form highly hydrated and viscous
matrices, HA imparts stiffness, resilience and lubricious faculty to various
tissues. The unique biophysical properties of HA are manifested in its
mechanical function in the synovial fluid, the vitreous humor of the eye and the
ability of connective tissues to resist compressive forces. HA is a major
constituent of the extracellular matrix, where it has a profound influence on a
variety of cellular events including cell migration and proliferation.
Its
biological properties make HA an ideal candidate for the development of
innovative biomaterials for various clinical indications. Purified medical-grade
HA is currently employed to reduce the incidence of post-operative adhesions, as
a viscoelastic aid in intraocular surgery, as a synovial replacement device, as
a component of wound healing formulations and in various cosmetic preparations.
Numerous other potential
applications are in reality precluded because of the physical nature of HA
itself. Unmodified HA exists only in the form of aqueous gel which has a short
residence time (i.e. it is rapidly degraded upon application) and is not
susceptible to being manufactured into stable three-dimensional configurations.
FAB has
optimized a number of well defined patented chemical modifications of HA that
have been successfully employed to produce novel range of viscoelastic
biopolymers
These HA-derivatives can range from soluble material to solid hydrogel. As the parent naturally occurring molecule, these HA-derivatives have demonstrated to offer the advantages of being recognized by cell receptors, of interacting with other extracellular matrix molecules and of being metabolized by intrinsic cellular pathways. Moreover, these biomaterials have prolonged and controllable residence time upon application compared to the natural HA.
Being
highly biocompatible, biointeractive, biodegradable and able to induce
angiogenesis these materials have demonstrated to be among the most suitable
candidates for the development of innovative 3-D scaffolds tissue engineering
application.
The cells
source:
The choice to use autologous cells as cells source for the artificial skin has the obvious benefit that there is little potential for rejection and the risk of disease transfer is minimal.
However, there is also the obvious need to scale-up and industrialize not just an of-the-shelf product but indeed a complete system/service for autologous skin tissue grafting.
This means that there is the need to set up the logistic for carrying out effeiently the following step:
-obtain a small biopsy from the patient and extract viable skin cells out of it
-expand the cell number in certified high-tech laboratories
-seed the cells on custom-made polymeric scaffolds
-and deliver back the cell-biomaterial construct to be grafted onto the same patient.
Within the CE funded project we have indeed demonstrated the feasibility of this process.
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Which is the market for such products ? Do you know similar, or nearly similar, product on the market ?
In the US alone, there are 100,000 hospital treated burns per year and 600,000 cases of surgical skin excision - burn wounds costing an estimated $100 million per annum. The age related problem of non-healing dermal wounds is far larger, with 11 - 12 million patients being treated in the US. This will become a greater problem as the population ages. Up to 15% of diabetic patients develop foot ulcers, leading to 50,000 amputations per year Almost half will die, or loose the opposite leg within 3 years. For the above mentioned pathologies Europe shows approximately the same numbers of patients. Clearly, the clinical problem of dermal ulceration is huge, and likely to grow in both Europe and the US.
Skin substitute have been an ongoing factor in the marketplace. Traditionally, patients’ own skin has been utilized in case where large amounts of skin have been lost to burns wounds (autograft). Biological skin (for example, cadaver skin) have also been widely used for years. Artificial skin based on the tissue engineering technology, on the other hand, is a relatively recent phenomenon but it is forecasted to grow rapidly in importance. These premium priced products will however need to prove their cost effectiveness in the marketplace, and they are expected to take several years from entry to gain acceptance.
However, ultimately, skin substitute will become a major factor in the marketplace, for sure the treatment of choice for severe wounds and much probably also for chronic non healing ulcers.
Although the production of artificial skin substitutes is a relatively new field, there are already some products and services available on the market. The great majority of this products have been developed by US based Companies, generally with the collaboration of famous academic departments. The leading products now available are:
·
Epicel marketed
in US and Europe by Genzyme Tissue Repair
· Dermagraft marketed in UK by Smith & Nephew
· TransCyte (Dermagraft TC) marketed in US and some European countries by Advanced Tissue Science and Smith & Nephew
· Apligraft manufactured by Organogenesis and marketed in US by Novartis Co.
Note: Of the above products only Epicel is based on autologous cells. All the others are allogeneic products, i.e. the cells are taken from donors. Donor cells can provide growth factors to stimulate healing but, as has been shown in this project, they cannot survive and integrate.
The strategic objective of the Consortium is to enter the competition in this marketplace.
Although the project is specifically aimed to the treatment of wounds, a broader understanding of many aspects of tissue engineering is likely to result from the project. The importance of the selection of the appropriate biodegradable scaffold and of its architecture is an issue that has relevance in other aspects of health-care ranging from orthopedics (the culture of chondrocytes for the repair of knee cartilage) to vascular surgery (the culture of endothelial cells for coronal and peripheral bypass). The market expectations for products based on the tissue engineering technology are impressive.
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have you already tested it on human beings ?
So far a first-generation system
base on a consecutive application of an autologous dermal graft and an
autologous epidermal graft have been developed and patented. Currently
on sale in Italy, they use the patient’s own cells, derived from a small
biopsy, expanded in culture, and seeded on hyaluronan biomaterials. Several
clinical tests are in progress - over 1000 patients have been treated with the
scaffolds to date. Hopes are high that the system will promote better healing of
problematic wounds and make burn treatment less disfiguring. However, the
delivery of two separate grafts is complicating the logistics of the system,
limiting the potential market of the system.
The aim now is to enable delivery of both cell types in a single step, i.e. to reconstruct a complete, entirely autologous tissue engineered skin.
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How was the repartition of work for your program between the different partners?
The
Consortium comprises six partners from 4 different European countries. Each of
the partner has complementary skills and experience. F.A.B. srl (IT), coordinator of the project, has pioneered the
development of biodegradable and biocompatible materials and devices for tissue
engineering. The other industrial partner, HDB
sa (BE), has an extensive knowledge on the engineering of machinery for the
textiles industry and is currently involved in the scale-up of the technology
for processing FAB’s biomaterials. PASTIS (IT), a recognized research organization in the field of
biomedical materials, has conducted mechanical testing and in vitro preliminary biocompatibility assessments. The most
promising device have been tested both in
vitro and vivo on appropriate
pre-clinical models by DKFZ (DE), RWTH
(DE) and LHMC (GB). In this respect, the
DKFZ is a highly specialized
center which has pioneered the study of dermo-epidermal co-cultures in an
organotypic manner. The center for cutaneous research LHMC has developed a unique, world wide recognized model for the
quantification of take rate of grafts using white large pigs. The department of
plastic surgery and burn center of RWTH
has a recognized experience in conducting research oriented clinical
investigations. LHMC and RWTH share the responsibility of conducting pilot
clinical trials. The multidisciplinarity and complementarity of the partnership
structure has been demonstrated by the expertise brought in by the different
parties, ranging from polymers synthesis to device engineering and
manufacturing, from physico-mechanical testing to in vitro cell viability test, from preclinical to clinical testing.
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The results ?
The principal achievements of the project to date can be summarized as follows:
·
SCAFFOLD ENGINEERING and
SCALE UP OF TECHNOLOGY
Ø identification of appropriate biodegradable hyaluronan based scaffold for seeding of dermal fibroblasts; optimization of manufacturing methods, definition of manufacturing equipment lay-out
·
IN VITRO TESTING OF DERMAL
AND EPIDERMAL SCAFFOLDS
Ø optimization of in vitro co-colture methodologies, identification of unique staining methods for esterified hyaluronan scaffolds. Understanding of in vitro degradation mechanism and degradation rates of cell loaded hyaluronan scaffolds.
·
IN VIVO TESTING OF DERMAL
AND EPIDERMAL SCAFFOLDS
Ø In vivo demonstration, in an immunocompetent pig model, that a allogeneic (i.e. non-autologous) dermal graft will not survive transplantation
Ø In vivo understanding of optimal clinical procedure for graft application and identification of secondary dressing of choice
Ø In vivo demonstration of efficacy of acellular hyaluronan scaffolds as dermal equivalents in an acute wound setting
· PILOT CLINICAL EVALUATION
Ø Protocol definition for conduction of pilot clinical evaluations in three acute wound models (burns, tattoo excisions, scar revisions), for testing of prototype dermal scaffolds developed.
Ø Ethics committee approval for clinical trial initiation.
The experimental data derived from the project to date seems to suggest that different products and different clinical protocols will need to be designed for the treatment of acute and chronic wounds respectively. Therefore, given the clinical expertise of the Consortium, as well as the timeframe available for the completion of the program, it was decided to focus all activities of project BE3524 in the identification of a treatment option for acute wounds.
Independent of EC funding, but nevertheless in close scientific collaboration with investigators in the Consortium, FAB has, in these years, completed the scaling up of its two stage dermo-epidermal autologous grafting procedure for the treatment of chronic wounds, based on its patented technology of chemical modification of hyaluronic acid. FAB has set up an 800 m2 facility for cell culture, developed CE accredited SOP’s for cell culture and graft delivery, organized 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 organization with CE approved products and one of the few companies worldwide generating sales with tissue engineered products.
The opportunity of close interaction with some of the most prominent investigators in cutaneous tissue engineering, as well as the access to community funding for the development of some of the aspects of this project has certainly significantly contributed to the success and international competitiveness of this initiative.
