WO2001025403A2 - Bio-artificial substrate for the production of animal and, in particular, human tissues and organs - Google Patents

Abstract

A substrate suitable for the survival, the proliferation and the correct differentiation and functioning of specialised tissue cells of the human and animal body, consisting of a material that is bio-compatible and bio-resorbable in pre-determinable times, which can be transplanted or implanted onto or connected with the body in order to achieve a complete integration of the transplanted, implanted or connected tissue with the other cell systems and their functions in the organism onto which the transplant or the implant or with which the connection has been made, and comprising a mixture and/or combination of natural and/or synthetic polymers in which fibroin is present.

Description

"BIO-ARTIFICIAL SUBSTRATE FOR THE PRODUCTION OF ANIMAL AND, IN PARTICULAR, HUMAN TISSUES AND ORGANS"

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TECHNICAL FIELD This invention, concerning the biomedical sector, refers in particular to a substrate for cell and tissue culture which favours the survival, proliferation, differentiation and correct functioning of cells of specialised tissues.

This substrate is ideally employed in the medical and surgical fields since it can be used for the regeneration ot organs and tissues to be transplanted in humans or for the setting up of bio-artificial organs to be functionally connected with the organism.

One particularly advantageous application ot this invention is the production of a regenei tmg artificial skin to be grafted onto patients with major burns or suffering from diseases that involve the degeneration of the cutaneous tissues.

BACKGROUND ART The skin is the most conspicuous organ of the human body, accounting for 16% of its mass and having the largest surface area, and it grows and renews itself at a constant rate. A complete renewal cycle of the superficial layer of cutaneous cells (epidermis) does in fact take place every three-four weeks.

Contrary to what may appear from a cursory analysis of its characteristics, the ikin is also one of the most complex organs of the human body since it consists of extremely specialised cell types that perform a series of very different, specific, and well-balanced functions ensuring the maintenance of the body's outer covering, protection against infection, heat and ultraviolet radiation, regulation of body temperature, water loss ( sweating), secretion of fats (sebum) and production of vitamin D

The outer layer of the human skin, which is known as the epidermis, contains no blood vessels and consists mainly of closely aggregated cells called keratinocytes. Other cells present in lesser quantities in the epidermis are melanocytes, Merkel cells and

Langerhans cells. The so-called "basal lamina", consisting of type IV and VII collagen. fibronectin. etc.. marks the boundary with the underlying dermis.

From a structural point of view, the epidermis consists of five cellular layers which from the surface downwards are the stratum corneiim. the stratum luciditm. the stratum gramtlosum. the stratum spinosum and the stratum basale or germinativum. and it is thanks to these layers that the skin can perform its role of mechanical protection of the human body. The natural history of any keratinocyte in the epidermis involves a progressive differentiation as it moves upwards from the stratum germinativum to the stratum corneum, passing through all the intermediate layers in sequence and finally being transformed into a dead corneocyte and being sloughed off by friction or during washing. The stratum germinativum is in direct contact with the basal lamina and is the site where new cells are generated. As already mentioned, the basal lamina represents the boundary between the epidermis and the subjacent layer of connective tissue known as the dermis or corium.

This layer is extremely well vascularised and also contains receptors connected with nerve fibres that allow the sensorial reception of mechanical, physical and chemical stimuli by the skin. The mechanical receptors include the Pacinian corpuscles and Meissner's corpuscles, the latter being particularly responsible for tactile sensitivity. Blood vessels and nerves are particularly abundant in the outermost layer of the dermis which is adjacent to or in contact with the basal lamina.

The dermis too contains cells, including fibroblasts. which are scattered within a matrix consisting of various fibrous proteins, such as collagen and elastin, and of components with an amorphous ground substance, such as glycosaminoglycans (GAG ) and proteoglycans.

Fibroblasts perform very important roles since, as well as producing the precursors of the collagen and elastic fibres and the components of the amorphous matrix, they also secrete growth factors and cytokines that are capable of affecting the functioning of the keratinocytes and endothelial cells of the blood vessels.

The high level of structural complexity of the human skin is undoubtedly the greatest problem to be solved when attempting to produce artificial tissues that reproduce the skin's morphofunctional characteristics. These tissues, commonly known as "artificial skin-grafts" by those working in the sector, should be particularly useful. and are thus studied very intensely, in relieving the suffering of patients with major burns or those affected by diseases involving significant dermatological implications. The sev eie scainng and psychological damage caused b> the sudden devastation ot the bod\ image as a lesult of majoi burns 01 to a lessei degree by diseases with significant dermatological implications, as well as the other functional consequences ot the damage to an oigan as important as the skin. ha\ e led researcheis thioughout the woi ld to strive toi alternativ e solutions that w ould favoui the healing processes and at the same time slow dow n the dev elopment of disfiguring scats and consideiablv impi ove the body image and psychological condition of the patients mostly thanks to the use ot skin-giatts in the injuied aieas, which piactically eliminate the nsks of rejection

These nsks deciease the more the artificial skin resembles the natuial skin and when, wheievei possible, the patient s own cells, or human cells of other origin, are used Betoie arm ing at the pioduction ot artificial cutaneous tissues a great deal of woi k had to be earned out in modelling the natural skin in ordei to leconstiuct the svnei gisms that take place both in the normal functioning of the oigan and in extreme conditions, such as immediately after the occurrence of a serious mjur\ and during the healing pi ocess

The leconsti uction of cutaneous skin tissues is not a single process but a combination of a senes of dynamic interactive processes that involve epidermal and dermal cells, subcutaneous tissue, and blood vessels These cells actively communicate with each othei bv l eleasing soluble messengei s - cvtokines - w hich intei act w ith specific membiane receptoi s

The most general classification of a tissue reconstruction process such as that ot the skin is made by identifying thiee distinct phases (Principles of Tissue Engineering,

Lanza, R P , Langei , R , Chick, W L 1997, Academic Press, R G Landes Company,

Austin, Texas, U S A ) of reaction of the tissue to the injury ( 1 ) inflammation, (2) tissue toimation and (3 ) tissue lemodelling

The injuied tissue is studded with sev ered and bleeding blood vessels It is also discontinuous in propoition to the severity of the injury The first ieaction that occurs is the formation of a blood clot The clot is rich in fibrin which blocks blood loss, evens out the surface of the injured tissue, and prevents the entry of harmful envnonmental agents The platelets in the clot produce mediators, including important grow th factors The fibi in also acts as an aggregating agent toi furthei platelets Numerous macrophages and neutrophil leukocytes arrive at the site of the injury, then action is impoitant since they cleai the injured area of toieign bodies and agents that may further haim the damaged tissues (e g bacteria) The neutrophils die spontaneousl) or due to the effect ot toxic factors and are in turn phagoc tized bv the macrophages that arrive at the site some hat later (see below)

The initial scenario described above also includes enzymes and oxidants that speed up the v arious leactions triggered by the interactions between the various paits in pla_\ and act as a buffei wheie the tissue is most vulneiable. I e where toieign bacteria endangei the piotective piocesses set into motion on the law suifaces bv the platelets and neutiophils

Togethei w ith the intiltiation ot the neuti ophils theie is also an accumulation ol monocyte-dei ived macrophages. attiacted by the local release ot paiticulai chemokines Via the mediating actions of lntegπn receptors, implemented ad hoc by the endothelial cells of the blood vessels, these leukocytes are "caught" locally and induced to cross the blood vessels, to penetrate the tissue matrix, and metamorphose into inflammatory macrophages in order to phagocytize cellular debris and residues of collagen and elastic fibres, the components of the extracellular matrix, the degenerated neutrophils and any pathogens

Re-epithehzation of the wound staits just a few houi s attei the ιn|ur\ The epithelial ceils that have survived the injury undamaged migrate from then initial site to the surface aieas covered bv the blood clot, giv ing use to a new epithelial la ei under hich further growth ot the connective tissue is possible To this aim, the epidermal cells undeigo structuial changes allowing then lateial mobilization by the preliminary dissolution ot the desmosomes At the same time, the epidermal cells surrounding the wound dev elop a motoi sy stem that allows their migratoiy activities In the w ound areas that lemain temporarily uncovered, the injured surface consists of a temporal y matrix of fibrin, fibronectin, tenascin, viπonectin and type I collagen

As the le-epithehzation process goes on, the proteins of the basal lamina leappear according to a well-defined sequence, staiting from the edges of the wound

Foui days aftei the lnjuiy so called gianulation connective tissue begins to form This piocess staits the lestoiation of the integument as a structuie consisting ot at least two distinct inteidependent layers, an outer epithelium and an innei dermis the latter being nch in capillaries macrophages and fibroblasts which cooperate activelv with each othei The maciophages aie in fact a source of important cytokines w hich play a role ot fundamental importance both in angiogenesis and in the production ot new collagen and elastic fibres by the fibroblasts that also regenerate the extracellular matnx that w ill legulate cellulai metabolism These cytokines endow ed with a high proliferation-inducing powei , aie commonly considered on the same level as growth factoi s The tempoiaiv extiacellular matrix is nch in substances that fuithei tissue dev elopment Examples of such substances aie fibionectin and collagen togethei with hv aluionic acid

At this point ot the tegenerative piocess the fibioblasts in the tissues that sunound the w ounded area piohfeiate intensely undei the mitogenic stimuli of the cytokines and start migi ating towaids the injuied aiea first adhering to and then penetiating into the blood clot thiough the fibronectin matrix

A mutual equilibrium is thus established between the fibroblasts and the extracellulai matrix, since on one hand the fibroblasts continuously synthesize and ennch the matnx with its fundamental components, their functioning being on the other hand legulated bv the matrix constituents Chemotactile-hke mechanisms regulate the migration of the fibioblasts which pioduce lamelhpods duected towards the source ot the stimuli creating new cellular surfaces and tnggermg complex mechanisms that result in true cell mobility

Pio feiation of the fibroblasts ceases when the system is sufficiently enriched with both cells and collagen matrix (the latter being by itself responsible foi blocking the piohferation ot these cells, contiary to the effects of fibnn and fibionectin matrix which favoui s fibroblast proliferation)

Tissue legenei ation is accompanied by a simultaneous pi ocess of neo- vasculansation chaiactensed bv the ever increasing branching ot piohferating capillanes and the neo-formation of capillary loops Angiogenesis is made possible by the local lelease of specific cytokines which stimulate the pro feiation of capillary endothehal cells Angiogenesis allow s the tissue to be permeated by oxy gen and by the nutritional substances that are indispensable toi its suiv iv al and conect functioning Only lecently has it become possible to identify and tiack the molecules that play a fundamental lole in angiogenesis These include, amongst others, angιopoιetιn- 1 (AP- 1 ). vascular endothelial growth factoi (VEGF). and keiatinocyte giowth factor (KGF)

Anothei phase in which the concerted reorganisation ot the tissues and the interactions between the various types of cells involved are particularly intense aie the contiaction of the gianulation tissue filling the wound and the reorganisation of the matnx dining which the w ound fibioblasts take on the lole and the features of the smooth muscle cells, thus becoming known as myofibroblasts and giving the gianulation tissue the ability to contract

This takes place at the same time as the compacting ot the connective tissue, w hich i caused bv the formation of cross-links that join together adjacent collagen fibeis A collagen fibei netwoik is produced which, thanks to the interactions with the matnx. can thus suppoit the contractile stimuli imparted by the myofibroblasts

The lemodelhng and catabolism of the collagen fibeis constitutes the driving phenomenon that leads to the formation of the scar This process, howevei . occurs lather slow ly and its functional result is only paitly satisfactory In fact, aftei the first thiee w eeks following the injury, when the accumulation of fibnllary collagen takes place at maximum speed, the scar tissue has acquired only thirty percent of the strength of the undamaged skin When it reaches its completion, the scar tissue achieves a lesistance only amounting to 70% as compaied with that ot undamaged skin

With a scenario such as the one reported above, which does not descnbe all the micro-phenomena involved in full detail, the production of artificial human skin using sy nthetic methods lepresents an extremely difficult challenge

The use ot human skin from living or cadaver donois for the tieatment of skin injunes has been piacticed foi quite some time, although it involves the lejection on the pait ot the host, not to mention the considerable nsk of infections that mav even be lethal ιe g hepatitis C, AIDS, etc ) The dermo-epideimal substitutes , moie commonly known as aitificial skin aie the lesult of meticulously perfected cell culture pioceduies on polymenc supports oi substiates often used foi subsequent applications onto injuiv sites The spaces left free by the mesh of the tabnc aie sufficient, once piohtei ation has begun, to favoui the population of the polymeric suitace as quickly as possible

The following relev ant dermal substitutes are av ailable on the maiket Deimagiaft®. Allogiatt® and Alloderm®

Allodeim® is a dermal substitute, consisting of human donoi dermis fiom which all liv ing cells ot epideimis have been remov ed, which can be pieseived at a low temperatuie thaw ed when lequued and be used in combination with a thin layer ot host epideimal cells The prepai atoiy pioceduie also involves the inactivation ot any vii us that might have been originally present in the tissue taken fi om the donor (genei ally a cadaver)

The basic piotein stiuctuie ot the dermis. albeit devoid ot cells, is preseived by the piepaiation process The material is le-hydrated w ith a saline solution pnoi to being grafted onto the bum wound site Clinical applications aie limited The possibility ot infections, due to the human origin of the product, cannot be completely excluded The production process is extremely complex and the quality level plays a highly critical

Dei magi aft® and Allograft® piesent the same pioblems as those descnbed tor Allodeim® Integia® is anothei dermal substitute It consists ot an innei lavei 2 mm thick, made up bv a combination ot bov ine collagen tibi es and a GAG c hondι oιtιn-6- sulphate, with 70-200 μm diameter poies, and is biodegiadable Its outei lav ei consists of a sihcone polymei that peimits water vapour transfer

Integra® is intended tor applications on deep burns, and the sihcone outer membi ane must be replaced after 2-3 weeks w ith an auto-graft of epidermal cells Clinical experience is rather limited at piesent the occurrence of suppurativ e processes immediately beneath the sihcone membiane has been frequently leported and the pioduction piocess is extiemely complicated and costly

In addition, the use ot collagenous matenals ot bov ine origin exposes the patient to the nsk of spongifoim encephalopathy, also known as "mad cow disease"

The document US-A-5.266.480 descnbes a three-dimensional cultuie of fibroblasts on a polymenc substrate (stromal matrix) made from one ot the following matenals nylon (polyamides), dacron (polyesters, polystyrene, polypiopylene, polyacrvlates. polyvinvl compounds such as polvvinyl chloride, polycarbonates, teflon, teimanox. nitrocellulose catgut, cotton, cellulose gelatin and dextiane With the exception ot cotton fibre, catgut, cellulose, gelatin and dextrane, these aie all polymers of sy nthetic origin, processed in such a way as to pioduce a fibre Pretieatment of the matrix betoie cell inoculation is also suggested, togethei with the enrichment of the cell cultui es with pioteins of \ anous kinds, such as collagen, glycopi oteins, glvcosaminoghcans (GAGs) and othei similai materials in oidei to speed up the cell piohteiation piocess This patent stiesses the impoitance of finding a substrate that acts as a suppoit in such a wav as to leave sufficient space to pievent the cells fiom being ti apped and horn stopping the pioduction of the giowth tactois necessaiy to sustain piohfeiative piocesses Thus, growth factors will not have all to be added to the matrix from the beginning, since the cells themselves will continuously produce them, ci eating right from the stait the steady-states that are propei of the skin The stiomal cells are explanted fiom organs and separated according to conventional methods One senous disadv antage ot this invention is the proposed use ot synthetic and non-ie-absorbable substiates, constituting to all extents and purposes foreign bodies light fiom the time of giatting w ith the consequent nsk ot rejection reactions to these matenals Anothei disadv antage ot this process is that it is extremelv complicated and requnes sev eral cell inoculation stages in ordei to obtain a fully formed cutaneous tissue consisting of the necessaiy amount ot different cells that carry out the v arious roles they would perform in healthv and fully developed tissue Paiticulaily as far as skm is concerned this inv ention also lequires the use of cells explanted fiom the toieskin of newborns. obtained bv means of cncumcision opeiations

The document US-A-5,902,741 descnbes three-dimensional cultuies of caitilaginous cells set up on a biocompatible matrix ot non-living material in the presence of TGF-β The stiomal cells cultuied according to this patent include chondiocytes, chondiocyte piogenitoi s. fibioblasts endothelial cells, maciophages, monocvtes, bone marrow stiomal cells and others These cells aie induced to proliferate by making them adhere to a ngid sti ucture consisting ot a biocompatible polymei until its surface is completely covered This rigid stiuctuie contains spaces that aie then filled by the stromal cells According to this patent, once these stromal cells have completed the formation of a solid tissue covering the biocompatible ngid structure, they begin to secrete growth factors and legulatory factoi s and aie therefore leady to act as a support toi the implantation of prev iously cultured ;// \ ιtι o cells, which will thus enjoy the ideal conditions to pio feiate and/oi diffeientiate according to the specific lequirements of the tissue regions in which they are located One disadvantage of this invention in order foi it to prove successful is the need to lesoit to sv nthetic materials, which do not decompose, dissolve or disappeai in any w av aftei implantation

A. tuithei disadvantage of this inv ention is that, although intended foi the leconsti uction of an organ oi ot one oi moie ot its parts, the process by which piolifei ation and differentiation ot specialised tissue cells is achiev ed is extremely complex since, before the desired cells are implanted, the synthetic material must always be coveied with the stromal cells This represents a tiansition phase that does not lead diiectly to the desired result and which takes a fairly long time, meaning that the product is not leadily available, which can sometimes be crucial in emergency situations The patent US-A-4.703 108 descnbes a pi ocess toi the prepai ation of biodegi adable matnces in the toi m ot three-dimensional sponges oi two-dimensional sheets toi which a type I or II collagen-based material is used, which are tieeze-dπed and tieated w ith a leticulation agent such as carbodπmide or a reactive succimidy c estei Aftei this leticulation leaction. the obtained material is subjected to extreme dehy diation conditions aftei which the spongy oi sheet-like material is obtained This material w ill also contain a chemical agent chosen from a group that also includes type IV and V collagen, fibionectin and laminin

An obv ious disadv antage of the process bi iefly descnbed abov e is that it is extiemely complex and quite lengthy It also involves tw o drying stages (the second taking place undei extreme conditions ) w hich must be earned out with gieat care since they can cause inepaiable damage to the material, making it useless and forcing the operators to start the procedure all over again, with obvious loss of time and effort.

This last problem makes it difficult to envisage this procedure being carried out at an industrial level or in any case to produce amounts of finished material that exceed those lequired for laboratory experiments.

A further problem of this procedure is that to obtain the desired result a very wide range of compounds has to be used, some of which are chemical agents that require particular caution.

Anothei problem in the piocedure described in the above-mentioned patent is that collagen is cited as the only material to be used. This material has limitations. how ever, since its properties and characteristics are fixed thus making it difficult to adapt to different situations.

Yet another problem ot this procedure is the need for collagen of bov ine origin which can be a vehicle of contagion for spongiform encephalopathy. and may be in any case allergenic. It is also enzymatically demolished, and thus re-absorbed, thereby offering no guarantee that this re-absorption will continue until the collagen has completely disappeared nor that the transplanted and regenerated tissue is restored to its original lull functional capability

The patent US-A-5.670.483 describes a series of stable macroscopic membranes that form by self-assembly due to the alternation of the amphophil peptides. hydrophihc and hydrophobic. tound in them. The range ot application tor these membranes is vast, including their use as artificial skin.

One disadvantage that these membranes present is that they are not biodegradable or bio-re-absorbable. Once implanted, their mechanical features remam unaltered and do not correspond exactly to those proper of the human skin; the membranes never achieve a complete integration into the tissue environment in which they are implanted or transplanted.

The document US-A-4.963.489 describes a cell and tissue culture system that can develop three-dimensionally. The procedure foresees a phase in which cells from a specific tissue are explanted. inoculated and cultured on a three-dimensional stromal matrix. In a first stage, the stromal cells w ill be giow n on the synthetic suppoi ting structuie until they reach confluence and completely covei its surfaces, thus pioviding an ideal scaffold toi the grow th ot the implanted cells The stroma will contain fibroblasts of foetal origin or tiom adults alternatively they w ill be explanted from a cadaver Once again the sti omal cells are cultuied on a thiee-dimensional synthetic matnx which pei se is not bio-resorbable The various options foieseen include the use of cotton and cellulose which aie natural fibres but extiemeh lesistant to the chemical attacks that can be undeigone in a natural system such as the human bodv The use ot gelatine is also anticipated which is none othei than denatuied collagen theiefoie w ith the same di aw backs and flaws as those reported tor the patent US-A-4703 108

The fu st shoi tcoming of the inv ention described abov e is the peimanence of the sv nthetic matenal used as a matrix, even after the culture ot the specialised cells has been completed This can lead to unpleasant consequences foi the patient In the event, foi example, of the reconstruction of a portion ot a functioning organ of the human body pait of its volume would be always occupied by the ineit synthetic material, thus leducing its functional capabilities

A tui thei drawback of the invention is the tact that it requires complex piepai ation pioceduies Meiely to tender moie biocompatible the synthetic material used as a matrix the lattei has to be covered with a lavei ot a suitable mattei such as e g collagen which is normallv of bov ine origin a time-consuming piocess that gieatlv complicates the pie-ti ansplant w oik

Foi some years now the scientific and technological world has been studying alternativ e uses ot silk and specifically of one ot its pioteins, I e fibroin, exploiting the pamculai pioperties of the le-naturated protein Its peimeability to oxygen ionic pei meability lesistance to pioteolytic enzymes and to acid and alkaline solutions, tianspaiency and mechanical pioperties have been evaluated

Applications hav e been proposed in the bio-medical field in the contact lens sectoi bandages for burns, artificial corneas and biosensors Various biocompatibility tests hav e been pei foimed. albeit ti agmentaπly hemocompatibility tests (H Sakabe et al /// \ ι\ o blood compatibility ot regenerated silk fibroin Sen-I Gakkaishi v ol 45. n 1 1 1989 487-490) cell cultures (Y Gotoh et al Svnthesis of polyethylene glycol )- silk tibioin conjugates and surface interaction between L-929 cells and the conjugates Biomatenals vol 18, n 3. 1997, 267-271 )

Fibroin' s resorbability is reported in the document US-A-5,606,019 which describes the use of an elastin and fibroin copolymer as a bio-resorbable material, with a bio-iesorption speed varying according to the relative percentages ot the two protein poly mei s in the mixtuie In the specifications, the resistance characteristics of the mixtuie aie stressed togethei with its easy resorbability in the conditions imposed by the specific legion of the human body for which the material should be produced and used The lange of the applications is extensive and includes fibres printed objects, membianes to pievent the formation of tissue adheiences. bandages toi wounds, suture thieads and clips

The first diawback ot this invention is that it involves a pioceduie foi the pi oduction ot the pioteins necessaiy to obtain the bioresoibable matenal based on genetic engineenng techniques, which are complex and include the manipulation ot plasmids. then transfer and amplification in prokaryotic cells, I e Eschet iclua toll, and phases ot refining and sepaiation of the finished product (work-up) that must be carried out w ith gieat care It can be noted that the document US-A-5,606,019, does not suggest the use of tibioin as a substrate for the culture, proliferation and differentiation of cells of anv ty pe In the document WO-A-98/57676 the biocompatibihty, tlexibihtv, and resistance to infections of fibroin and of sencin aie undei lined, and their use tor the production ot matenal to cov er wounds is descnbed Foi such purposes fibroin is used in an non- cr stalhne form, I e with a degree of crystallisation that remains below 10% or in a powdei toim The patent US-A-5.932.207 describes a transplantation system, suitable tor the pioduction ot biological paits. particularly oigans of living organisms which basically consists ot a scaffold, a component that favoui s the covering ot the scaffold with specialised cells, a set of adhesion materials ensuring a sufficient cellulai adherence to the stiucture and a maturation factor ensuring that the specialised cells that ha e become pait of the new system mature and differentiate when necessary

Yet again, due to the nature of the adhesion materials, it is necessary to lesort to lengthy degradation and lesoiption piocesses that aie not always successful

Fuitheimoie, the processes necessaiy to obtain the tiansplantation-ieady system are complex it is not easy to envisage how it would be possible to pioduce such a system in amounts exceeding those needed for laboratoiy use The document WO-A-96/25961 describes the use of collagen fibres for the pioduction ot a bio-resoibable extracellular matrix, and document EP-A-0913 162 descnbes a specific use of collagen in the form ot fabric, suitable for the lepan ot tissue defects

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W . Schillei W R . Hansbiough, J F , Hickeison. W L . Himel, H N , Kealey G P . Twomey J . Missavage, A E . Solem, L D , Davis. M . Totoπtis, M . and Gentzkow. G D A multi-centei clinical tnal ot a biosynthetic skin leplacement, Dermagiatt-TC, compaied w ith ciyopieseived human cadaver skin toi tempoiary coverage ot excised bum wounds J Burn Caie Rehabil , 18 52-57, 1997

Soejima K . Nozaki. M . Sasaki, k . Takeuchi M . and Negishi \ Tieatment ot giant pigmented nevus using aitificial dermis and a secondaiv skin giatt horn the scalp Ann Plast Suig 39 489-494. 1997

Sprague. K U The Bombw mori silk proteins characterization of large polypeptides Biochemistry, 14 925-931, 1975

Whitfield, J F . Isaacs, R J , Jouishomme. H . MacLean, S Chakrav aithy, B R Moi ley P . Ba soni. D . Regalia, E , and Armato. U C-teiminal fiaginent of parathv i oid hormone-related piotein. PTHrP-( 107- 1 1 1 ), stimulates membi ane- associated piotein kinase C activity and modulates the prolifeiation ot human and muπne skin keratinocytes J Cell Physiol. 166 1- 1 1, 1996

DESCRIPTION OF THE INVENTION

The fust aim ot this invention is to overcome the pioblems and limitations of the known technique by pioposing the use of a natural matenal as such oi combined with biodegiadable and non-biodegiadable materials, ot synthetic or natural ongin. as the substrate suitable for the dev elopment, proliferation and correct diffeientiation of specialised tissue cells of the human body This material should be biocompatible and stable oi bio-resoibable in piedefined times, in such a way as to obtain tissues, structui es and organs that can be transplanted and/oi implanted in oi externally connected to the human body w ith their partial oi complete structural and/oi functional integiation theiein or thereout

The second aim of this invention is to piopose the use of a substi ate matenal that is suitable toi the development ot the abov e mentioned cell types, which can be produced easily and iapidly even on a large scale

The most impoitant features ot this invention are described in detail in the main claim

The suboidinate claims describe paiticulai ly advantageous realisation forms of this invention

The afoiesaid aims aie achiev ed thanks to the use ot natuial tibioin — e g obtained fiom but not exclusiv ely fi om the Bombw man silkwoi m and also appropnately woiked upon and/oi mixed and/or co-polymeπsed and/oi covenng substances ot natuial and/oi synthetic origin — as a substrate that is bio-compatible and bio-sorbable in piedetined time lags and that is suitable to promote the survival, the piolifeiation. the matuiation, and the differentiation of cells torming specialised tissues oi oigans of the human body, I e tissues consisting of cells with a high degree of ditfeientiation. e g such as. but not limited to those making up the skin

The cells whose surv ival, proliferation and differentiation is piomoted by the substiate obtained fiom tibioin in accordance with this invention can give rise to both tw o and thiee-dimensional structures, according to the foim of the substrate matrix on or in which they are cultured

It is in fact known fiom the Literature that cells that have to piohferate on a three-dimensional scaffold, when appropriately stimulated by the addition of suitable factois find themselves in a condition veiy similar to their natural one. as long as they are piov ided with a propei substrate

Accoiding to anothei particularly advantageous aspect of this invention, the substrate obtained as per this invention, appropriately worked upon and/or mixed and/or co-polymensed. is particulai ly suitable to favour the surv iv al, the piolifei ation, the matuiation and ditfeientiation ot normal human adult osteoblasts onginating fi om both the maxillai v bones and the othei bones ot the skeleton, of mesothe al cells isolated fiom the normal human adult peritoneum, of normal astrocytes isolated fiom the cortex of the human adult tempoi al lobe, of hepatocytes and liv er stem cells, ot hvei biliaiy epithelium, ot tenocytes (or tendon fibroblasts), ot chondiocytes (oi cells isolated trom cai tilaginous tissues ), ot endothelial cells of the tunica intima of blood v essels of steioid-secieting cells ot the adienal cortex, ot smooth muscle cells of the tunica muse it Uu i s of the intestine and blood vessels, of squamous epithelium ot the oral cavity and the conjunctiv a/cornea and of human pie-adipocytes of the w hite adipose connective tissue

Detailed studies vveie earned out on the interactions taking place between the fibiom bio-membrane according to this invention and normal human adult dermal fibioblasts

A senes ot additional tests were pertoi med on the interactions of the fibroin membiane as pei this invention with the main cell type f ound in human dermal connectiv e tissue Strains ot normal fibroblasts obtained from samples ot deimis taken from tout different individuals weie isolated in in vitro cultures These cells were then seeded on samples of the bio-membrane as per this invention, contained in the lectangulai wells of in x iti υ culture plates, and the fibroin bio-membranes holding the cells w eie incubated at 37°C foi three weeks

At predetermined time intervals the culture medium was sampled to evaluate the cellulai consumption of glucose and the production of lactic acid Moieovei . in oidei to obtain moie piecise infoimation about the events occuning at the lnteitace between the human fibi oblasts and the tibioin bio-membrane as per this invention, samples ot this bio-membiane that had been in contact with the cells foi predefined time lengths were examined, aftei removal of the fibroblasts without damaging the membrane, under a scanning electron microscope (SEM), an instrument that in expert hands leveals the finest details of the surfaces of the materials examined

The lesults of these studies demonstrate that the human fibroblasts obtained fiom samples ot deimis taken trom tour different individuals and seeded on the fibioin bio-membi ane as per this invention not only multiply v ery actively . as was to be expected ( data not repot ted ), but at the same time also consume increasing amounts ot glucose the fav oui ite cell fuel, simultaneously secreting ever-increasing amounts of lactic acid the metabolite ot anaerobic glycolysis into the giow th medium The cumulatn e curv es ot the consumed glucose and ot the ptoduced and secteted lactate have a fairly similar exponential trends which are in agreement with a simultaneous and quite lemarkable expansion ot the population of human connective tissue cells on the fibtoin bio-membrane as pei this invention These curves also demonstrate that, albeit w ithin the tiamewotk ot an acceptable biological variability (human subjects aie not inbied) once seeded on the bio-membiane, the tibtoblasts isolated from tour ditfeient individuals behave accoidtngly

Thus, hav ing tuithei tested fibroblasts derived riom five ditfeient individuals ovei two six-month penods, it is leasonable to assume that the bio-matenal as pei this inv ention can establish veiv good biological inteiactions with the fibioblasts ot most human subjects Thetetoie. it seems to be paiticulaily suitable as a substiate tot tissue ieconsttuction in the clinical setting

The SEM studies carried out on our specimens levealed a series of very inteiesting details on the nature of the interactions taking place between the human dermal fibioblasts and the fibroin bio-membrane as per this invention When there was no contact with the human connective tissue cells, the surface of the bio-membrane appealed spotted in a totally casual way, with veiy small rises and hollows (Figuie 10) Aftei a w eek' s contact w ith the human fibroblasts and once these had been appropnatelv temoved. the surface ot the bio-membrane appeared v eiy dttterent. exhibiting a senes of gioov es mostly parallel to each othei and with small pits oi niches as shown in Figure 1 1 (marked by arrows) During the first few days of //; x itro cultuie the cells ev idently "dig burtows^' in the bio-membiane, presumablv thanks to the secietion of lytic enzymes in the extracellular environment Aftei tw o weeks of contact w ith the fibioblasts and once these had been lemoved, the ' niches ' in question weie no longet visible on the surface of the bio-membrane even when observed at greatei magnification (Figure 12 ) The suiface of the membrane no longei presented niches, but piotuberances. some small, others large (Figure 12, arrows) It is obvious that in the meantime the cells had pioduced and secreted into then environment the components of the amoiphous extracellular matrix which had filled the "niches" and accumulated onto the adjacent surfaces Thus, it appears that, aftei an initial phase of "settling on the tibioin bio-membrane, the human fibroblasts start to sv nthesise the matenals necessary to ieconstiuct an env ironment that is tamihai to them, l e the amoiphous extiacellulai matnx of the dermal connective tissue In slightly less scientific terms, the human cells do all they can to "make themselves comfortable^" By observing Figure 13 it can be noted that alter three weeks ot contact with the fibioblasts and once these had been appiopπately removed, the surface ot the bio-membrane had taken a glassy appearance, since a homogeneous and "tianslucent" material coveted all its lπegulaiities

SEM images also show that bundles ot fibtes with a slightly undulating pattern protiude moie or less above this glassy surface, these aie presumed to be bundles of ne lv totmed collagen fibres shown bv atrow s in Figute 13 Thus, once adequate amounts of amoiphous extracellulai matnx had been totmed, the fibioblasts pioduced and extta-cellulaily secteted the piecuisors of the collagen fibtes. which w eie then assembled togethei as normally occurs in

o The de novo pioduction of collagen fibtes indicates that the human fibioblasts placed in contact with the fibioin bio-membrane as per this invention not only actively proliferate but also reach a fairly high level of differentiation and functional maturation

Finally, immunohistochemical studies showed that, as occurs also in \ ι\ o, the protein vtmentin constitutes the bulk of the intermediate filaments making up the cytoskeleton of the human fibroblasts cultured on the fibroin bio-membrane

In bt tef these observ ations convincingly demonstrate that the fibroin bio- membiane as per this invention constitutes a scaffold capable of interacting almost peifectly . trom a biological point of view, with normal adult human fibioblasts. allowing them not only to proliferate actively but even to mature functionally The end tesult is that the fibroin bio-membrane as pet this invention becomes inhabited by a population ot normal matuie human fibroblasts that pioduce and externally secrete both the components ot the amoiphous extiacellulai matnx and the piecuisois ot collagen ftbies (and perhaps elastic fibres too), just as occurs in normal in \ n o connective tissue In conclusion, from a biological standpoint the tibioin bio-membrane as per this invention has pi oven itself to be a substrate suitable to act as a scaffold for the creation ot a bio-artificial dermis apt foi clinical applications

Fin thei more, in order to experimentally validate the suitability the fibroin bio- membrane as per this inv ention also as a scaffold tor a composite, dermo-epidermal svstem studies w eie earned out on the intei actions occuri ing between the bio- membi ane accoidtng to this invention and noi mal adult human kerattnocy tes d e epideimal cells)

As seen earhei , keratinocytes form the most superficial epithelial layei , usually know n as the epidermis, of the skin By definition, keratinocytes are epithelial cells since at neat v ariance with fibioblasts, they adhere to each other veiy closely by means ot membtanal specialisations, so that the extracellular space between two adjacent epidei mal cells is minimal Fuithermote, the epidermal cells pioduce and outw aidly seciete numei ous cytokines thtough which they intensely communicate w ith the adjacent keiatinocytes via leceptoπal signalling systems ///

o only the the sti atum i>ernιιnatιxum of the epidermis, I e the layer in close contact with the basal lamina (which marks the boundaiy with the connective tissue) contains piecisely spaced stem cells, horn the division of which and of their progeny the keratinocytes pioper denve In the remaining layers of the epidermis the keratinocytes undergo a complex and coordinated set ot subcellulai ptocesses leading to their progressive ditfeientiation and apoptosis ( "diffpoptosi s") at the end of which thev aie eventually transtoi med into lifeless hornv scales ("corneocy tes^' ). which are still capable ot mechanical protection and w atet loss prevention Human keratinocytes can be cultured in \ ιtι o. but they lequne a continuous contnbution ot exogenous growth factors that in vivo are partly produced by the fibioblasts lodged in the undeilying dermis. pattly secreted by the keiatinocytes themselves, and partly derived from the blood cnculating in the deimal capillai tes The ability to tepioduce at least a good part of the inteiactions that occur in o betw een the keiatinocytes and the dermal fibroblasts would constitute the main advantage of clinically using dermo-epidermal substitutes (sheets of aititicial skin) with respect to grafting sheets only consisting of keratinocytes

The keratinocytes isolated by means of enzymatic dissociation from the epidei mis of thiee ditfeient subjects weie seeded on portions of the fibi otn bio- membiane accoiding to this invention ( / ) alone, (2) in the presence on the same surface ot human fibroblasts that had been previously irradiated ( i.e. incapable ot proliterativ e activ ity but still aliv e and functioning at least for a certain time), and (3) in the presence on the opposite side of the bio-membrane of human fibroblasts that had or had not been prev tously irradiated. It should be remembered that the aim of the co-culture with the fibroblasts was to recreate a situation similar to that in vivo, in which the two types of cells are subjected to reciprocal interactions. The in vitro culture of the bio-membranes as per this inv ention seeded with keratinocytes with or without fibroblasts was earned on for at least three w eeks. The results obtained demonstrate tor the first time that normal human keratinocy tes seeded on samples of the fibroin bio-membrane in the absence of fibioblasts promptly adhere to it. forming fairly small cellular clumps. Three days after seeding, the keratinocytes exhibit a typical epithelial appearance and are possessed with normal cytological features. However, the absence of the fibroblasts makes itself felt. since the keratinocytes grow rather slowly and even nine days after seeding the cellular islets are a long w ay from confluence (Figure 16). These observations demonstrate that the fibroin bio-membrane allows for the adhesion, survival and growth (albeit a slow growth ) of the normal adult human keratinocytes: an extremely encouraging preliminary lesult Ev en after seeding onto one of the surfaces of the bio-membrane previously cov ei ed in part by pre-iπadiated ( not proliferating but aliv e ) human f ibroblasts. the human keratinocytes promptly adhered to the substrate. Three days after seeding, numerous keratinocy tes were present, forming fairly extensive islets betw een which the tapered forms of the fibroblasts could still be identified; even in this case the cytologic features of the keratinocytes appeared normal (Figs. 17 and 18). Nine days after seeding, the initially noted difference with the bio-membranes seeded with keratinocytes alone was even more evident: the keratinocyte islets were much larger, while most of the pre-irradiated ( incapable of replicating) fibroblasts had disappeared (Fig. 19 ). After 15 days, the pre-irradiated fibroblasts had completely disappeared. while the keratinocyte islets had merged together to form a continuous layer of cells, nearly all ot which were still very small and. therefore, still actively proliferating (Fig. 20) On the othei hand, human keiatinocytes co-cultuied w ith pie-inadiated fibroblasts on d plastic sin fac e had formed aftei nine days modeiately sized islets, hich were on average considei ably smaller than the islets of the same age on the fibroin bio- membiane as per this invention Moreovei , again on a plastic suifac c pait ot the epideimal cells had in the meantime giown consideiably in size, therebv abandoning then pio fei ative activity to lrieveisibly move along the "chffpopto s i s^" pathway Theiefoi e. the simultaneous presence on the fibioin bio-membi ane ot pi e-u radiated human fibioblasts is capable ot significantly stimulating the piohfeiative activity of the human keiatinocy tes thereby pi ev enting the onset ot the c ffpoptosis phenomenon Thanks to this the human adult keiatinocytes lapidly multiply, thus leconstituting in just 15 dav s a continuous sti uctuie similai to the geiminativ e lavei of the ;//

o epideimis

Fmallv ev en when seeded on the suitace opposite to the one to w hich the eithei pie π i diated oi untieated human fibioblasts had adhered, the human kei atinocytes had toimed numeious clearly distinct islets already aftei three days, consisting ot small proliferating cells endowed with normal cytological features Within nine days these islets had expanded considerably and aftei 15 days they had formed a continuous layer of cells that weie still all small and thus actively proliferating It can be concluded that it the fibioblasts aie piesent even on the surface opposite to that occupied by the kei atinocytes and thus w ith no dnect intercellulai contact intervening between the two cell tv pes thev keep pioducing pow eitul mitogenic stimuli foi the epideimal cells

Taken togethei these iesults clearly demonstiate that the simultaneous piesence on the two opposite sides of the tibioin bio-membiane of the tw o main types ot skin cells, l e keiatinoc tes and fibioblasts. is not only technically possible but leads also to the establishment ot positive reciprocal interactions w hich, by effectively prev enting the piocesses leading to chffpoptosi s. induce the keratinocytes to prohfei ate intensely and to toi m in approximately two weeks the equivalent of the sti atum

of the ///

It is theiefoie appaient that the tibioin bio-membrane as per this invention is a neai ly ideal scaffold foi the pioduction of sheets of bio-artificial skin, in which both keratinocy tes and human fibroblasts can be simultaneously piesent and activ e ILLLSTRATION OF DRAWINGS

Fuithei teatuies and adv antages of this inv ention will be ev ident trom leading the follow ing description of one foim of leahsation giv en here as an example with the help ot the tiguies enclosed heiein. in which 5 Figure 1 shows a light micioscope picture of the fibioin bio-membrane obtained in accoi dance with this invention, on the 12^lh day ot staying in x itro in the total absence of human cells The fine folds that inciease the suiface area of the membrane are paiticularly evident.

Figuie 2 show s noimal human adult fibioblasts cultured in x iti o and obsei ved 10 undei the fluorescence micioscope after staining with the fluorochrome acridine orange, hich binds to nucleic acids A metaphasic plaque of chiomosomes in a div iding cell can be easilv seen at the centre ot the lllustiation.

Figure 3 show s noimal human adult fibroblasts cultured on the fibioin bio- membiane obtained in accoidance with this invention, three days aftei seeding Gioups 15 ot cells close to each othei can be noted,

Figuie 4 show s noimal human adult fibioblasts in culture on the membiane obtained in accoidance with this invention, six days aftei seeding Cell accumulation and the two veiy fine folds that cross the bio-membrane along the horizontal axis can be obseived. 20 - Figuie 5 show s a light microscope pictuie ot human fibioblasts in cultuie on the bio-membrane as pei this invention. 21 days aftei seeding The high density achiev ed by the monolayei of cells and the formation of a notable cellulai aggregate consisting of sev eial layers, v isible on the light ot the liguie. aie worthy ot note,

Figuies 6 and 7 aie graphs iespectively illustrating the accumulation of uiea and 5 ot lactate in the giowth media to which the normal human fibioblasts cultuied on the bio-membiane as pei this inv ention were exposed,

Figuies 8 and 9 aie two diagrams of the cumulative curves ot the consumption ot glucose and the accumulation ot lactate pioduced and secreted bv noi mal human adult dermal fibioblasts taken from tout different sub_jects, during 2 1 day s of //; \ ιtro ι⁽⁾ cultuie on the bio-membiane piepaied accoiding to this inv ention,

Figuies 10. 1 1 . 12 and 13 show the ultrastructural changes undergone by the suitace ot the bio-membrane as pei this invention The bio-membiane is shown as it appeal s pnoi to cell seeding and attei one. two and three weeks ot contact with the noimal adult human fibioblasts The images were obtained with a scanning electron micioscope ( SEM ) aftei remov l ot the fibroblasts by means ot a technique that does not damage the bio-membiane in any way.

Figures 14 and 15 both show light micioscope pictuies of normal human adult keiatinocv tes seeded on a bio-membiane obtained in accoidance with this inv ention and cultui ed ;/; \ ιtι o in the absence ot fibi oblasts, lespectively show ing then typical epithelial appeaiance and then noimal cytological charactenstics, - Figui e 16 shows a light microscope picture of normal human keratinocytes seeded on a bio-membrane obtained in accordance with this invention and cultured ;/^; \ iti o in the absence ot fibioblasts. 9 days aftei seeding Cell islets still a long w ay fiom confluence can be seen

Figuies 17 and 18 show two light micioscope pictuies of normal human adult keiatinocytes thiee days aftei seeding on a substrate consisting of a bio-membiane as pei this invention, which had prev iously been partially covered with pre-irradiated

( 6000 Rad) human fibioblasts, the normal cytological features of the fibroblasts can be seen in Figuie 18.

Figuies 19 and 20 show the noimal human adult keiatinocytes lespectiv ely 9 and 15 dav s aftei seeding on the substi ate obtained as per this inv ention pi ev iously inoculated ith pie π iadiated fibi oblasts that appeal scatteied.

Figuies 21 and 22 show noimal human adult keratinocytes co-cultured with pre- ni adiated fibi oblasts on a standaid plastic sui tace. nine days after seeding of the keiatinocytes. the cy tological features of the cells can be seen in Figure 22, - Figuies 23 and 24 show two light microscope pictuies of the sui tace ot the substiate obtained as pei this invention, this surface being opposite to the one on w hich noi mal human adult pre-in adiated and not pre-n radiated fibi oblasts had been piev louslv inoculated, thiee days after seeding,

Figuie 25 is a light mici oscope pictuie of what is described in Figuies 23 and 24, 9 dav s aftei seeding of the keratinocytes.

Figure 26 is a light micioscope picture of what is described in Figuie 23. 15 days aftei seeding ot the keiatinocytes

DETAILED DESCRIPTION OF THE INVENTION

Information will now be given regarding fibroin, the expenmental method used to obtain it and the tests earned out on three types of membiane in oidei to verify the effectiv eness in achieving the aims of this invention, the results ot w hich made it possible to determine the most suitable type of membrane

Details ill then be given concerning the methodological proceduies employed toi the ;/; » iti o cultuie and the piohferation assay of specialised tissue cells of various types ot the human body The follow ing paragiaph will theretoie. in certain detail describe fibioin. a natui al piotein contained in silk thus pioduced by the domestic silkw oi m Bυmbw man and cuiientlv also obtainable bv means ot genetic engineering techniques The use of this material as a substrate foi the surviv al, piohferation matuiation. and ditfeientiation of tissue cells, ditteientiated oi otherwise, of the human body, constitutes the grounds foi the patentability of this invention

The silky filament of the domestic silkworm consists of two monofilaments of tibioin, a fibrous protein, cemented together by seπcin, a gelatinous piotein Fibioin is foi med by a sequence of amino acid residues consisting mainly of glycine (glv ) alanine (ala). seπne ( sei ) and tyiosine (tyr) These amino acids can account for up to 90% of the entne molecule

In shoi t the sti uctuie ol the cry stalline component precipitated horn raw fibioin as identified aftei incubation with chemoti ypsin, and constituting the fraction kno n as Cp. is

Gly-Ala-Gly-Ala-Gly-Seι-Gly-Ala-Gly -[Ser-Gly -(Ala-Gly)„jVTyι The i emaining 10% is an irregular mixture in terms of composition and sequence ot almost all the iemaining amino acids, including low amounts of pioline and hydioxy-piohne, w hile cystine is absent or nearly so

These featuies distinguish fibroin, tor example, from keratin and from collagen, and explain some ot its contrasting mechanical pioperties, particular impoitance being given to its inextensibility . whatevei the envπ onmental conditions in which it finds itself The moleculai weight ot the macromolecule is 370 kD and is foimed bv a chain know n as L. l e light, w eighing 25 kD, and a laigei chain known as H. I e heavy weighing 350 kD These two chains aie joined togethei by a disulfide budge Fibroin piesents in three different possible phases - the amorphous phase, also known as iandom coil" which is soluble in water.

- the α-hehx phase, also known as "silk I which is metastable and also soluble in watei . and which, as such, is easily subject to a phase tiansition towards the β-sheet phase, also known as

- silk II which is not hydio-soluble and is foimed by paiallel chains joined togethei by hydiogen bonds with a high order level

The natuial tiansition ot any system fiom a metastable toim to a stable form, in this specific case horn the "silk I ' form to the silk II ' toim. takes a veiy long time. since it is meiely the lesult ot the leorgamsation of each individual atom in the constituent molecules fiom a high energy state to a lowei , and hence moie stable, eneig state

This is why nature ensured that this tiansition occuired during the silkworm^'s secretion phase An analysis of the fibroin secreted by the silkwoim iev eals the piesence ot a 55% of ciystals in the β-sheet form, which are stable and scatteied in an amoiphous matnx, obviously, the reveise process only is possible v ia piotein degradation

Bv dissolv ing the fibioin it is also possible to obtain a totally amorphous matenal theretoie entirely soluble in water

The complex secondary moleculai structuie, where this retei s to the dev elopment of the molecules in the three-dimensional space, ot silk can be used to check specific interactions in ditfeient chemical and mechanical envnonments

The piesence and fiequency of the v arious ciystalhne structuies and of the amoiphous zones in the fibioin molecule can be modified by stietching, by compression oi by means of v anous chemical methods The aims of these processes that lead to confoimational changes consist in the preparation of stable membranes acting as ban lers and substrates to immobilise and store enzymes See on this topic A Kitamura et al Application of silk fibioin to functional membranes lelationship between conditions ot membrane piepai ation, membi ane potential and ion pei meability Published in Sen-I Gakkaishi. v ol 44. no 4. 1988. pages 193- 197

The invention descnbed here is not limited by the following accomplishment examples The Literatuie cleaily show s that the conditions lequired foi the pieparation ot the tibioin membi ane by means of ca sting, in other w ords the concentiation, the solvent, the substiate, the temperatuie. etc , aie all linked to the properties of the lesulting fibioin membranes These properties are the degree of ciy stalhnity, the absoiption of watei and the conformation On the basis ot this lelationship. the vanous prepaiation parametei s weie studied in order to obtain a material that overcomes the initial problems of fragility and instability in a watery env nonment that aie ty pical of such a matenal. as seen abov e

Once perfected, the piepaiation conditions and procedures were standaidised to make them totally lepioducible and to obtain samples that aie always homogeneous Obtaining the fibroin

The fibroin is obtained thiough a normal piocess of degumming law silk (fibioin and seπcm) in fabric form was placed in a wateiy solution of Maiseilles soap at a concentiation ot 10 g/1 with a fibioin bath/soapy solution ot 1 100, at a temperatuie of 98°C foi an houi The samples weie then washed lepeatedly in distilled watei and any impurities and fatty substances weie extiacted in petroleum ethei

The efficiency ot the degumming process was checked by observ ing under the light micioscope a sample stained with Coomassie blue stain

Preparation of the fibroin membranes The pi ocedure toi the pieparation of the fibroin membranes is based on piotocols alieady existing in the Liteiature, such as toi example the procedure defined bv \ Minouia et al in then aiticle entitled Physico-chemical pioperties of silk fibroin membi ane as biomatenal Published in Biomatenals. Vol 1 1 , August 1990 pages 430- 434 These pi otocols w ere appiopnately modified in ordei to pei fect the level of biological inteiaction of the membianes with the cellulai env nonment w ith w hich they aie placed in contact The degummed silk obtained as descnbed in the pievious example w as dissolved in a w atei y solution of 9 M of lithium bromide at a weight/volume l atio ot 1 g/ 10 ml with a 10% solution, at a temperature of 65°C foi 3 houis

The solution obtained was filtered through a no 1 porous septum and diluted with distilled watei to bung the solution to a 5% weight/volume ratio

The solution was dialysed against watei toi 2 days in order to achiev e the total elimination ot salt

Spectia/Poi dialysis tubes manutactuied by Spectrum - MWCO 3.500 w ere used. 14 mm in diametei and w ith a v olume/length ratio of 1 0 ml/cm The dialysis membi ane selected had a low molecular cut in older to retain small peptides in the solution that became detached during the prev ious phases and which seem to favoui

Casting of the watery solution at 5% (w/v ) was pei formed at loom tempei ature with a poly styiene mould This polymei was chosen for the mould since it was found not to inteifeie w ith the structuie ot the matenal (formation of a material with a high peicentage of crystallinity on the glass ), and also because it does not modify the suiface morphology of the membiane in contact (as polyethylene does)

The pioduct obtained is an amorphous state membrane, thus completelv soluble in w atei Ti ansition h orn the l andom-coil toi m to the lequiied stable and oi dei lv β- sheet toi m w as achieved bv Heating the membiane w ith a wateiy solution ot methanol ( 80 100) bv means of a piocess already known in the Liteiatuie and descnbed by N Minoura et al in "Fine structure and oxygen permeability ot silk fibroin membiane treated with methanol", Polymer , Vol 31 , February 1990, pages 265-269 Evaluation of the stability of the materials treated in a watery solution of methanol

This w as pei formed by assaying the proteins released in a saline solution ( PBS ), ev aluating the molecular weights by HPLC analysis

The materials used foi companson are a conti ol material, an untieated oi amorphous, material and a matenal physically treated with heat

The table shows that the treatment with methanol is more effective in inducing a change in protein conformation and in inducing it to acquire the β-sheet confomiation, which makes the membrane extremely stable in water since it causes the establishment of strong intermolecular hydrogen bonds.

Characterisation The characterisation parameters ot the fibroin molecule obtained by means of the procedure described in the preceding examples are as follows: a) molecular weight of the degummed fibre and of the fibroin after the solubi sation and dialysis phases; b) molecular weight of the lyophilised dry residue released in distilled water at different times by membranes obtained with different preparation methods; c ) angle of contact: d) water absorption measurements: e) physical-mechanical tests and dynamic mechanical thermal analysis ( DMTA): f) Differential Scanning Caloπmetry Determination of the molecular weight of fibroin in steric-exclusion chromatography The fibroin membrane samples were analysed by means of chromatographv. ith the following instruments:

- HPLC Waters consisting of a model 510 pump, a U6K in_jector, a UV/Vis model 490 detector. The analysis conditions and the data acquisition and processing were handled by the Maxima 820 chromatographic software;

- Shodex Protein KW-804 column. Fibroin

Analysis of the results does not reveal anv significant differences in the distnbution of the moleculai weights between the fibroin samples analysed (fibie aftei degumming and fibioin aftei solubihsation with LiBi )

In both cases the denatuiating conditions of the mobile phase led to a great inciease in the hydrodynamic volume of the fibroin molecules, placing them at the uppei limits of the tiactioning lange ot the column used (average appaient moleculai weight 650-700 kD) This is demonstrated by the asymmetrical form ot the elution curv e w hich piesented a lapid ascent in coirespondence with the exclusion v olume of the column, followed by a giadual descent towards the region ot low molecular weights \oι can it be excluded that mtei molecular aggiegation phenomena occui in the solution How ev en fiom the lesults obtained it can be concluded that solubihsation with lithium bionude does not appeal to have led to significant modifications to the stiuctuie ot the fibi oin the moleculai w eight being practically unchanged w ith lespect to the initial fibre Residues The piotein matenal that was detached orn the films preseived in w atei for the times adopted is chaiactei istic. horn a chromatogiaphic point ot view since it contains mainlv piotein fragments ith a medium-low molecular weight

It is believed that these aie fragments of fibioin, in paiticulai peptides onginating trom the low moleculai weight subunit L (Light chain) which were detached horn the film immei sed in watei as the result of the interaction with v arious tactoi s that act in a concentrated way . such as the swelling action ot the w atei ana extiaction-solubilisation of the more soluble protein fractions, the piobably hydrolytic action ot the w atei . the piobable vanations in pH and the fiagments that are detached horn the chain dunng the phase of fibroin solubihsation in lithium salts In tact, the alkaline conditions of degumming can induce partial hydrolysis ot the disultide budge that in natural fibroin binds the subunits H (Heavy chain) and L This hy pothesis is leinfoiced by the presence of a shouldei that emerges in the low moleculai w eight legion of the fibie elution curv e, as well as by prev ious expenence ( unpublished data) Angle of contact

The dynamic angle ot contact (advancing and leceding) was measured using a Cahn model 322 miciobalance and the Wilhelmy technique Moist samples (steady state ) measunng 20x5x0 15 mm were analysed with 3 cycles ot immersion and emergence at 150μm/sec in distilled water by HPLC (Merck) at a temperature of 25°C The samples in question did not show any appieciable phenomena ot hysteresis, and the aveiage angle of contact measured was 55° ± 3°

Samples comparable to the previous ones but with a lowei degree of watei absoiption showed much highei angles of contact with lespect to the value iepoited above

It should in any case be pointed out that the expenmental value to be considered is the one measuied on fibroin membianes in a steady state, since it repioduces a more co ect phy siological situation

Absorption tests in distilled water The membranes weie weighed after stove drying at 60°C and then at incieasing incubation times in distilled water (15, 30. 60 minutes. 2 houis. 4 houis, 8 houis and trom one to 45 days)

Maximum water absorption is evaluated at aiound 40% in weight attei 2 days in a wateiy envnonment

Mechanical tests Measuiements weie taken relative to the mechanical chaiactenstics of the membianes at different degiees ot hvdiation

The curv es seem to show an increase in the lesistance of the material as the watei absoiption incieases

It is possible that theie is a loss of substances with a plasticising effect and/oi the toimation ot intei -chain bonds that confei to the membiane supenoi mechanical characteristics, w ith a simultaneous structural anation in favoui of the β-sheet oi silk II civstalhne phase

Comparative test

Three membranes weie produced, tw o ot which, membianes B and C, made trom non-puπtied fibroin thus containing negligible fractions of impurities, and membrane A. consisting ot a copolymer of D, L-lactic acid at 60% in weight and of ε- capiolactone at 40% in weight, and each ot them pioved to be suitable for use as a substiate in accoidance with this invention

The proceduies employed to obtain the three membranes are described below Membrane A

D L-lactic acid ( Boehnngei-Ingelheim) w as punfied by leciystalhzation fiom a solution in ethv lacetate and dned at 45 C undei v acuum toi 24 houis

The ε-capiolactone (Aldnch) w as distilled undei leduced piessuie and pieseived undei nitiogen piessuie

The polymer was produced by causing the monomei s to react in nitiogen atmospheie using stannous ethyl hexoate as a catalyst, in a glass leactoi agitating at 120°C Aftei polvmensation, the copolymei was purified by dissolution in acetone and subsequent piecφitation in methanol The membrane w as piepaied bv solv ent evaporation fiom a solution of the copol y mei punfied in acetone, subsequently eliminating the solvent by oven vacuum treatment Membrane B

The degummed silk w as dissolved in a 9 molai solution of lithium biomide in w atei . at a tempeiatuie ot 65°C foi three houis at a concentiation ot 0 1 g/ml

The solution w as then filtered through a porous ceramic filtei and diluted with distilled water until it reaches a concentration ot 0 05 g/ml. and then dialysed against w atei foi tw o days to eliminate the salt, using dialysis tubes (foi example Spectra/Poi manutactuied bv Spectrum - MWCO = 3500, diametei = 14 mm. Volume/Length = 1 5 ml/cm)

The solution w s then poured into polystyrene containers and the membiane was obtained by ev apoiation ot the watei at room temperatuie

The membiane as then immeised in a solution of 80% methanol in watei for 60 seconds to lendei it crystalline and thus insoluble in water Membrane C

The degummed silk w as dissolved in a 9 molai solution of lithium biomide in atei at a tempeiatuie of 65 ^CC toi thiee houis, at a concentration ot 0 1 g/ml

The solution was then filtered on a porous ceramic septum and diluted with distilled watei until it teaches a concentration of 0 05 g/ml, and then dialysed against watei foi two days to eliminate the salt, using dialysis tubes (for example Spectra/Por manutactuied by Spectium - MWCO = 3500, diameter = 14 mm, Volume/Length = 1 5 ml/cm)

The solution w as then brought lapidly to freezing temperatuie in liquid nitiogen and then tieeze-dned

The tieeze-dned solution w as dissolved in isopiopanol fluoiide. obtaining a 5% ( weight/v olume) solution of fibioin

The solution was then pouted into polystyrene containeis and the membrane was immei sed in a solution ot 80% methanol in water tor 60 seconds to make it crystalline and thus insoluble in w atei

The thiee membi anes weie stenhsed in ethylene oxide placed in contact with noi mal human fibroblasts horn previously lnfotmed adults, and incubated at 37°C in a growth medium toi thiee weeks

Obseivattons w eie then made using an inveited microscope, both under normal and tluoiescent light, ot the in x itt o cultures obtained, w hile the growth media were changed at legulai intei v als and the cell-conditioned media weie sampled and stored at - 80°C toi subsequent biochemical analy sis

With paiticulai leteience to the Figures, it can be seen how the suiv iv al and piohlei ation ot the normal adult human keiatinocytes in the cultuie grown on the membiane B obtained in accoidance with this invention is particulai lv evident, and Figuies 17-25 also show how the pnoi seeding ot pie-niadiated and non-pie-iπadiated normal adult human fibroblasts, in order to prevent then proliferation, w hich takes place twice as quickly compared with the proliferation rate ot the keratinocytes. did not prev ent oi in any way condition the crowding of the substrate as pei this inv ention by the epidei mal cells, ev en when they were seeded on the opposite suiface of the membiane

Moieov ei the images cleai lv show how the cv tological featuies of the cells making up the cultuies giow n on the suiface ot the substiate aie completelv normal

The lesults obtained and the comparisons of membiane B as per this in ention with the othei two membi anes tested for efficiency led to the following surpnsing conclusions membrane B as per this invention and membrane C a) allow a more intense cellulai growth with lespect to membiane A, b ) allow veiy intense synthesis metabolic processes to take place without significantly activating proteocatabohc processes Moieovei. membrane B as per this invention in companson with membrane C can be pioduced in considerable quantities at lower costs thus hav ing not only typical biological advantages, but even economic ones and thus also being easiei to maiket

Protein expression Although the morphology of the keratinocytes and then ways of aggregating cleai lv ditfei horn those piopei of the fibroblasts and can be seen ev en by a non- expenenced eye. it seemed appiopnate to demonstrate that on the bio-membiane as per this inv ention they express specific cytoskeletal proteins propei of the intermediate filaments l e the cytokei atins (not expiessed by the fibroblasts) Sheets ot the bio-membiane obtained as described in Example X. on which kei atinocytes had been cultivated, were challenged with a ' pan" antibody dnected against the cytokeratins and then incubated with a secondary antibody marked with

FITC or bound to alkaline phosphatase. We thus could demonstrate that the keratinocytes contain cytokeratins filaments in their cytoplasm. On the other hand, an antibody directed against vimentin, the typical protein of the intermediate filaments of the fibroblasts. decorated the intermediate filaments of the latter cells very clearly without reacting with the keratinocytes in any way. The two cell types thus maintain cvto-specific characteristics even when cultured on the bio-membrane as per this invention.

IN VITRO CELL CULTURE METHODS Example I

In vitro culture method of normal squamous epithelial cells of the human epidermis, oral cavity, conjunctiva and cornea and of fibroblasts of the underlying connective tissues The above-mentioned cells can be isolated with comparable techniques from biopsy samples of the corresponding tissues (skin, oral mucosa, conjunctival mucosa and cornea). As an example for them all, the methods applied to the epidermis and the dermis obtained from human skin biopsies will be described.

On reaching the laboratory, the skin sample is incubated at 4°C overnight in a dispase solution (ϋ.25% w/v). The attenuated enzymatic digestion makes it possible to detach the epidermis (as a single lamina) from the underlying connective tissue (dermis and subcutaneous tissue) with no difficulty; the two tissue samples obtained in this way undergo separate treatments.

(A )Dermis and subcutaneous tissue

The aim of the procedure is to isolate the fibroblasts, which are the cells assigned to maintain the structure of the connective tissue and which can be cultured separately or co-cultured with the keratinocytes { 'feeder layer' method).

The connective tissue sample is cut into fragments thanks to the scissor-like action of two scalpel blades in a solution of trypsin (0.25% w/v) and EDTA (0.02% w/v); enzymatic digestion is then performed in an incubator at 37°C with the help of a magnetic stirrer maintained at low speed (50 rpm) for 30 minutes, followed by centrifugation at 600 rpm for 10 minutes a 4°C. The supernatant is decanted, the pellet is lesuspended and the liv ing cells are counted in a Neubauei chamber The fibroblasts. pre-inadiated with gamma rays (6000 rad) or non-pre-iπadiated. are then seeded (B) Epidermis

The aim of the piocedure is to isolate the epidermal keratinocytes, which aie the main type ot epithelial cells ot the skin The thm epidermal lamina is carefully and quickly fragmented in a trypsin solution (0 25% w/v ) A specific trypsin inhibitor is then added and the solution is centπfuged at 600 rpm toi 10 minutes at 4°C The supernatant is decanted, the pellet is lesuspended and the living cells are counted in a Neubauer chambei The keiatinocytes aie then seeded Supports used for the in \ itro cultures

1. Plastic tlasks w ith tieated oi untreated surfaces to mciease the adhesiv eness

2. Plastic tlasks w ith suifaces as abov e, coated oi not with a la ei of pre-inadiated fibioblasts

3. Fibioin membianes (puie or combined with othei compounds) w ith the uppei suitace coated or not with a layei of normal fibioblasts or fibroblasts pre-irradiated with gamma rays (6000 rads)

4. Fibioin membianes (pure oi combined w th other compounds) w ith the lower surface ( l e the one opposite the uppei suiface) coated oi not with a layei ot noimal fibioblasts oi fibioblasts pie-inadiated w ith gamma ray s (6000 rads) Culture medium

The medium 3MCDB 153 1 ( consisting of three pai ts of Dulbecco s Modified Eagle Medium [DMEM] and one pait of Medium F 12 ) is normally used, to which foetal bov ine seium (FBS 10% v / ). antibiotics (solution ot penicilhn-streptomvcin 1 % w/v). epideimal giowth f actoi (EGF, 0 1 μg /ml ), insulin (20 ng/ml). pituitary extiact (PTE, 30 μg/ml ) and hydrocoi tisone (0 5 μg/ml) are added The medium is leplaced ev ei y day oi every othei day with a fiesh medium ot the same composition Evolution of the cultures

The noimal human fibioblasts piohfeiate rapidly , toiming fust a continuous monolayei of cells paiallel to one anothei and then a senes of layers with cells that aie not always in pai allel In the meantime, the fibroblasts produce and seciete incieasing quantities of extiacellulai matrix components and of collagen fibie precuisors the lattei ones being then integiated in bundles ot fibres which max even be quite latge in size The fibioblasts aie avid consumeis ot the glucose piesent in the culture medium and typicalh seciete considerable quantities ot lactic acid into the medium The fibioblast cvtoplasm contains both intermediate filaments consisting of vimenttn and their cytot pical matker decortn

The normal human keratinocytes proliferate rapidly, starting trom minute clustets and fotm a single layei of small and highly adherent epithelial cells They have a mitottc cycle time of about 48 hours and then cytoplasm contains intermediate filaments totmed tiom the so-called ' light ^' cytokeratins (while the cells aie piohferating actively) or 'heavv cytokeiatins (when the cells differentiate) If the keratinocytes reach the suitace of the giowth medium, they toim a series of layers whose suiface cells end up bv entenng the phase ot terminal postmitotic ditfeientiation {chffpoptosis) Dφpυptosis is tnggeied even if EGF is withdrawn fiom the cells foi longer than 48 consecutive houis The giowth rate of the keratinocytes is much fastei if human oi todent pre- iiiadiated ot non-pie-iπadiated fibroblasts aie present on the bottom ot the flask ot on the levetse suiface of the cultuie scaffold

Example II

In vitro culture method of normal human adult astrocytes

Astiocvtes aie neuron supporting cells and can be isolated from tntra-opeiative biopsy samples (eg the cerebial coitex of the temporal lobe) On teaching the laboratotv. the neivous tissue biopsx is cut into minute fiagments subjected to a bland enzvmatic digestion with trypsin (025% w/v) in Hanks Basal Salt Solution (BSS) at room temperatuie (18°C) and, finally mechanically dissociated by repeatedly triturating the tissue fiagments with Pasteur pipettes endowed with progressively smaller bores The isolated cells ate then seeded in cultuie flasks where they pio ferate in an ad hoc medium (see below) Initially then giowth is vetv slow Once they teach a 70% confluence the nerve cells are detached with a solution of trypsin (025% w/v) and EDTA (002% w/v) in BSS and seeded into new flasks The piocedute is tepeated seveial times ovei a number ot months Supports used for in vitro cultures

1. Plastic flasks with treated oi untreated surfaces to increase cell adhesiveness 2. Fibroin membranes (pure or combined with other compounds) Culture medium

Dulbecco^' s Modified Eagle Medium (DMEM) is normally used, to which FBS ( 10% v/v). antibiotics (solution of penicillin-streptomycin 1 % v/v). basic fibroblast growth factor (bFGF; 20 ng/ml), insulin-like growth factor- 1 (IGF- 1 : 20ng/ml). platelet-denved growth factor (PDGF; 20 ng/ml). epidermal growth factor (EGF; 10 nM). β-estradiol ( 10 nM ) and cholera toxin ( 10 nM) are added. The medium is replaced ev ery 2-4 days with a fresh medium of the same composition. Evolution of the cultures When actively proliferating, normal human adult astrocytes appear as small polvgon-shaped cells with an epithehal-hke appearance, which may subsequently differentiate, rather than continuing to proliferate, moving towards the phenomenon ot stellation. i.e. the emission of numerous richly arborized cytoplasmic extensions. Changing the medium frequently prevents stellation from appearing and maintains an intense proliferative activity; changing the medium very rarely has diametrically opposed effects. The astrocytes do, however, express cyto-typical markers, such as glial fibrillcin acid protein (GFAP) and the GAP-43 protein, which can be detected by lmmunohistochemistry and Western immiuioblotting. In protracted cultures, human astiocy tes toim extremely intricate networks of finely arborized cellular extensions which are superimposed on one another in several layers.

Example III In vitro culture method of normal squamous epithelial cells (mesothelial) of human adult serous membranes Human mesothelial cells are isolated by means of enzymatic dissociation from mtra- operativ e biopsies of the serous membranes (pleura, pericardium, peritoneum) As an example tor them all, we will refer to the procedures applied to a biopsy sample of greater omentum (peritoneum).

On reaching the laboratory, the biopsy fragment is thoroughly washed in a saline solution. Enzymatic digestion is then performed with a dissociating solution containing tr^ypsin (0.125% w/v) and EDTA (0.02% w/v) in Hanks' Basal salt Solution ( BSS ). Digestion is carried out in an incubator (at 37°C) stirring the tissue slowly (40 rpm) for 25 minutes. The supernatant obtained is then centrifuged at 1700 rpm for 5 minutes at 4°C. This is followed by re-suspending, counting and seeding of the epithelial cells forming the pellet. Additional cycles of enzymatic digestion of the tissue remaining after the first dissociating cycle can be carried out . However, this involves the risk of also isolating the omental fibroblasts rather than the mesothelial cells alone. At the end of these cell dissociation cycles, the remaining tissue is cut into minute fragments which are also inoculated into the culture flasks. Supports used for the in vitro cultures 1. Plastic flasks with treated surfaces to increase cell adhesiveness 2. Fibroin membranes (pure or combined with other compounds) Culture medium

The culture medium used to culture the human mesothelial squamous cells has the following composition: Ham's F12 enriched with FBS ( 10% v/v), antibiotics (solution of penicillin-streptomycin 1% w/v), insulin (0.5 μg/ml), glutamine (2 mM), iron- saturated human transferrin (0.5 μg/ml), hydrocortisone (0.4 μg /ml) and cholera toxin ( 10 ng/ml). The medium is renewed every 4 days with a fresh medium of the same composition.

Evolution of the cultures The epithelial cells of the human peritoneal mesothelium have initially a small size and group together in tiny clusters sporadically interspersed by fibroblasts. The cells proliferate actively, generating polygon-shaped elements endowed with a cytoplasm rich in organelles that grows in size as time passes and ends up by undergoing a strong vacuolization. Among the increasingly larger and often binucleate epithelial cells, which form more and more extensive sheets but always remaining in a monolayer. in which some nests of small actively proliferating cells persist. These nests may join with one another thereby forming a network. The few initial fibroblasts do not proliferate but die due to apoptosis unless their growth is favoured.

Example IV In vitro culture of normal adult human osteoblastic line cells Intra-operative biopsy fragments of bone tissue from the maxillary bones or from other bones of the human skeleton are repeatedly washed in phosphate buffered saline solution A (PBS/A) befoie undergoing two pre-digestion cycles with type II collagenase (0 5 mg/ml) toi 45 minutes at 37°C The bone fiagments aie chopped ith a bone cuttei and cultuied in clustei-type plates with 12 wells each Supports used for in vitro cultures 1. Plastic flasks with treated or untieated surfaces to increase cell adhesiveness 2. Fibioin membranes (pure oi combined with othei compounds) Culture medium

The giowth medium used tot the in x itio culture of the human osteoblastic cells has the follow ing composition Dulbecco s Modified Eagle Medium (DMEM ) ennched with FBS ( 10% v/v ). antibiotics (penicillin-streptomycin solution 1 % w/v ). sodium ascoibate ( 50 ug/ml ) and boiosihcate glass shveis The medium is lenewed ev eiv 4 day s w ith tiesh medium w ith the same composition The addition of β-glyceiophosphate and agents such as v itamins A and D to the above-mentioned medium exeits a differentiating effect (rather than a merely mitogenic one) on the osteoblastic cells Evolution of the cultures

Aftei the fust two weeks of staying in culture, the cells that migrate out of the bone fragments are detached by trypsintzation (trypsin at 0 25% w/v) fiom the boiosihcate glass shveis to which they mainly adhered and seeded into plastic cultuie flasks again using DMEM enriched with FBS and ascorbate After a turthei lound ot tiypsinizatton, the cells can be placed onto any othei type of suppoit The osteoblastic cells first form single lavei s in w hich the anangement of the v arious elements leads to the cieation of voittces which become moie and more marked as the cells, progressively increasing in numbei form sev etal layei s The proliferating cells do not initially expiess their specific plasmalemmatic maikei, I e the alkaline phosphatase enzyme This appeals at a subsequent stage and continues to be expiessed with varying intensity by the sev eral osteoblastic cells, paralleling then cunent degree of ditfeientiation The most matuie cells piactically stop pioltferating but secrete the collagen fibre piecuisots and the extracellulai matrix components, including osteocalcin and osteonectin. into the medium Eventually, the calcification processes of the extracellular matnx. w hich aie first focal and then moie widespiead. can be so intense as to lead to the /// x itio toimation ot structures very similat to cancellous bone Example \ In v itro culture of normal neonatal rat liver hepatocytes

Neonatal tat hepatocytes aie isolated by means of combined enzymatic and mechanical dissociation of the hvei ot animals not more than 4 days old Aftei the animals have been saciificed using a painless technique, the livets are removed using a special suigtcal table that is cooled in order to limit the consumption of oxygen by the hepatic tissue to a minimum Once extracted, each hvei is chopped into pieces measuiing aiound 2-3 mm The pieces of tissue ate washed several times in saline solution Digestion is then earned out with a solution consisting of type II collagenase (5 mg/ml). ovine testtculai hyalutonidase (5 mg/ml) and tiypsin (0 02% w/v ), in w hich the pieces ot hv ei ate slow ly sti ed tor 15 minutes At the end ot the fust lound ot enzvmatic digestion tne liquid phase is lemoved and the activity of the enzymes is blocked by adding piotease tnhibitots and FBS (20% v/v) before stonng it at 4°C The iounds of enzv matic dissociation are lepeated four-five times, stonng the conesponding supematants at 4^JC The pieces ot hvei "softened" by the incubation with the proteases aie tnturated several times with Pasteur pipettes of increasingly smallei bores This mechanical action almost completely frees the cells from the connective tissue stroma The tractions obtained by enzymatic and mechanical dissociation are mixed and centπfuged at 600 rpm tot 5 minutes at 4°C The cells of the pellet obtained by this pioceduie ate resuspended and counted Finally, the hepatocytes aie sepatated from the stiomal cells bv using a Peicoll™ giadtent betore seeding them onto the cultuie supports

Supports used for the in vitro cultures 1. Plastic flasks with tieated or untieated surfaces to increase cell adhesiv eness 2. Fibioin membranes (pine ot combined with othei compounds) 3. Lltiathin (37 5 μm thick) porous discs of non-toxic polyethylene Culture medium

The giow th medium used foi the neonatal rat hepatoc_\ tes is the HιWo,Ba _m medium, to which FBS ( 10% v/v) oi no FBS and antibiotics (cephaloπdin-stieptomycin solution 0 1 % w/v ) ate added in the fust few houis Evolution of the cultures sing the Peicoll™ cultuies aie set up in which the neonatal hepatocytes make up at least 98% of the total cell population The cells gioup togethei in homogeneous islets in monolayei and generally, alieady aftei 24-48 hours show the reconstitution of the bihaiy canaliculi A small ti action ( around 15% ) of the hepatocytes prohtei ate spontaneous in the cultures and aiound 8 % of the cells die ev eiy day //^; x iti o due to spontaneous apoptosis This means that in basal conditions the in \ ιtι o hepatocellulai population maintains an almost constant size oi gi o s only slightly ev en toi moie than 30 days Ennching the seium-tree HιWo,Ba_2ϋ00 with giowth factors, such as epideimal qi ou th fat toi (EGF). tι cιnsformιni> gi m th fa toi -a (TGF-α). hepatocλ te gi oxxthjactoi (HGF). pancieatic hormones (insulin and glucagon), phorbol estei s and cyclic adenosine monophosphate 5 ( cAMP) incieases the size ot the proliferating hepatocellulai ti action and. at the same time, ot the hepatocellulai population as a whole The gi owth ot the tew sti omal cells present at the start in the cultures is prevented bv using the selective medium HιWo_sBa_^ooo that does not contain the essential amino acid aiginine, which the connective tissue cells aie unable to synthesize The neonatal l at hepatocytes produce ex novo cyto-specific markers, such as albumin, fibnnogen and other plasma globulins and secrete them into the growth medium as well as specifically uptaking H-bilnubin horn the medium dining foui weeks of staying in in x iti cultuie Example VI

Culture of steroid-secreting cells of normal human adult adrenal cortex Portions ot normal adrenal tissue are taken, after obtaining informed consent, fiom adult patients during operations for the simultaneous removal ot kidney and the adrenal gland on the same side The glandulai tissue is decapsulated to eliminate the zona glomei losa and then placed in a Universal type container (volume = 25 ml ) containing 10 ml of Eagle Minimum Essential Medium (MEM) enriched w ith FBS (20 % v/v ) inactivated at 56^ϋC for 30 minutes and. finally sent to the laboratory in a Dew ai flask containing melting ice The procedures for the culture ot the adrenal cortex tissue begin ithin 30 mιnutes-6 houis of remov al of the sample The tissue is transferred to a Petn dish (diametei 10 cm) containing 10 ml of cold (4°C). stenle phosphate buffet ed saline solution A (PBS/A ) The tissue is cut into pieces 4-5 mm in size w hich are lepeatedlv washed with sterile PBS/A to lemove the blood cells, and finally tiansteired to a 50 ml Erlenmeyei flask containing a solution ot trypsin (0 2% w/v ). type I collagenase (0 5% w/v ) and ovine testiculai hyaluronidase (0 4 % w/v ) The pH is adjusted to 7 3-7 4 by adding a small amount ot NaHCO solution (8% w/v ) The histolytic mixtuie is heated to loom temperature ( 18°C) The pieces ot tissue aie left to rest without any agitation in the disaggiegating solution foi 30 minutes Then they undergo toui subsequent lounds ot magnetic staring ( 100 ± 20 ipm) lasting 20 minutes each The vanous supernatants are lemoved, mixed with piotease inhibitors and inactivated FBS (20% v/v ) and stored at 4°C The iemaining pieces ot tissue aie tiansfened to a Petn dish wheie they are chopped with two knives The supernatants and the fiagments of tissue aie mixed togethei and centntuged tw ice at 50 iζ The cells in the iemaining pellets aie mixed togethei pnoi to seeding Supports used for the in vitro cultures 1. Plastic flasks with tieated or untreated suifaces to increase cell adhesiveness 2. Fibroin membranes (puie or combined with othei compounds) 3. Lltia-thin (37 5 μm thick) porous discs of atoxic polyethylene Culture medium

The cultuie medium used is Eagle MEM. enriched with inactivated FBS (20% v/v) cephalondine (50 μg/ml), penicillin and streptomycin (50 μg/ml) and ny statin (25 Ul/ml) Alternatively the seium-hee HιWo_ςBa,₀₀₀ medium is used with the addition of the above-mentioned antibiotics The medium may also be ennched w ith a tiophic- ditteientiative factor specific toi the adrenal coitex cells, I e the adrenocorticotiopic honnone (ACTH) in its entne (amino acids 1-39) oi shortened form (amino acids 1 -24) Evolution of the cultures In the absence of ACTH the cells ot the onae fasticulcita and l eticulai is of the adrenal coitex resemble fibioblasts, lose many ot their typical ultrastructuial and functional featuies and may move towaids apoptosis The addition of ACTH to the medium stimulates the pro feiative processes and leads to marked effects of ultiastructural ditfeientiation (prolifeiation ot the smooth endoplasmic reticulum, ditfeientiation ot the tubulo-v esiculai ci istae of the mitochondria, hypertrophy of the Golgi apparatus, and leduction of the size ot hpid dioplets) and to an intensification of the steioidogenetic piocesses and of the secietion ot steioid hoimones Aftei a 7- day tieatment ith ACTH the /// \ ιtι o cells appeal to have an ultrastiucture completely similai to that of the coπesponding zones of the adrenal cortex in x ivo

Example VII Culture of normal human adult tenocytes (tendon fibroblasts)

The aim ot the proceduie is to isolate the tenocytes or fibioblasts lesident in the tendons - stiuctuies consisting of densely packed highly oideied collagen fibies w ith quite tew inteispeised blood vessels The tendon sample taken intia-opeiatively with the infoimed consent ot the patient is maintained at 4°C until it leached the laboratoiy It is then immediately tianstened to a Petn dish and chopped thanks to the scissoi action of tw o very shaip kniv es The fiagments aie subjected to enzy matic digestion in a solution ot tiypsin (0 25% w/v ). type I collagenase (3% w/v ) and ov ine testiculai hvaluionidase (0 25% w/v j in an incubatoi at 37°C with the help ot a magnetic stirier maintained at low speed ( 50 rpm) toi 30 minutes The tiaction is then centrituged at 600 rpm toi 10 minutes at 4°C The supernatant is decanted, the pellet is resuspended and the living cells are counted The tenocytes aie then seeded Supports used for the in vitro cultures 1. Plastic flasks with tieated or untreated surfaces to inciease cell adhesiveness 2. Fibioin membianes (pine or combined with othei compounds) Culture medium

The cultuie medium used is Dulbecco's Modified Eagle Medium (DMEM ) ennched with inactiv ated FBS (20% v/v ). cephalondin (50 μg/ml), stieptomycin (50 μg/ml) and nvstatin (25 Ul/ml) The medium can also be enriched with platelet dem ed gi ow th factoi (PDGF) and fibi oblast i>ι

th f ctoi (FGF) Evolution of the cultures

The noimal human tenocytes piohfeiate rapidlv. fust forming a continuous single layei ot cells that aie arranged parallel to each othei and then a series of layeis w ith parallel cells that form fairly long vortices In the meantime, the tenocytes produce and secrete onto the suppoits consideiable amounts ot type I collagen fibre precuisors w hich are then integrated into relatively large fibre bundles The cytoplasm contains type I collagen fibie precursors, deconn and intermediate filaments made up of vimentin Like othei fibioblasts, the tenocytes are avid consumeis of the glucose present in the culture medium and, typically . secrete considerable amounts of lactic acid into the medium

Example VIII Culture of normal human adult chondrocytes

Fragments of articulai cartilage are obtained fiom patients who have giv en their informed consent The tissue is maintained at 4°C until it reaches the laboiatory It is then tianstened to a Petn dish and cut to fiagments measuring 1 -3 mm in volume by two veiy shaip kniv es The chondrocytes (oi caitilaginous cells) aie contained in a highly viscous extracellulai matnx The cartilaginous fragments aie theiefoie incubated in 5 ml ot a solution ot ov ine testiculai hvaluionidase (0 5% w/v ) in Hanks Basal Salt Solution (BSS) foi 15-20 minutes at room temperature ( 18°C) The fragments aie then washed in BSS and tiansferred to an Erlenmeyei flask containing 5 ml ot a trypsin solution (0 2% w/v) which is slowly stirred for 30 minutes The supernatant is decanted and a 5 ml solution ot collagenase (0 25% w/v) in BSS is next added After a 30-mιn incubation at 37°C. the supernatant is decanted, a further 5 ml of collagenase solution (0 25% w/v ) in BSS is added and the fragments are incubated for 120 minutes at 37°C The pie-digested fragments are triturated with Pasteur pipettes having giadually decieasing bores The supernatant is lemoved and centrituged at 600 g for 9 minutes The sedimented cells are centrituged again after their le-suspension in BSS The isolated cells aie then le-suspended counted and seeded onto the cultuie suppoits Supports used for the in vitro cultures

1. Plastic flasks with tieated oi untreated suifaces to inciease cell adhesiveness

2. Fibioin membianes (pure or combined with other compounds) Culture medium

The culture medium used is Ham^'s F12 with the addition of FBS ( 15% v/v ). penicillin ( 100 U/ml). streptomycin ( 100 U/ml) and Mg^'+ (2 3 mM) Evolution of the cultures

The human chondrocytes cultuied on two-dimensional substrates appear as spindle- shaped cells that proliteiate i apidly, forming confluent monolavers of cells that are mainly airanged in paiallel to one anothei On the same substiates the production of the exti acellulai matnx components and of type II collagen is extremely limited Onlv the cultui e on thiee-dimensional suppoi ts allows the cells as then pi ohteiating activ itv giadually attenuates, to differentiate and produce considerable quantities ot histo-typical matenals (glycosaminoglycans. etc ) proper of the extracellular matrix and of type II collagen fibies

Example IX

Culture of normal human adult pre-adipocytes

The sample ot white adipose tissue, taken intra-operativ ely aftei obtaining an informed consent fi om the patient, is repeatedly washed with phosphate buffered saline solution A ( PBS/A ) to lemove most ot the blood cells it contains, and then it is cut into pieces w eighing aiound 10 mg each The tissue pieces aie subjected to a first l ound of enzv matic digestion with a disaggregating solution consisting of phosphate buffered salin solution A (PBS/ A, l OmM ). type I collagenase ( 1 5 mg/ml ) and bov ine seium albumin ( BSA. 20 mg/ml) toi 30-40 minutes at 37°C. undei continuous stirring The latio between adipose tissue weight and disaggregating solution volume is lg/4 ml The pie-digested pieces of adipose tissue are then filtered thiough a nylon mesh w ith 250 μm diameter pores, subjected to a second round ot enzymatic digestion w ith the disaggiegating solution tor a further 30 minutes at 37°C and filtered again through a nylon mesh as descnbed above The two cellular suspensions are then mixed and centntuged at 200 g tor 10 minutes To eliminate the erythrocy tes, which at this point constitute the main contaminating cell type, the cellulai suspension is tieated w ith an en thioc ue sing buffei ( ELB NH₄C1 154 mM. KHCO 10 mM. EDTA 0 1 mM) at loom tempeiatuie ( 18-20O foi 10 minutes, and subsequently filtered thiough a nylon mesh w ith 150 μm diametei pores Aftei repeated w ashings and centnfugations (at 200 g foi 10 minutes ), the isolated cells are resuspended counted and seeded onto the cultuie suppoits Supports used for the in vitro cultures

1. Plastic tlasks w ith tieated oi untieated surfaces to inciease cell adhesiv eness

2. Fibioin membianes (puie or combined with other compounds) Culture medium

The cultuie medium used foi the human pre-adipocytes is a defined ratio combination of Dulbecco^'s Modified Eagle Medium (DMEM) and Ham's F12 ( 1 : 1 v/v). containing penicillin ( 100 U/ml). streptomycin (0.1 mg/ml) and FBS ( 10% v/v). Evolution of the cultures

To remove the cellular debris, the cultures are washed with PBS/A after a 16-20 hour incubation period. Fresh culture medium for pre-adipocytes is now added (DMEM/ Ham^'s F 12 1 : 1 v/v enriched with NaHCO_., 15 mM. HEPES 15 mM. biotin 33 μM. pantothenic acid 17 μM, iron-saturated human transferrin 10 μg/ml. penicillin 100 U/ml and streptomycin 0.1 mg/ml). The pre-adipocytes have a fibroblast-like morphology but they emit numerous, often velamentous, cytoplasmic expansions. They proliferate, at first slowly and then more rapidly, but in the conditions described so far they do not differentiate. To induce and maintain adipose differentiation, which implies an irreversible block of the proliferative activity and the progressive accumulation of lipids in the cytoplasm until a signet ring appearance is achieved, typical of the mature adipocytes, the pre-adipocyte cultures are treated with the following compounds added to the growth medium: cortisol ( 100 nM), insulin ( 66 nM). triiodothyronine (0.2 nM) and, for the first three days, with isobutylmethylxanthine (IB MX, 0.25 mM).

Example X Culture of human adult smooth muscle cells The smooth muscle cells can be isolated from the tunica muscularis of hollow viscera (e.g. the intestine) or from blood vessels (e.g. arteries of various diameter and site and veins from the lower half of the body of adult subjects). The visceral or vascular tunicae musculares are isolated from intra-operative samples (after obtaining an informed consent from the donors), placed in Dewar flasks at 4°C and sent to the laboratory. The samples are then repeatedly washed with phosphate buffered saline solution A (PBS/A) to remove the blood, cut into pieces of 1-2 mnr volume and, finally, subjected to repeated rounds of enzymatic digestion. The dissociating solution consists of Hanks^" Basal Salt Solution (BSS) containing trypsin (0.25% w/v) and collagenase (5% w/v). Dissociation is carried out at room temperature ( 18°C) with a slow but constant stirring ( 60 rpm). The supernatants are enriched with anti-protease agents and FBS (20% v/v ). stored at 4°C. mixed together and finally centrifuged at 100 g for 4 minutes. The pellets are washed in BSS and centntuged again several times The isolated cells aie then resuspended. counted and inoculated on the culture supports Supports used for the in vitro cultures

1. Plastic tlasks w ith treated or untreated suitaces to inciease cell adhesiveness 2. Fibioin membranes (pure oi combined with other compounds) Culture medium

The cultuie medium used is Dulbecco's Moditied Eagle Medium (DMEM ) w ith the addition of penicillin ( 100 U/ml). streptomycin (0 1 mg/ml ) and FBS ( 10% v/v ) Evolution of the cultures The smooth muscle cells have a spindle-shaped appearance, they have a single nucleus and piohferate actively /// x itio foiming confluent monolayers ot cells Thev express type-specific isotoims ot actin They can be easily sub-cultured after detachment with a trypsin solution (0 25% w/v) in Hanks' BSS

Example XI Culture of normal endothelial cells of blood vessels

The endothelial cells form single layer lamina of squamous epithelial cells that covei the entne suitace of blood vessels of various diameters The human endothelial cells aie geneially isolated fiom intra-operative samples (after obtaining informed consent) of umbilical vessels, dermal tissue, adipose tissue, etc Here, we w ill limit oui selves to descnbing the method foi isolating them trom the umbilical vessels

Ten cm sections of these asepticallv lemov ed vessels aie placed in Dew ai tlasks w ith melting ice and taken to the laboratory One end of the vessel is fixed to a svnnge which tii st injects phosphate buffeted saline solution A (PBS/A) into the vessel The umbilical vessel is then filled with dissociating solution (containing collagenase 0 25% w/v in Hanks^' Basal Salt Solution [BSS]), the free end is clamped and it is left to incubate foi 20 minutes at room temperatuie ( 18°C) The clamp is removed, the dissociating solution is diained off and pouied, together w ith the PBS/A used to wash out the vessel, into a conical test tube and centntuged foi 5 minutes at 4°C at 100 g The isolated cells are resuspended in BSS and centntuged twice Finally, the spun down cells aie counted and applied to the cultuie supports

Supports used for the in vitro cultures 1. Plastic tlasks with tieated oi untieated surfaces to inciease cell adhesiveness

2. Fibioin membianes (pine oi combined with othei compounds) Culture medium

The culture medium generally used for endothelial cells is Dulbecco's Modified Eagle Medium (DMEM ) with the addition ot penicillin ( 100 U/ml), stieptomycin (0 1 mg/ml) and FBS ( 10% v/v ) It is also possible to use Medium 199 oi medium RPMI, with the same additions The human endothelial cells also require the presence of hypothalamus- denved endothelial mitogens (25 μg/ml) and heparin (90 μg/ml) in the cultuie medium Evolution of the cultures The human endothelial cells in cultuie have a triangulai or polygonal shape with raised maigins w hich confeis them then typical halo due to the associated light diffi action Thev piohfeiate veiy intensely if human serum (up to 20% v/v ) is added to the medium togethei w ith the seveial othei components mentioned above, instead of bovine serum The cells toim a continuous monolayer that eventually coveis the entne suiface of the cultuie suppoit The cells can now be sub-cultured by means of a standaid try psinization (tiypsin 0 25% w/v in BSS) The human endothelial cells typically produce factoi VIII and express specific surface antigens, all detectable with lmmunocy tochemical techniques They also express the conv erting enzyme, angiotensin and uptake the low density poproteins. as can easily be detected with biochemical methods Proliferation of the endothelial cells stops spontaneouslv if thev aie allowed to leach confluence (an example ot contact inhibition ot growth)

The invention described abov e lefers to some of its particular toims of embodiment

It is howevei evident that the limitation is not limited to these forms of embodiment but includes all the modifications and variations that can be consideied and w hich do not lequne the application of any inventive eftoit thus without going bev ond the tiamewoik of this invention as claimed

In accordance with othei pieferred forms of leahsation. the substrate consists of a mixtuie ot bio-iesorbable polymers in which fibioin is present in a quantity v aiymg fiom 0% to 100% in weight

Claims

1. Substrate suitable for the survival, the proliferation and the correct differentiation of specialised tissue cells of the human body, said substrate consisting of a material that is bio-compatible and bio-resorbable in pre- determinable times, in order to achieve complete integration of the transplanted tissue and optimum integration with the other cell systems and their functions in the organism in which the transplant or the implant has been made, or with which a functional connection has been established, said substrate comprising a mixture and/or combination of natural and/or synthetic polymers, wherein this mixture and/or combination includes fibroin.

2. Substrate according to claim 1. wherein aid fibroin is of natural origin.

3. Substrate according to claim 2. wherein said fibroin is secreted by the Bombyx mori silkworm.

4. Substrate according to claim 1. wherein said fibroin is of synthetic origin.

5. Substrate according to anyone of the preceding claims, wherein said mixture contains fibroin in pure form.

6. Substrate according to anyone of claims 1 to 4, wherein said mixture contains fibroin in mixed form.

7. Substrate according to anyone of the preceding claims, wherein the fibroin covers a supporting scaffold advantageously and uniformly, said supporting scaffold consisting of a ceramic, a metal and/or a svnthetic and/or natural polymeric material and by any of their combinations.

8. Substrate according to claim 7, wherein the fibroin is deposited on said supporting scaffold.

. Substiate accoiding to claim 7 wherein the surface of said suppoiting scaffold is coveied with fibroin

10. Substrate accoiding to anyone of the preceding claims, vvheπn the tibioin is piesent in said substiate in a quantity varying trom 20% to 80% in weight

11. Substi ate suitable toi the survival, the proliferation and the coπ ect ditfeientiation of specialised tissue cells of the human body said substiate consisting of a matenal that is bio-compatible and bio-iesoi bable in pre- deteiminable times, in order to lemtegrate the lost tissue oi impaiied functioning by transplant oi implant or proper connection with the best possible integration w ith the other cell systems and their functions in the organism in w hich the transplant or the implant or with which the connection has been made, whereby said substiate consists of pure fibroin

12. Substiate according to anyone of the preceding claims, wheiein human body tissue cells aie seeded on it with the aim of favouring then growth, said tissue cells including those of the skin and ot the liver, mesothelial cells, astrocytes. human skeleton osteoblasts tenocytes or human tendon fibi oblasts chondiocv tes oi cells isolated from cartilaginous tissues, endothelial cells ot blood vessels, steioid-secreting cells of the adienal coitex. smooth muscle cells ot the tunica musc ulat is of the intestine and blood vessels, squamous epithelial cells of the oral cavity or the conjunctiva/cornea and human pre-adipocytes of the white adipose connective tissue

13. L se ot fibroin for the production of a substrate according to anvone of the pieceding claims, said substrate being suitable for the survival, the piohferation and the conect differentiation and functioning of specialised tissue cells of the human body both inside and outside ot it

14. L se of fibioin accoi ding to claim 12, wherein the human body tissue cells include those of the skin and of the hvei , mesothelial cells, astrocytes, human skeleton osteoblasts, tenocytes or human tendon fibroblasts, chondrocytes or cells isolated trom cartilaginous tissues, endothelial cells of blood vessels, steroid-secreting cells of the adrenal coitex. smooth muscle cells ot the tunica mu scularis of the intestine and blood vessels, squamous epithelial cells ot the oial cav ity oi the conjunctiv a/cornea and human pie-adipocytes of the white adipose connective tissue

15. Process foi the pioduction of a substrate accoiding to anyone of the pieceding claims w heiein degummed silk is dissolv ed in a solution ot lithium biomide in w atei at a tempeiatuie highei than loom tempeiature and at a standaid pressuie the solution so obtained being then filtered thiough a poi ous ceiamic filtei . diluted w ith distilled watei , dialysed and left to evaporate in polystyrene containers, wherein the membrane so obtained is immersed in a solution of methanol and water to make it crystalline and hence insoluble in water