Compositions for repairing and regenerating human dura mater

BACKGROUND OF THE INVENTION

The dura mater is a functionally important structure in the anatomy of the central nervous system, forming a system of membranes covering the entire central nervous system and protecting it from external influences.

A dura mater tire may need repair for a variety of reasons including trauma, inflammation or cancer, surgery or birth defects. The need to close dura mater defects, especially after surgery and post-traumatic fistulas, has become an impulse to look for the perfect replacement for dura mater.

Such defects may cause postoperative complications, especially cerebrospinal fluid leakage, infections and subsequent cerebral seizures. Some forms of tire transplantation are necessary in almost 30% of craniotomy cases. Since primary closure of the tire defect is often ineffective, the availability of a substitute tire to prevent the above complications is of great practical importance.

A permanent fluid-tight closure of the dura mater is necessary to prevent cerebrospinal fluid leakage after skull trauma or surgical interventions to remove malignant tumors in the brain or spinal cord. Currently, neurosurgeons use absorbable or non-absorbable dura mater substitutes and usually attach them to the dura mater in the skull or spinal cord with sutures and / or fibrin glue. Examples of absorbable materials include a human dura mater, human fascia, bovine pericardium, cross-species collagen sponges and implants of woven materials made of absorbable polyesters (polyglactin and / or poly-p-di-oxanone). Examples of non-absorbable tire replacements include materials made of polytetrafluoroethylene (PTFE) or polyester urethane.

Almost all previously tested tire transplants are associated with complications, sometimes serious ones. The main complications that have been described are chronic inflammation and rejection reactions and the formation of meningocortical adhesions causing, among others, the formation of epileptic foci. Hematomas and cerebrospinal fluid fistulas are also observed, which in turn provide the gate for disease-causing organisms.

In the search for the perfect dura graft over the past decades, many materials and methods including metallic materials, implants, synthetic materials, autologous tissue transplants and conserved human dura mater have been evaluated. Most of these materials are inappropriate because they are associated with post-operative complications, some of which are serious. Examples of complications include chronic inflammation and rejection reactions, the development of meningocortical adhesions, haemorrhage and dural graft closure in thin layers of connective tissue. Earlier studies with individuals with favorable dura mater replacement have shown that early transplant uptake, associated with the formation of a new endogenous tire, is the main prognostic factor for uncomplicated and permanent union of tires.

In the past, several types of autologous tissue have been used as a substitute for dura mater. In 1911

Kostling used a patient's hernia bag to create a tire transplant. Kostling, W., Med. Wochenschr, 58, 1042 (1911). Later other autologous tissues such as temporal fascia, broad thigh fascia and periosteal lobes were used. Barrow et al. successfully reproduced large damage to the dura using an endogenous larger network. Barrow et al., J. Neurosurg. 60; 305-1 (1987). The advantage of autologous transplants is that there is no risk of pathogen transmission or tissue rejection. However, additional tissue removal intensifies surgical trauma and prolongs the already complicated surgery.

For many years, a preserved human dura mater tire was used as a replacement for dura mater replacement tires. These preparations contain interwoven connective tissue fibers just like the body's own dura mater. It is believed that after the application of dura mater dermatitis in neurosurgery, it forms a fluid-tight closure similar to the body's own dura mater and, possibly after a longer period, is replaced by the body's own tissue. The corpse material is preserved by freeze drying (lyophilization) and sterilization by gamma rays (Lyodura, B. Braun Melsungen Aktiengesellschaft, Melsungen, Germany) or by a multi-stage chemical process (Tutoplast® process; Tutoplast® Dura, Tutogen Medical GmbH, Neunkirchen am Brand, Germany). However, dura mater transplants have a significant risk of transmitting viruses and prions that can cause fearful disease, spongiform encephalitis (Creutzfeldt-Jakob disease or Gerstmann-Streussler syndrome). Due to the many deaths that occurred after transplantation of human dura mater, the use of human dura mater has been restricted or banned in many countries.

Human broad fascia and pericardial preparations were also used as a dura mater replacement with a lower risk of transmitting infectious agents than with human dura mater. Although these formulations carry a lower risk of disease transmission, they are absorbed more slowly over months or years, which can cause scarring and encapsulation of the dura mater replacement.

Tire substitutes were also obtained from non-human sources, such as bovine or porcine collagen, isolated from skin or tendons and bovine pericardial tissue. It is thought that, similar to human sources, some bovine meninges can transmit diseases, namely bovine spongiform encephalopathy (BSE), to a patient receiving a dura mater transplant. However, the use of porcine tire substitutes caused adhesions to underlying brain tissue.

Narotam et al., US 5,997,895, discloses tire substitutes derived from processed bipolar collagen in the form of a porous collagen sponge, felt or film. Collagen treatment inactivates contamination with viruses and prions so that the replacement does not contain infectious amounts of viruses and prions. It has been disclosed that the porosity of the tire replacement is necessary for the infiltration of the replacement through blood vessels, cells and meningeal tissue. However, the use of available porous materials in clinical practice has disadvantages because shape stability and primary fluid tightness are not always guaranteed. Narotam et al. Also discloses tire replacement materials that are sandwich combinations of two or more collagen sponge forms, felt or films, of which at least one form is sufficiently porous for ingrowth of meningeal tissue.

Absorbable polyesters are also available in clinical practice, but they have the disadvantages of low elasticity and slow degradation. In special situations, these implants cause wound healing problems and can increase the number of infections.

Foils or sheets containing metal such as gold, platinum, silver, nickel, tantalum or steel, or polymers such as polytetrafluoroethylene (PTEE) or other polyesters have also been used as substitutes for dura mater. Although these substitutes are not absorbed by the patient, they are surrounded by a layer of durable connective tissue and remain in the body throughout the patient's life as a foreign body, without being replaced by the body's own structures. The porous structure of PTEE membrane films can cause an increased risk of germs growing in the internal pores, which cannot be controlled by the body's own defense mechanism.

Collagen based products are becoming increasingly popular. Chemical processes can be used to modify connective tissue-rich structures, such as the pericardium or dermis, so that only the acellular, antigen-free collagen framework remains. There are products that completely consist of collagen fibrils or synthetic materials coated with collagen. In both cases, the collagen fiber network acts as a matrix for the growth of endogenous connective tissue.

Chaplin et al. Studied products derived from guinea pig skin (XenoDerm, Lifecell Corp., The Woodlands, TX) in an animal model. Neurosurgery, 45: 2, 320-7 (August 1999). They were compared with autologous pericardium. The epidermis, all cellular components and other potentially antigenic or infectious components were chemically removed in the manufacturing process. Collagen fibers and the spatial arrangement of the skin remained unchanged. The product has been reported to be rapidly incorporated into the surrounding meninges in the presence of a mild cellular response. Infiltrating fibroblasts were mainly observed in the implantation area. It was reported that the graft and own dura mater were almost indistinguishable at the end of the 6-month follow-up after surgery.

Following these results, Warren et al. (2000) investigated AlloDerm® (LifeCell Corp., The Woodlands, TX) to replace tires in humans. Neurosurgery 46 (6): 1391-96 (2000). During the study, two hundred patients received an AlloDerm tire transplant. This material was obtained from human skin. The manufacturing process was the same as for XenoDermu and an acellular collagen biological matrix was obtained, free from major histocompatibility antigens (MHC). Seven out of 200 patients developed postoperative complications such as cerebrospinal fluid (CSF) infection and fistulae, but none of these cases were described as caused by the transplant itself. Surgical revision has been reported to indicate that none of these patients developed adhesions or recoil reactions at the site of tire transplantation. The material was reported to be very similar to the surrounding tire on macroscopic examination. The results of long-term research on this product are not yet available.

Filippi et al. (2001) described experiments using solvent-preserved, gamma-sterilized, bovine pericardium (Tutopatch®, Tutogen Medicel GmbH, Neunkirchen, Germany) to replace a tire in 32 individuals. Filippi et al., Neurosurg. Rev., 24: 103107 (2001). It was reported that the postoperative course was uncomplicated in all, but one patient died of cardiac reasons shortly after the procedure. Transplantation has been reported to be easy to handle, durable and cheap. Long-term studies to determine the risk of late complications are not yet available.

CoUins ^and in. (¹⁹99) J. Biomed. Mat.^Res. ^{25, 267-276}disclosed a product of cross-linked, reconstituted collagen for use as a replacement for tires.

WO 02/22184 (Organogenesis Inc.) discloses a cross-linked wound dressing product for the treatment of wounds such as ulcers.

US 6,312,447 (Bio-Vascular Inc) discloses a cell-free product made from human skin from corpses for use as a replacement for tires.

Collagen products are suitable for many applications as a biomaterial: in chemotactic interaction, in which they are involved in facilitating the rapid infiltration of endothelial cells and fibroblasts, resulting in the production and deposition of new collagen fibers; in the accompanying limited lymphocytic inflammatory response in surrounding structures, they promote the absorption of the biological collagen matrix. Collagen also has haemostatic properties that are of therapeutic use. Plaque deposits on the collagen structure themselves break down and release coagulation factors that facilitate the formation of fibrin in combination with plasma coagulation factors.

Well-known tire replacement materials and associated methods of use have not provided fluid-tight, absorbable dura mater replacement replacement to prevent encapsulation, scar formation or adhesions with brain tissue, and also with a low risk of transmitting germs, viruses and prions that can cause spongiform encephalitis or other diseases. An ideal dura mater replacement should not involve an immune or inflammatory response and must be non-toxic. It should be absorbed quickly and at the same time should enable the reconstruction of the connective tissue architecture so that a new tire forms. The transplant should not adhere and attach to the brain or bone tissue during this process. The material should be tear resistant, should retain its shape and be resistant to the penetration of cerebrospinal fluid. The dura mater replacement material should also be constant in volume and shape, i.e., it should not expand or contract after implantation. Other criteria considered are viral and prion security, ease of use, and cost-effective manufacturing.

SUMMARY OF THE INVENTION

Among the various aspects of the present invention is to provide a dura mater replacement material that is absorbable, fluid-tight, flexible, constant in volume and shape, and provides an excellent safety profile including risk of disease transmission.

Briefly, therefore, the present invention relates to a method of repairing and / or restoring dura mater tissue in mammals. The dura mater tissue is contacted with equine collagen foil, including a biological matrix of collagen fibrils.

Equine collagen film is made by a process in which a suspension of collagen fibrils precipitates to form a multilayer film from collagen fibrils and in which the collagen fibrils are not crosslinked with chemicals or radiation.

In another aspect, the invention relates to a method of repairing dura mater tissue in mammals comprising contacting the dura mater tissue with a substantially non-porous equine collagen foil, including a non-naturally occurring multilayer biological collagen fibrous matrix, wherein the equine collagen foil is essentially composed of acellular elements, and collagen fibrils are not crosslinked with chemicals or radiation.

In a further aspect, the invention relates to a method of repairing and / or restoring dermatitis tissue in mammals comprising contacting dura mater tissue with a substantially non-porous equine collagen foil constructed essentially of a multilayer collagen biological matrix in which the collagen biological matrix is ​​not crosslinked with chemicals or radiation .

Other aspects and features of the invention will become partly clear and will be partly demonstrated in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a SEM (scanning electron microscope) photograph illustrating the surface of the dry equine collagen foil. Collagen fibrils are clearly visible. There is basically no surface pores in the image.

Figures 2A and 2B are photographs taken under ESEM (environmental scanning electron microscopy) conditions, which indicate almost natural conditions in a mildly humid atmosphere, illustrating the upper surface, visible from the side of the equine collagen foil. Basically no pores are visible in the photo.

Figures 3A and 3B are photographs taken under ESEM conditions illustrating the bottom surface of the equine collagen foil. Collagen fibrils are illustrated in Fig. 3A. The principal lack of pores is evident from the photographs.

Fig 4 is a SEM photograph illustrating the surface of hydrated equine collagen foil. Collagen fibrils are clearly visible in Fig. 4. In the photograph, essentially no pores.

Figures 5A, 5B and 5C are photographs taken under conditions

ESEM (humid atmosphere) illustrating the cross-section of equine collagen foil. The material has a structure similar to a pile of sheets packed very tightly. The picture shows the spacing between collagen layers.

Figures 6A and 6B are SEM photographs illustrating a cross-section of a dry equine collagen foil. Multilayer collagen layers and the spacing between collagen layers are visible in these photographs.

Fig. 7 is a photograph illustrating the intraoperative aspect of left-sided tire injury after insertion of equine collagen foil; the edges of the surrounding tire are covered with blood clots.

Fig. 8 is a photograph illustrating a general view in microscopic Trichromium staining of the surgical site (frontal section) showing cortical structures and a dura underlying with two grafts (equine collagen foil on the right and Tutoplast® Dura on the left) (magnification 8X ).

Fig. 9 is a photograph illustrating dura mater transplants eight weeks after surgery. On the left side, Tutoplast® Dura, a dura mater transplant looks unchanged with well visible edges. Other parts of the connective tissue membrane covering the graft can be seen. On the right side, the transplant of the biological matrix from equine collagen foil looks fully absorbed into the surrounding tire. Black spots are caused by small blood clots in the new tire.

Fig. 10 is a photograph illustrating the macroscopic aspect of a transplant of a biological matrix from equine collagen foil in contact with the bark, eight weeks after surgery. The graft appears smooth, mobile and completely embedded in the surrounding tire. No cortical changes can be seen.

Fig. 11 is a photograph illustrating the macroscopic aspect of a tire transplant from a Tutoplast® Dura corpse coming into contact with the cortex, eight weeks after surgery. The graft looks smooth and homogeneous, but there are no signs of graft incorporation. Cortical meningitis is absent.

Fig. 12 is a photograph illustrating two multinucleated giant cells with intracellular fragments of a biological matrix collagen equine foil transplant, two weeks after surgery (hematoxylin-eosin (HE) staining; 800X magnification).

Fig. 13 is a photograph illustrating fibroblasts and phagocytic cells infiltrated with a transplanted biological matrix from equine collagen foil four weeks after surgery. Cancerous capillaries with erythrocytes are also visible (HE staining; 600X magnification).

Fig. 14 is a photograph illustrating fragments of a transplanted biological matrix from equine collagen foil surrounded by phagocytic cells and moderate lymphocyte inflammation, four weeks after surgery (HE staining; 600X magnification).

Fig. 15 is a photograph illustrating a Tutoplast® Dura corpse tire four weeks after surgery. The corpse tire shows minimal signs of cellular infiltration or graft remodeling. The concentration of the inflammatory lymphocytic reaction can be seen above and below the transplant (HE staining; 250X magnification).

Fig. 16 is a photograph of the microscopic aspect of a new tire eight weeks after surgery, illustrating newly formed layers of collagen fibers, fibroblasts and the remains of a transplanted biological matrix from equine collagen foil (Trichrom staining, 150X magnification).

Fig. 17 is a microscopic photograph of a new tire sixteen weeks after implantation of equine collagen foil illustrating the density of collagen fibers and newly formed capillaries filled with erythrocytes (van Gieson staining, 200X magnification).

Fig. 18 is a drawing illustrating the device used to measure the water resistance, tear strength and elasticity / flexibility of the dura mater replacement material. To determine the degree of elasticity / flexibility, the extent of material convexity was measured based on the specific height of the water column. In order to determine water tightness, the amount of water squeezed by the tested materials was measured.

Fig. 19 is a graph showing the extent to which the equine collagen foil is prominent (collagen content: 5.6 mg / cm²) under the influence of increased hydrostatic pressure (height of the water column).

Fig. 20 is a graph showing the extent of collagen foil accumulation (collagen content: 4 mg / cm²) under the influence of increased hydrostatic pressure (height of the water column).

Fig. 21 is a graph showing the extent of DuraGen prominence under the influence of increased hydrostatic pressure (water column height). DuraGen broke at 200 cm H2O hydrostatic pressure.

Fig. 22 is a graph showing the coverage range of several dura mater replacement products under the effect of increased hydrostatic pressure (water column height).

Fig. 23 is a graph showing the extent of prominence and loss for water for equine collagen foil (collagen content: 5.6 mg / cm2) under the effect of increased hydrostatic pressure (height of the water column).

Fig. 24 is a graph showing the extent of water bulge and loss for collagen film (collagen content: 4 mg / cm2) under the effect of increased hydrostatic pressure (water column height).

Fig. 25 is a graph showing the extent of DuraGen bulge and water loss under the effect of increased hydrostatic pressure (water column height).

Fig. 26 is a graph showing the extent of bulge and water loss for several dura mater replacement products under the effect of increased hydrostatic pressure (water column height).

Fig. 27 is a graph showing tear resistance / maximum voltage for various collagen implants. Sample E is equine collagen foil

DETAILED DESCRIPTION OF BENEFITS OF PERFORMANCE

In accordance with the present invention, it has surprisingly been found that a substantially non-porous film composed of non-naturally occurring equine biological collagen fibrils can be effectively used as an absorbable dura mater replacement for repair, regeneration and restoration of the tire in mammals, including humans, laboratory animals and like them. The equine collagen foil of the present invention is fluid tight and provides a high level of security against the risk of transmission of viruses or prions. In addition, equine collagen foil is flexible and flexible in nature, while maintaining high tensile strength. This foil, hereinafter referred to as "equine collagen foil", after implantation corresponds to the most important properties of a human dura. Equine collagen foil serves as the core of the biological matrix for in vivo cell ingrowth and is replaced by a new tire during regeneration and recovery.

In one embodiment, the biological matrix of equine collagen foil comprises connective tissue proteins that are essentially collagen fibrils. Preferably, the biological matrix of equine collagen foil comprises connective tissue proteins that are collagen fibrils. More preferably, the biological matrix of equine collagen foil comprises connective tissue proteins that are type I collagen fibrils.

In addition to building with collagen fibrils, equine collagen foil may contain an excipient, preservative, growth factor or additive to increase the flexibility and flexibility of the final product.

Equine collagen foil

The equine collagen foil of the present invention is a biological matrix of collagen fibrils treated to remove cellular elements and produce a sheet of collagen fibrils.

The equine collagen foil used in one embodiment of the present invention is a non-naturally occurring multilayer collagen film consisting of numerous multidirectional intertwined collagen fibrils. An illustration of the dry equine collagen foil can be seen in Fig. 1. The micrograph (SEM) illustrates the surface of the equine collagen foil in which collagen fibrils are embedded. In Fig. 2A-2B, you can see a photo of the top surface of the equine collagen foil under ESEM (environmental scanning electron microscopy) conditions in which a poorly humid atmosphere provides almost natural conditions. Collagen fibrils are visible on the surface. The surface appears smooth and essentially non-porous. Photographs (ESEM) of the lower surface of the equine collagen foil are shown in Figures 3A and 3B. A photograph of the bottom surface also illustrates the major non-porosity of the equine collagen foil.

Before using equine collagen foil to repair mammalian dura mater tissue, the dry equine collagen foil material may be hydrated. Fig. 4 is a SEM photograph illustrating the surface of hydrated equine collagen foil on which collagen fibrils are clearly visible. The basic surface non-porosity is clearly visible in the photo.

The unique orientation of collagen fibers in two-dimensional directions in many layers is mainly responsible for fluid tightness, even at high hydrostatic pressure and provides high strength and high flexibility. Due to the many thin parallel layers of collagen fibrils in the equine collagen foil, this material is suitable for temporarily replacing the body's own dura mater while covering cavities after implantation, to obtain a leak-proof cerebrospinal fluid closure and provides the framework for the biological matrix for cell ingrowth and making a new tire. This property is important for the wound healing process because it reduces the patient's risk of developing a fluid state.

Equine collagen foil structure and absorption characteristics

Equine collagen foil is absorbed in mammals in which it has been implanted. It is believed that this property is enhanced by the structure of the equine collagen foil. In the process used to make equine collagen foil, stacked layers of collagen fibrils are formed.

There are gaps between the layers into which the patient's cells and vessels can migrate and form tissue of the new tire. Each layer of collagen fibrils is essentially non-porous. The few pores that may be present are typically isolated from each other and do not merge through multiple layers of collagen fibrils. The multilayer structure of the present invention enhances the liquid-tight characteristics of the equine collagen foil. Images from the scanning electron microscope in Fig. 1 to 4 illustrate the non-porous nature of equine collagen foil.

While the equine collagen foil is substantially non-porous, there are gaps between the layers of collagen fibrils. Intervals and layered characteristics can easily be seen in Figs. 5A, 5B and 5C, which are photographs of cross-sections of equine collagen foil under ESEM (humid atmosphere). Figures 6A and 6B are SEM photographs of dry equine collagen foil. Thus, equine collagen foil is analogous to a stack of pages, in which each page is substantially smooth and non-porous, with spaces between the pages. When the film is in dry form (Figs. 6A and 6B), the gaps are more pronounced. Intervals decrease when equine collagen foil is observed under near-natural conditions in a mildly humid atmosphere. Figs 5A, 5B and 5C are cross-sectional images of the equine collagen foil in a humid atmosphere, which illustrates the reduction in spacing in the equine collagen foil.

In addition to enhancing fluid-tight properties, numerous parallel-oriented thin layers of collagen fibrils in equine collagen foil also serve as the core of the biological matrix for the ingrowth of cells forming de organ's own tire. Previously, it was widely believed that the structure of a porous stroma was necessary to promote the ingrowth of autonomic tissue and blood vessel networks into the dura mater tissue to be replaced. It has been surprisingly found that the non-porous, layered structure of equine collagen foil enhances the ingrowth of cells, blood vessels and the creation of new collagen structures in the equine collagen foil and in the spaces existing between its numerous layers, creating a new tire with a typical layered structure of a natural tire within weeks of implantation . As further described below and in Example 1, the ingrowth of cells, blood vessels and new collagen structures is increased so that, within weeks of surgery, the new tire is difficult to distinguish from the patient's pre-existing meningeal tissue. Within about four to eight weeks after surgery, the cellular organization of meningeal cells ranges from about 40% to 70%. After about sixteen weeks, the transplant is fully organized (100%).

Animal experiments show a rapid cellular infiltration around the multilayer equine collagen foil. Histologically, dense infiltration of the collagen biological matrix with lymphocytes, macrophages and fibroblasts was observed within 14 days of implantation. Capillaries formed in the transplant later. The continuous transition zone between the equine collagen foil and the surrounding tire is easily noticeable due to the formation of collagen fibers.

After only 4 weeks, the equine collagen foil is partially replaced by the loose structure of the body's own tissue. After 24 weeks, it is difficult to distinguish the pre-existing patient's tire from the newly created tire-like connective tissue architecture replacing the implanted equine collagen foil.

Transmission of diseases / immune response

A significant advantage of using the equine collagen foil of the present invention is the generally low risk of transmission of the disease to the patient into whom it is implanted. A process for producing equine collagen foil in which collagen fibrils are treated with acids (e.g., hydrochloric acid, acetic acid and the like) and bases, such as sodium hydroxide, favorably inactivating or reducing the level of infectious, possibly present, bacteria, viruses and prions. Treatment of biological material with hydrochloric acid, sodium hydroxide, ethylene oxide (ETO), etc. has been indicated by government agencies in drug and biological material regulations as an acceptable way to inactivate prions and viruses. This treatment may, according to some regulations, reduce official requirements for testing equine collagen foil batch by batch. Thus, treatment of collagen fibrils in the manufacturing process increases product safety and reduces the risk of transmitting the disease to the patient.

Horse material subjected to the processing described above was not found to transfer any pathogen to the patient. Thus, in addition to the manufacturing process, the use of horse-derived collagen further reduces the risk of transmitting spongiform encephalitis, as previously described for replacement materials from human corpses. The use of horse-derived collagen, such as collagen from horse Achilles tendons, reduces the risk of transmitting transmissible spongiform encephalopathy (TSE), which is also called bovine spongiform encephalopathy (BSE) or scrapie. Transmission of this disease has been associated with the use of biological material derived from ruminants (e.g. biological material from cattle, goats, sheep and the like).

The equine collagen foil of the present invention, in which the collagen is derived from a horse and is treated (e.g. with enzymes) further reduces the risk of eliciting an immune response. No immune response has been described for ten years during which horse-derived collagen was used in tissue replacement procedures (for tissues other than dura mater).

Foil from horse collagen also causes a reduced inflammatory response. When compared to dura mater replacement materials containing collagen from sources such as human fascia, the number of inflammatory cells resulting from implantation of equine collagen foil is significantly lower. Inflammatory processes caused by implantation of equine collagen foil are also significantly shorter compared to dura mater replacement from other sources. These properties significantly reduce the risk of rejection of the equine collagen foil transplant by the patient's immune system, thereby increasing the success of neurosurgical procedures requiring dura mater replacement.

Stability of volume / linear dimensions

Problems can arise when said dura mater after hydration begins to significantly expand or contract. Porous collagen dura mater replacement products, according to prior art, have in some situations tended to shrink considerably after hydration. In such conditions, the inserted dura mater may tighten the seams that attach it to the patient's dura mater causing damage to the implant, the patient's own dura mater and the operating field. Other complications include the pressure exerted on the surgical field with persistent dural expansion after implantation and the application of undesirable pressure to the surrounding nervous tissue.

The change in volume of the equine collagen equine film of the present invention is slight or insignificant after hydration. Unlike porous replacement products, equine collagen foil generally retains its size and shape during irrigation, has excellent shape stability, remains biologically stable even after hydration, and does not cause problems with swelling or contraction in the brain after implantation. When hydrated and implanted, the equine collagen foil does not expand or contract significantly in area or thickness, to the extent that it would break the surgical sutures or break fibrin glue that attach the equine collagen foil to the patient's dura .

In one embodiment, the shrinking or swelling area of ​​the dry equine collagen foil can vary from about -5% to about 20%, when completely hydrated. In a further embodiment, the area of ​​the dry equine collagen foil can vary from about -5% to about 10%, when completely hydrated. In a further embodiment, the area of ​​the dry equine collagen foil can vary from about -5% to about 5%, when completely hydrated. In a further embodiment, the area of ​​the dry equine collagen foil increases by no more than about 4% with complete hydration.

In one embodiment, the thickness of the collagen film increases to about 4 times after complete hydration, compared to dry film. In another embodiment, the thickness of the collagen film increases to about 3 times after complete hydration, compared to dry film. In another embodiment, the thickness of the collagen film increases about twice after complete hydration, compared to dry film.

The thickness of an adult human tire varies from about 5 mm on the base of the skull to about 2 mm. The thickness of the dura may also depend on the age of the patient, as it is generally believed that children and adolescents have thinner dura tissue than adults. The thickness of the equine collagen foil of the present invention can be adjusted depending on the desired area of ​​use and the patient being treated.

In one embodiment, the equine collagen foil of the present invention, in dry form, has a thickness of about 0.01 mm to about 3.0 mm. In another embodiment, the equine collagen foil has a thickness of about 0.02 mm to about 2.0 mm.

In another embodiment, the equine collagen foil has a thickness of about 0.03 mm to about 1.5 mm. In another embodiment, the equine collagen foil has a thickness of about 0.05 mm to about 1.0 mm.

In yet another embodiment, the equine collagen foil has a thickness of about 1.0 mm or less.

The weight of the dry equine collagen foil depends on its desired thickness. In one embodiment, the weight of the dry equine collagen foil is from about 1 mg / cm² up to about 50 mg / cm². In a further embodiment, the weight of the dry equine collagen foil is from about 1.5 mg / cm2 to about 30 mg / cm2. In a further embodiment, the weight of the dry equine collagen foil is from about 2 mg / cm2 to about 20 mg / cm2. In a further embodiment, the weight of the dry equine collagen foil is from about 2.5 mg / cm2 to about 15 mg / cm2. In another embodiment, the weight of the dry equine collagen foil is from about 3 mg / cm2 to about 10 mg / cm2.

In one embodiment, the weight of the equine collagen foil increases to about 15 times after complete hydration, compared to dry foil. In another embodiment, the weight of the equine collagen foil increases to about 10 times after complete hydration, compared to dry foil. In another embodiment, the weight of the equine collagen foil increases to about 7 times after complete hydration, compared to dry foil. In yet another embodiment, the weight of the equine collagen foil increases to about 5 times after complete hydration, compared to dry foil.

Implanted replacement materials suitable as a dura mater replacement material should not be flaccid, but should have high stability / tensile strength, even after hydration. The equine collagen foil of the present invention preferably has high tensile strength, which improves and supports the use of equine collagen foil for surgical purposes and provides increased mechanical stability after implantation. Comparative experiments are presented in the examples below, where the tensile strength of the equine collagen foil was higher compared to porous collagen preparations (e.g. collagen foam). In addition, increasing the thickness of the equine collagen foil can significantly increase tensile strength.

The tendency of equine collagen foil to tear under the effect of pressure can be measured as its "maximum tensile load" or "maximum tensile force," hereinafter referred to as "maximum tensile force." The maximum tensile force of equine collagen foil can be determined by exposure to pressure a strip of equine collagen foil having a specific width and measuring the pressure applied that causes the damage (e.g. tear or tear) of equine collagen foil. The maximum tensile force can be calculated from the following equation:

"Maximum tensile force" = applied force / width of the equine collagen foil strip = Newtons / cm strip.

In one embodiment, the equine collagen foil withstands a maximum tensile force of about 1 to about 30 Newtons / cm-strip, preferably about 1.5 to about 15 Newtons / cm-strip, preferably about 2 to about 10

Newtons / cm-strip, more preferably about 3 to about 6 Newtons / cm-strip.

Although the collagen film of the present invention has a high tensile strength, it remains flexible and flexible when hydrated. This feature of equine collagen foil allows optimal adaptation to the anatomical conditions (e.g. curvatures) present at the implantation site.

In the hydrated state, the equine collagen foil can easily be moved around the operating field and optimally modeled to the shape of the damage in which it is to be implanted. After implantation, the equine collagen foil graft remains smooth and mobile. After some time, cells and blood vessels migrate through the equine collagen foil, possibly replacing it with a tire-like new tire. After colonization of the meninges, equine collagen foil does not adhere to the nerve tissue, brain, skull or spinal cord.

Manufacture of equine collagen foil

The equine collagen foil of the present invention can be made from high molecular weight collagen fibrous suspensions in a controlled drying process. The gradual precipitation of the collagen fibrous suspension results from the evaporation of water and the simultaneous increase of pH. As a result of the controlled drying process, a multilayer collagen film structure is obtained, which can be implanted by neurosurgeons as a replacement material for the human dura mater. Multilayer collagen film constructions provide numerous properties described above that are beneficial for dura mater replacement and biological matrix for regeneration of living tire tissue.

In one embodiment, in the process of producing the equine collagen foil of the present invention, all cellular components are removed, producing an equine collagen foil consisting of collagen fibrils containing substantially acellular components.

Using the procedures established for collagen chemistry, collagen-containing tissue is used as the starting material for producing the equine collagen foil of the present invention. In one embodiment, horse tendons are used as the starting material. In a further embodiment, the Achilles tendons are used as the starting material.

In one embodiment, the starting material, for example horse Achilles tendons, was ground and treated for at least 1 hour with 1 N sodium hydroxide and neutralized with hydrochloric acid. The starting collagen material is treated in an acidic medium at pH 2. The acid used may be hydrochloric acid, acetic acid and the like. Then, non-collagen proteins and cross-crosslinking intermolecular bonds present in the starting material are enzymatically degraded with pepsin to form a collagen suspension.

The suspension is then neutralized. In one embodiment, the suspension is neutralized to a pH from about 6.5 to about 8.0. In another embodiment, the suspension is neutralized to a pH from about 6.9 to about 7.5. In another embodiment, the suspension is neutralized to a pH of about 7.

The collagen suspension is centrifuged, the supernatant discarded, and the pellet is resuspended in acetic acid at a pH of about 24.5. In this way, non-collagen proteins are effectively removed from the collagen suspension.

The steps described above can be repeated depending on the need to remove non-collagen proteins present in the sediment.

The surprising effect of the equine collagen foil production process is that the controlled increase in the pH of the collagen suspension in acetic acid is achieved by thorough removal of water, by evaporation for a prolonged period of e.g. 24 hours. A specific increase in pH causes the precipitation of multidirectional entwined collagen fibrils in the form of bidirectionally oriented layers, forming a multilayer equine collagen foil construction. In one embodiment, the process is carried out in a drying oven at a temperature from about 20 ° C to about 55 ° C, with steam removal equipment and simultaneous steam neutralization of acetic acid. In a further embodiment, the process is carried out in a drying oven at a temperature from about 30 ° C to about 45 ° C.

The equine collagen foil resulting from the manufacturing process is considered dry when the water loss is undetectable or insignificant. The water content in the "dry form" of the equine collagen foil is typically from about 2% to about 18% by weight. The relatively large residual water present in the "dry form" of the equine collagen foil prevents or inhibits the denaturation of collagen molecules that make up the equine collagen foil.

The process described above is responsible for the deposition of collagen fibrils from the suspension, because the low solubility components fell out at the beginning of the process when raising the low pH. This technique causes collagen fibrils to deposit during the evaporation of water and at the same time increases the pH.

During the precipitation process, natural crosslinks are formed between the fibers as the fibers precipitate out of solution to form a collagen film. Unlike cross-links produced between collagen fibers by chemical means or radiation (e.g. ionization or ultraviolet irradiation), which can cause a longer absorption time, enabling the formation of natural cross-links, promotes a reduction in the absorption time after implantation of equine collagen foil. The natural cross-links of the filaments in the collagen film used in the invention appear as a result of the action of natural, physiological-like agents. Originally, these natural crosslinks are formed as a result of non-covalent interactions (e.g. van der Waals interactions or dipoldipol) or by forming easily dissociating Schiff base type bonds between side chains of amino acids in the collagen molecule. Intermolecular collagen crosslinks are responsible for physical and chemical stability. The important step in the formation of collagen crosslinks depends on the enzymatic conversion of lysine or hydroxylysine residues and results in aldehydes, allysine and hydroxyallysine. These aldehyde groups spontaneously react with reactive amino groups, resulting in Schiff base components containing labile aldol condensation products with labile aldimine bonds (-CH = N-). Thus, the filaments of the product of the present invention can be dissociated by treatment, for example, with a weak acid. Cross-links resulting from the use of chemical cross-linking agents can be detected by the presence of stable, covalently cross-linked cross-linking residues. Typically, this is accompanied by the use of a Schiff base reagent (e.g., glutaraldehyde) to form Schiff base reaction products and then, stabilizing the bonds either by Amadori rearrangement or by reducing conditions. In addition, collagen can be cross-linked with various bifunctional carbodiimide reagents. Cross-bonds generated under the influence of radiation can be detected by the presence of stable covalent bonds between collagen fibrils, created by the reaction of free radical residues formed during irradiation. On the other hand, the filaments in the product of the present invention are essentially transversely bonded by stable covalent bonds and have not been treated in any way, neither chemically nor by radiation. Thus, any connection between the fibers in the product of the invention is substantially non-covalent or easily reversible, so the fibers are not stably crosslinked. Chemicals such as cyanoamide, glutaraldehyde, formaldehyde, acrylamide, carbodiimidione, diimidates, bisacrylamides, etc. have been used in the past to chemically form collagen fibril cross-links in dura mater replacement materials. However, the use of such chemicals may have posed a risk of toxicity related to the inevitable contact of brain tissue with residual compounds in dura mater replacement materials. In contrast, the precipitation process prevents the risk of toxicity of cross-linking chemicals and longer absorption, associated with cross-linking of collagen fibrils with chemicals or radiation.

The resulting dry, precipitated collagen composition forms a horse collagen film consisting of a high molecular weight multilayer collagen film consisting of numerous naturally multidirectional intertwined collagen fibrils. Equine collagen foil contains mainly interstitial Type I collagen. Equine collagen foil is essentially free of pores and is essentially fluid tight. Immunodiffusion tests can be performed on the product to confirm the absence of foreign proteins.

The process described above used to make equine collagen foil for use in the present invention is also used by Resorba Wundversorgung GmbH &

What. KG, Nuremberg, Germany, for the production of commercially available collagen films from Baxter AG, Vienna, Austria. Commercially available films are used as hemostatic agents, transient tissue replacement materials, for wound covering and as carriers for fibrin glue.

The thickness of the equine collagen foil for use in the present invention may vary depending on the needs of the particular application. For example, in the repair of children's dura mater tissue, thinner equine collagen foil can be used, while thicker equine collagen foil can be used to repair adult dura mater tissue.

The thickness of the equine collagen foil can be controlled by varying the amount of starting material used to make a specific size of equine collagen foil.

The equine collagen foil is sterilized by gas, using ethylene oxide (ETO) or similar gas for gas sterilization or irradiation.

Attachment procedure

Before use, the equine collagen foil can be hydrated, e.g. in physiological saline. In one embodiment, the saline contains a 0.9% sodium chloride solution. In another embodiment, the equine collagen foil is hydrated in solutions containing excipients or drugs. The time it takes to hydrate the equine collagen foil depends on the thickness of the foil. The equine collagen foil is watered until it is evenly thick throughout. In one embodiment, the equine collagen foil is hydrated for about 5 seconds to about 1 hour in physiological saline. In another embodiment, the equine collagen foil is hydrated for about 5 seconds to about 30 minutes in saline. In another embodiment, the equine collagen foil is hydrated for about 5 seconds to about 20 minutes in saline. In another embodiment, the equine collagen foil is hydrated for about 5 seconds to about 10 minutes in saline. In yet another embodiment, the equine collagen foil is hydrated for about 1 minute to about 6 minutes in saline. In another embodiment, the equine collagen foil is hydrated for about 5 minutes in physiological salt. In another embodiment, the equine collagen foil is not hydrated prior to implantation.

Equine collagen foil can be attached to the patient's dura mater by accepted surgical methods, e.g., fibrin glue, tissue glue, surgical sutures or compression fitting by surgical methods. Alternatively, natural attachment between the equine collagen foil and dural tissue can be used to attach the equine collagen foil to dura mater tissue without the use of any binders, adhesives or compression fitting methods. After rehydration, the equine collagen foil can be cut slightly wider than the surgical opening in the patient's dura mater. Thus, the equine collagen foil may slightly overlap the patient's dura mater to which it is to be attached. In one embodiment, the hydrated equine collagen foil is cut to approximately 0.5 cm to about 1 cm excess relative to the tire. The amount of excess can vary depending on the preferences and experience of the neurosurgeon.

In one embodiment, according to the well-known interaction between collagen and fibrin, the equine collagen foil can be attached to the dura mater with fibrin glue approved for use in neurosurgery. Examples of fibrin glues approved for neurosurgical use include Tissucol and Tisseel fibrin glues (Baxter AG, Vienna, Austria). Alternatively, tissue glue approved for use in neurosurgery can also be used. A fibrin or tissue glue can be applied on a continuous line along a portion of the equine collagen foil crossing the dura to create a fluid tight seal. As described above, fluid-tight connection is an advantage in preventing complications associated with cerebrospinal fluid loss, such as fluid flow.

In another embodiment, the equine collagen foil creates a fluid-tight connection when attached to its own dura with a continuous line of fibrin or tissue glue.

In another embodiment, the equine collagen foil that exceeds the dura may be sprinkled with fibrin or tissue glue to attach to the dura.

In a further embodiment, the equine collagen foil is attached surgically to the dura mater as soon as it fits into the desired implantation site. Although this embodiment can be used to attach the equine collagen foil to the patient's own dura mater, however, the sutures may create additional channels, which can result in fistula formation and cerebrospinal fluid leakage. If equine collagen foil is to be sewn, use non-tension sewing methods to avoid tearing the foil. It is recommended to seal the sewing line, for example with fibrin glue.

In a further embodiment, the equine collagen foil is fitted and implanted according to compression fitting methods known in the art. In this technique, the equine collagen foil is fitted to the desired implantation site and held in place by the natural pressure present inside the skull or spinal cord. Thus, the transplant remains in place without the use of surgical sutures, fibrin or tissue adhesives.

In a further embodiment, the equine collagen foil is fitted and implanted without the use of any fibrin, tissue glue or compression fitting techniques. In this technique, the equine collagen foil is adjusted to the desired implantation site and held in place by the natural attraction or adhesion that appears between the equine collagen foil and dural tissue.

The equine collagen foil of the present invention can be used as a dura mater replacement graft to repair human dura mater tissue due to congenital conditions, birth defects, diseases, injuries, tumor removal or other surgical procedures that tear or penetrate the patient's dura mater or in any other condition in which the hard tire needs repair. Equine collagen foil can also be used to repair dermatitis tissue of other mammals, including but not limited to sheep, monkeys, horses, laboratory animals or other mammals. Equine collagen foil can be used to repair dura mater tissue in the skull or along the spinal cord.

The present invention further relates to a kit comprising equine collagen foil and instructions for its preparation and use as a replacement for dura mater.

Contraindications

A patient with a known allergic reaction to equine or equine products has contraindications to receiving equine collagen foil.

Other contraindications may include a patient who has undergone radiation therapy shortly after surgery. For example, a patient for radiation therapy shortly after brain tumor resection is not a good candidate for the recipient of the equine collagen foil of the present invention. Radiation can slow or stop the growth of a new tire that contains intensely dividing cells in which equine collagen foil is absorbed. In such situations, non-absorbable dura mater replacement materials such as Teflon will be preferable.

An experienced surgeon will recognize situations where you may need to use non-absorbable replacement materials.

Definition "Equine collagen foil" means a biological matrix (ie matrix of biocompatible material) from equine collagen fibrils treated to remove cellular components and form a collagen fibrous sheet. The term "equine collagen foil" does not include a composite foil of one or more substantially non-porous collagen fibrous sheets joined to one or more porous collagen sheets.

'Tire tissue' means the mammal's own dura tissue.

"Non-naturally occurring biological matrix" means a generated matrix or framework containing collagen fibrils made of (1) material existing in nature (i.e. natural material) that is treated and processed in a way in which the collagen fibrils contained in the natural material move or set up again in relation to their natural arrangement in the collagen structure of natural material; or (2) material not found in nature (i.e. unnatural material) treated or processed with collagen fibers. For example, a non-naturally occurring biological matrix can be made from collagen-containing starting material that is processed mechanically or chemically (e.g., milling, cutting, etc.). On the contrary, the collagen biological matrix, which is produced by treating or processing the starting material in a manner that preserves the structure of the collagen framework, is not a naturally non-naturally occurring biological matrix (e.g. epidermal tissue treated to remove cellular components while maintaining the naturally occurring collagen structure).

"Substantially non-porous" means that the pores that are present in the equine collagen foil as a result of precipitation of collagen fibrils to form a collagen sheet are substantially spaced apart. The pores that can be joined together do not connect in a way that would penetrate the entire thickness of the equine collagen foil. Mechanical punctures that form holes in equine collagen foil are not pores. Preferably, the material appears to be substantially free of pores that could be seen in an electron microscope at 1500X magnification.

The following examples further illustrate the invention.

Example 1

This example shows the results of sheep experiments carried out to assess the suitability of equine collagen foil as a dura mater replacement material used to repair dura mater tissue and as a biological matrix for tire regeneration.

The experiment was carried out to assess the properties of equine collagen foil when used as a skull replacement dura mater, which was tested on a sheep model. Equine collagen foil contains native equine collagen fibrils (5.6 mg / cm²) cleared of chopped horse Achilles tendons and does not contain cellular components.

Human reference tire (Tutoplast® Dura) was used as reference product. Both products were attached in place with fibrin glue alone (Tissucol Duo S Immuno, Baxter AG, Vienna, Austria).

The following tests were carried out:

the macroscopic aspect of incorporating two transplants;

Reactions of surrounding tissue structures (inflammation, adhesion, fibrosis, necrosis) and histological evaluation of the process of incorporation and organization of connective tissue.

The purification process for the production of equine collagen foil began with at least 1 hour of treatment with a sodium hydroxide solution of the tendon starting material, followed by neutralization with hydrochloric acid. Then pepsin was used to tendon decay. The colloidal collagen thus formed precipitates as fibrils. Drying and gas sterilization give equine collagen foil with 5.6 mg of native collagen fibrils per square centimeter. Nothing else is added and no artificial methods of cross-linking (i.e. involving the use of chemicals or radiation) are carried out. Immunodiffusion tests confirm the absence of foreign proteins.

MATERIAL AND METHODS

Experimental animals

The study was conducted on 25 adult sheep. Sheep came from mixed farming of domestic animals used in agriculture. The animals weighed an average of 53.0 kg at the time of surgery and their average age was 2 years. All animals were females. The animals were kept in an animal yard at Luebeck Medical University, equipped with roofed structures and fenced open areas. The animals received standard mixed feed. The animals were divided into groups of five animals for histological studies and characterization of the different stages of graft incorporation (Group 1 - Group 5).

Groups survival was 2, 4, 8, 16 and 24 weeks.

Test product

The tested equine collagen foil was made from native equine collagen fibrils (mainly interstitial type I collagen). One square centimeter of material contained

5.6 milligrams of collagen fibrils, without cellular components. The comparison (Tutoplast® Dura, Tutogen Medical GmbH, Neunkirchen a. Brand, Germany) is a human corpse tire preserved in a tissue-saving process.

Tissucol Duo S fibrin glue was used to attach the grafts to the dura mater. This bi-component adhesive consists of a pre-filled syringe containing human plasma proteins, fibrinogen, coagulation factor XII, plasma fibrillin and aprotinin, and a second pre-filled syringe containing thrombin and chloride calcium.

Anesthesia

Animals were premedicated by intramuscular injection of xylazine hydrochloride (Rompun 2%, Bayer AG, Leverkusen, Germany), dose: 0.1 mg per kg body weight, (S) -ketamine (Ketanest S, Parke-Davis GmbH, Karlsruhe, Germany) dose : 2 mg per kg body weight and 0.5 mg atropine, 1 ml solution for injection, (Atropinsulfate Braun 0.5 mg, B. Braun Melsungen AG,

Melsungen, Germany), in an injection cocktail. The arterial and venous line was inserted into the right ear. For anesthesia, 1 mg / kg body weight Propofol (Disoprivan 2%,

AstraZeneca GmbH, Wedel, Germany). The animals were intratracheally intubated (ID 7.0 mm) and 100% oxygen was administered for controlled standardization (Sulfa 808V respirator,

Drager ,, Luebeck, Germany).

Propofol, (S) -ketamine and sevoflurane were administered to maintain balanced anesthesia. During the procedure, pressure and tidal volume in the respiratory cycle were monitored, fractions of inhaled oxygen (Oxydig, Drager, Luebeck, Germany), final expiratory carbon dioxide content (Kapnodig, Drager, Luebeck, Germany), electrocardiogram and blood pressure by means of the blood method.

Preoperative antibiotic prophylaxis

Immediately before surgery, each animal received an intravenous injection of 2.0 g cefazolin (Basocef 2.0 g, Curasan AG, Kleinostheim, Germany). Antibiotic prophylaxis was maintained for the next 4 days after surgery, via two subcutaneous doses of the depot product, Strepdipen-Suspension (1.0 ml contains 100,000 IU benzylpenicillin benzatin and 100,000 IU dihydrostreptomycin sulfate; dose 1.0 ml per kg body weight). Subcutaneous injections were given immediately after the procedure and again 48 hours later.

Surgical technique

Already intubated animals were placed in the left lateral position. Then the head was rotated clockwise and kept upright by being attached to the operating table.

Then the skull was shaved thoroughly, the skin was degreased with gasoline and then disinfected. A sterile kerchief with an opening for the surgical field was attached and the whole animal was covered with sterile sheets.

The first skin incision was made 1.5 cm from the midline and widened by about 6 cm. Each capillary bleeding was coagulated with bipolar forceps. A hook was used and the skull bone was exposed in the temporo-parietal region by moving the bonnet apart and apart. Two holes (0.8 cm in diameter) were drilled about 5 cm apart with a hand drill. Then, with a saw, an elongated oval disc was removed from the skull bone between the drilled holes.

Any bleeding from the skull bones was blocked with bone wax. An incision of a dura mater approximately 0.5 cm in length was made with a scalpel. Tire shears were then used to cut an oval dura fragment with approximate dimensions of 3x2 cm along the bone boundaries. Special attention was paid to damage to the spider tire. TachoComb® haemostatic agent (Nycomed Austria GmbH, Linz,

Austria) was used to stop bleeding from meningeal vessels.

Then an oval fragment of equine collagen foil (3.5 x 2.5 cm) was cut and immersed in sterile 0.9% saline for 5 minutes. To close the defect, the equine collagen foil was put overlapped under the brim of the surrounding dura and fixed with spotting fibrin glue. See Fig. 7.

The skull bone disc was then attached again using two mini plates (Bioplates, Codman, Nordestedt,

Germany). The bonnet was closed using absorbable sutures (Vicryl 2.0) and the skin was sutured with Ethilon 3.0.

Tutoplast® Dura was used in a similar manner on the right side of the skull. See Fig. 8. Both wounds were finally treated with an aerosol dressing (Hansaplast Spruhpflaster, Beiersdorf AG, Hamburg, Germany).

The average procedure time was 120 minutes. An average of 60 minutes elapsed from the time of induction of anesthesia to the beginning of the procedure, and the average time from the end of sewing to the end of anesthesia was 5 to 10 minutes.

Postoperative animal observation

The animals were allowed to rest in their enclosure about 30 minutes after extubation. The animals were regularly monitored by a surgeon, veterinarian and animal carer for signs of inflammation or neurological disorders. Animals were released into the open enclosure eight days after surgery.

Animal euthanasia

Animals were killed for sampling after a predetermined survival time, 2, 4, 8, 16 and 24 weeks after surgery. Prior to killing, the animals were sedated by intramuscular injection of 1 mg per kg body weight 2% Rompun. An ECG monitor was connected and the arterial and venous line in the right ear was connected. Deeply sedated animals were then killed by intravenous injection of T-61® (Hoeschst Roussel Vet, Somerville, New Jersey); 1 ml solution for injection contains 0.2 g embutramide, 0.05 g mebezonium iodide and

0.005 g tetracaine hydrochloride; 0.3 ml per kg body weight). The process was monitored by ECG and blood pressure measurement.

Sampling

The animals' heads were then shaved and arranged as described for the surgical procedure. A round incision was made on the skin about 9 cm in diameter around both surgical scars. The tendon head was moved away so that large areas of the skull cover were visible and a hole was drilled in the right frontal region. Using a saw, a round disk was cut from the skull bone measuring about 8 cm. The entire transplant site including bone, meninges and cerebral parenchyma was obtained by excision along the bone boundaries with a scalpel. Then the meninges and brain tissue were carefully separated from the bone above and fixed in formalin for histopathological examination. The graft sample had a diameter of approximately 7 cm.

Histological methods

The graft samples were examined macroscopically and divided into frontal sections. Both locations of the procedure were prepared in parallel. Five sections approximately 2-3 pm thick were taken from each sample.

Changes were evaluated using standard staining methods, including Hematoxylin-Eosine for cellular components, Elastica van Gieson for interstitial structures and Trichrome to assess the production of new collagen fibers and iron staining to determine the extent of bleeding.

Results

The procedure and postoperative course

The period of anesthesia, surgery and post-operative follow-up was quiet in most animals except two. One animal died during induction of anesthesia as a result of cardiac arrhythmias. Another animal died suddenly and unexpectedly 14 days after the surgery, after being calm until then. Microscopic examination of the brain showed extensive cortical necrosis with scarring features. The most likely cause of death is prolonged cerebral ischemia of unknown etiology.

There were only a few intraoperative bleeding, mild and most cases from small dural vessels. Bleeding was quickly controlled with bipolar forceps or hemostatic agents.

None of the animals showed neurological disorders during postoperative follow-up. Similarly, none of the animals showed signs of inflammation, cerebrospinal fluid leakage, or impaired wound healing.

Macroscopic examination

During sampling from treatment locations, the following parameters were tested and calculated:

- Creating adhesions between the skull and the tire;

- Cerebrospinal fluid leakage or inflammatory lesions;

- Visible changes in meningeal transplants and

- Meningocortical adhesions and cortical response.

Microscopic examination

Histological cross-sections were examined systematically in the following scope:

- Description and estimation of the inflammatory response in the transplant area (epidural, epidural, in the transition zone between the tire and the transplant);

- Degree of connective tissue organization in transplantation;

- range of reaction to a foreign body;

- Changes in the subarachnoid space (inflammatory processes, fibrosis vs. open subarachnoid space) and

- Changes in the bark (inflammation, necrosis)

Macroscopic and histological results

The histological results described below are identical in terms of cell composition, but differ in intensity in different frontal sections from the same animal and in all animals in the same group.

Macroscopic assessment of graft incorporation

Removal of the skull bones after a period of 2 weeks revealed minimal bilateral adhesions between the fibrin glue residue and the bone above. Adhesions were ripped off easily. There were no signs of inflammation or CSF leakage around both transplants. Both transplants were dotted with single blood clots several millimeters in diameter.

In the left hemisphere, equine collagen foil is still demarcated as such and appears to be less translucent compared to its original glass transparency. The collagen product maintained a connection to the edges of the tire when lifting tires from above the bark. There were several very weak adhesions that were easily broken without damaging the bark.

The Tutoplast® Dura in the right hemisphere is unchanged for the naked eye. Tutoplast® Dura detaches in places where the tire contacts the graft when removing the product. There are some subarachnoid adhesions when the tire rises from the bark, but it tears easily with tweezers.

Four weeks after surgery, there are still single adhesions between the edges of the bone above and the tire below, caused by fibrin glue residue. The bone could easily be pulled away from the dura mater without damage, without causing damage to the tire or graft. In place of the equine collagen foil in the left hemisphere, the boundary line between the dura and the graft was no longer clearly visible. The transplant is less transparent than before and has acquired a pale red color. The part of the graft coming into contact with the brain surface is homogeneous, smooth and mobile. Epidural adhesions are no longer present. Single blood clots are visible.

Tutoplast® Dura seems unchanged to the naked eye again. Inspection of the graft and tire contact area reveals an inadequate, easy to tear connection.

Eight weeks after surgery, the transition region between the tire and equine collagen foil is no longer present. The structural continuity of the tire is visible on both sides of the tire. The area around the application of equine collagen foil is only visible as a thinner film with a slightly reddish tinge. See Figures 9 and 10. The Tutoplast® Dura graft in the right hemisphere is at the same time covered on both sides with a thin membrane of connective tissue. See Fig. 11. The edges of the graft are still clearly visible under the surrounding tire.

A weak pull is sufficient to pull Tutoplast® Dura away from the tire shown.

After 16 weeks, the creation of a new tire in place of the equine collagen foil went on and the tire and new tire are barely distinguishable.

Surrounding the Tutoplast® Dura graft capsule in the right hemisphere becomes more pronounced.

After a period of 24 weeks, the cross-sections of both graft sites do not differ macroscopically from those in the previous group.

Microscopic assessment of graft incorporation

Two weeks after surgery, as expected, the equine collagen foil graft area reveals extensive areas of inflammatory lesions. The entire subarachnoid space is closed by adhesions resulting from abundant exudate from lymphocytes, segmented granulocytes and macrophages. Some areas of very extensive inflammatory exudate are visible above the transplant. In addition to the lymphocytic and monocytic components, there are also small bone fragments, with a corresponding response to a foreign body of multinucleated giant cells.

The equine collagen foil transplant itself presents the disappearance of a homogeneous structure and infiltration with inflammatory cells, heart-shaped or extensive. See Fig. 12. In several cases, ischemic necrosis (associated with surgery) of superficial cortex layers occurs.

Tutoplast® Dura also has an extensive inflammatory response, especially in the subarachnoid space. The graft itself is not infiltrated with inflammatory cells, but inflammatory lymphocytes and monocytes and foreign body response can be identified at both ends.

After a period of four weeks, inflammatory changes in the area of ​​the equine collagen foil graft were significantly reduced, but carpet lymphocytic and monocytic exudate is still present. See Figures 13 and 14. Multinucleated giant cells are more common as a foreign body reaction, particularly close to bone fragments. Numerous fibroblasts can be observed inside the primary equine collagen foil transplant. The typical homogeneous graft structure is not visible on the HE (hematoxylin-eosin staining) sections. EVG (Elastica von Gieson) and trichrome stained samples show increased formation of new collagen fibers at the former graft location. The dura mater itself gives a smooth path to tissue with a loose structure containing newly formed collagen fibers, showing inflammatory infiltration. The previously observed soft tire adhesions are no longer present. The subarachnoid space is again a visible, evident gap. The soft tire still has small spots of lymphocytic monocytic infiltrate.

Connective tissue organization is not observed in the Tutoplast® Dura area. Inflammatory exudate is present above and below the implanted tissue and inflammatory infiltration is also present in the transition zone to the tire. The subarachnoid space is detectable and unobstructed. See Fig. 15.

Eight weeks after the procedure, in the group with equine collagen foil, a further reduction of the inflammatory process in the new tire is observed. There are still few clusters of lymphocytic and monocytic infiltrate. The subarachnoid space is clean and again only small foci of inflammatory activity are present. The EVG and trichrome stained sections show continuity between the endogenous tire rich in collagen fibers and the new tire's emerging collagen fibers. This new film is variable in thickness and exhibits loose structure in places. See Fig. 16.

Inflammatory activity was also cleared around Tutoplast® Dura. Embedding into an adjacent tire is absent in places, and there are signs of inflammatory infiltrate and adhesions with the adjacent tire in other places.

After 16 weeks, in the group with equine collagen foil, clusters of nodular lymphocytic and monocytic infiltrate are still visible. There was no change in the continuity between the collagen-rich primary tire and the new tire, with a structure that did not show major changes compared to 8 week finds. Collagen fibers of varying thickness are present, with local loosening of the structure.

The area around Tutoplast® Dura again shows inflammatory changes and adhesions with the surrounding tire, these findings are particularly intense.

After 24 weeks, in the group with equine collagen foil, apart from further withdrawal of the cellular inflammatory response, there are no clear differences in the extent of graft incorporation compared to 16 weeks. See Fig. 17.

Histological results

Quantitative assessment of the inflammatory response, connective tissue organization, and extent of foreign body responses around the transplant and epigastric and epidural space are provided below ^Tand^be^{li 1}.

Inflammation (inflammatory reaction):

- no visible signs of an inflammatory reaction, only limited inflammatory infiltrates + mild inflammatory response ++ significant inflammatory response +++ severe inflammatory response

Transplant organization:

- connective tissue organization absent or mediocre + individual fibroblasts in transplantation limited tissue organization (40-70%) u / K tissue organization> 70%

K visible continuity between new tire and tire, full organization (100%) of the transplant

Reaction to a foreign body with multinucleated giant cells:

- no reaction to a foreign body only limited reaction to a foreign body + mild reaction to a foreign body ++ clear reaction to a foreign body +++ extensive reaction to a foreign body

The results of the histopathological examination of the subarachnoid space around both grafts are provided in Table 2 below.

Inflammation

- no inflammatory response + mild inflammatory response ++ pronounced inflammatory response

Severe inflammatory reaction

Closed SAS (subarachnoid space)

- diffuse inflammatory cells + SAS closed by cellular infiltration in isolated inflammatory infiltrates in SAS pF / F partial fibrosis / SAS fibrosis

Ciwarta ^SAS :

- SAS generally clean, with isolated cell groups + clean subarachnoid space

Discussion a

Assessment of surgical methods and support for both transplants

Equine collagen foil has been characterized as being easy to use intraoperatively. Rehydration in saline for 5 minutes gave a very strong thin film, about 2 mm thick, which maintained its shape, did not clump and was easy to cut. The material could easily be placed on the tire defect using pliers and a blunt hook. Due to the mobility of the equine collagen foil, it was easy to improve its position on the surface of the brain before fixing it in place. There was no need to sew the film because the graft was attached quickly and easily to the tire border with fibrin glue. Previous studies have shown that fibrin glue is a sufficient bonding agent for tire closures.

The experimental application of sutures for attachment caused the equine collagen foil to tear with the lightest pull, indicating that fibrin glue is a better approach for this graft.

When the product was later removed, it was found that in some cases too loose application of fibrin glue led to local adhesions with the bone above. These adhesions had to be removed carefully with a rotor to prevent the tires and bark from being pulled. Prevention of this problem was to use a sufficient amount of fibrin glue. When the product was removed, it became clear that the mere application of fibrin glue produced a secure closure of the meninges and prevented CSF leakage. This was evident from the 2nd week after surgery, when none of the animals developed subcutaneous CSF leakage or cerebrospinal fluid fistulae.

Tutoplast® Dura can also be easily used after a short hydration for seven minutes, but is more rigid and has greater thickness variation.

The fibrin glue produced sufficient adhesion to pre-hold the Tutoplast® Dura in place, but the connection between the primary tire and the graft was loose during secondary opening of the treatment area. It was also found 24 weeks after the procedure, because Tutoplast® Dura itself was not combined with the autologous dura. Therefore, it seems that the practice of attaching this graft with separate sutures has an advantage over the use of fibrin glue, because as observation indicates, otherwise insufficient embedding will not occur. There were very few adhesions in the transition zone between Tutoplast® Dura and the cortex and were easily removed with a raspator.

The technique used to introduce equine collagen foil between the tire and the cortex is somewhat uncomfortable in certain surgical situations. In particular, it is difficult to achieve adequate fixation in procedures for large defects of the interstitial brain, e.g. cancer cavities.

Macroscopically, the animals presented effective closure of the tire defect, without any evidence of transplant rejection. Small adhesions between the implant and cortical structures developed in several cases, probably due to minor spider tire injuries during the procedure. Excessive use of fibrin glue has generated small areas of easy-to-release adhesions with the bone above in several animals.

Histologically, the dense infiltration of the equine collagen foil with lymphocytes, macrophages and fibroblasts was visible 14 days after implantation. Concomitant inflammatory changes in the subarachnoid space and under / above the dura mater and in the transition zone between the transplant and the duraemia resolved well from the 4th week after surgery. The continuous transition between grafting of equine collagen foil and the surrounding tire due to the formation of new collagen fibers was also clearly visible at that time.

This new tire, induced by equine collagen foil, is not as thin as the original tire 24 weeks after surgery, probably because the dura foil only provides the original thickness. It is possible, however, this difference disappears later as the amount of collagen fibers increases.

Two weeks after the procedure, Tutoplast® Dura macroscopically covers the product with a thin layer of connective tissue, which becomes thicker over time. Again, all animals exhibit adequate tire closure without CSF fistulas and single adhesions between graft and cortex are present.

Despite similar inflammatory reactions in the structures surrounding the transplant, there are no signs of post-operative organization processes and there are poor symptoms of cellular infiltration and graft recovery.

None of the animals developed neurological disorders or wound infections in addition to the predicted local inflammatory response involving lymphocytes and macrophages.

Example 2 - Edema capacity of equine collagen foil

Studies on the edema capacity of equine collagen foil were carried out as follows:

1) First, the equine collagen foil was cut into 1 cm square fragments².

2) Samples of these fragments were examined in a regular scanning microscope to determine the basic morphological consistency and thickness as a reference for edema procedures.

3) The fragments were placed in plastic culture vessels.

4) Fluid uptake capacity and edema capacity were then tested by gradual titration with saline, administered with Eppendorf micropipettes, using reduced fluid volumes ranging from 10 / cm2 to 150 / cm2 (see Ta^be^land ³).

Results :

The amount of liquid soaked in the equine collagen foil according to the above procedure was determined after 1, 2 and 3 hours.

A fragment of equine collagen foil with an area of ​​1 cm²absorbed 1 μΐ of saline completely, without significant increase in thickness through edema.

ΐΐ of saline was completely absorbed by a 1 cm2 fragment of equine collagen foil, which led to a mild increase in material thickness.

Only minimal increase in the thickness of equine collagen foil was observed throughout the series. It is estimated that the maximum increase in thickness is only almost twofold compared to the initial volume, even after 3 hours.

No significant increase in thickness or absorption of fluid was seen after the first hour.

Example 3 - Increasing the length of hydrated equine collagen foil

Seven dry 1.0 cm2 equine collagen foil fragments were hydrated for 1 hour in isotonic sodium chloride. The average increase in length due to hydration of the dry fragments was 3.4 percent.

Table 4

<td>Fragment No.</td><td>Dry length (mm)</td><td>Length hydrated (mm)</td>

<td> 1</td><td> 17^,5</td><td> 18</td>

<td> 2</td><td> 17^,7</td><td> 18^,3</td>

<td> 3</td><td> 17</td><td> 17^,9</td>

<td> 4</td><td> 17^,8</td><td> 18^,5</td>

<td> 5</td><td> 18^,1</td><td> 18^,7</td>

<td> 6</td><td> 18,. 6</td><td> 19^,l 5</td>

<td> 7</td><td> 17^,7</td><td> 18^,2</td>

<td>Average</td><td> 17,8</td><td> 18^,4</td>

Example 4 - Increased weight of hydrated equine collagen foil

Seven pieces of equine collagen foil with an area of ​​1.0 cm² hydrated for 1 hour in isotonic sodium chloride. The hydrated pieces of equine collagen foil weighed about five times more than when dry.

Table 5

<td>Fragment No.</td><td>Dry weight (mg)</td><td>Hydrated weight ⁽mg)</td>

<td> 1</td><td> 8^,4</td><td> 40^,2</td>

<td> 2</td><td> 7^,6</td><td> 37^,9</td>

<td> 3</td><td> 8</td><td> 39^,l 3</td>

<td> 4</td><td> 8^,1</td><td> 39^,l 6</td>

<td> 5</td><td> 8^,l 6</td><td> 41^,9</td>

<td> 6</td><td> 8^,7</td><td> 46^,8</td>

<td> 7</td><td> 7^,7</td><td> 38^,3</td>

<td>Average</td><td> 8^,2</td><td> 40^,6</td>

Example 5 - Tensile strength and elasticity / flexibility of equine collagen foil

The tensile strength of several dura products and equine collagen foil was measured. The samples were mounted on the lower end of the pipe and the tensile force was increased by continuously increasing the water column to a maximum of 300 cm. Fig. 18 provides an illustration of the test chamber.

The test chamber was suitable for determining the strength of dura mater replacement products by determining the point at which the product broke due to pressure exceeding the tensile strength of the product. Equine collagen foil (collagen content: 5.6 mg / cm²) according to the present invention and collagen foil (collagen content: 4.0 mg / cm2) were compared with Duragen (Integra NeuroScience, Plainsboro, NJ).

The test results show that the equine collagen foil (collagen content: 5.6 mg / cm2) and collagen foil (collagen content: 4.0 mg / cm2) withstand a water column pressure of up to 300 cm without breaking. See Figs. 19 and 20. For comparison, the pressure exerted on a healthy skull does not exceed more than about 15 cm water column; in pathological conditions, the pressure can rise to about 50 cm.

DuraGen had significantly lower tensile strength, it was broken even at a pressure of 200 cm water column. See Fig. 21.

The test chamber was also suitable for comparing product flexibility by comparing the extent to which the product could stretch under pressure. The product's flexibility / elasticity was therefore measured by measuring the product's prominence under the weight of the water column.

The flexibility / elasticity of the equine collagen foil was compared (collagen content: 5.6 mg / cm²) and collagen film (collagen content: 4.0 mg / cm2) with DuraGen, test collagen film, Tutoplast Fascia lata (Tutogen Medical GmbH, Neunkirchen am Brand, Germany) and Etisorb Dura Patch (^eth^andcon ^GmM &amp; Co. KG^Nordstedt, ^NIEMCA^s) . ^Pand^tgovernment ^FIG. 22-2⁵

Compared to other products, equine collagen foil (collagen content: 5.6 mg / cm2) showed mixed significant tensile strength combined with flexibility / flexibility. This allows her to withstand the pressure exerted on her as a substitute for the dura mater and will remain flexible and flexible, enabling her to adapt to the contours of the brain and skull. In contrast, Ethisorb and Tutoplast showed high voltage strength in the water column experiment, but had much lower flexibility / flexibility.

Example 6 - Liquid-tight properties

Measurements of fluid-tight properties of dura mater replacement products were tested using the same experimental set-up as discussed in Example 5. In this experiment, water drop development and volume of water lost were measured relative to the height of the water column. The results of this experiment are shown in Figs. 23-26.

In this experiment, equine collagen foil (collagen content: 5.6 mg / cm2) remained fluid tight even with a water column over 300 cm. Collagen foil (collagen content: 4.0 mg / cm²) showed a loss of tiny water droplets under a 300 cm water column. Due to its porous structure, DurGen was not water-tight, but showed good visible water loss even at low water pressure. Similarly, Tutoplast Fascia Lata was also not watertight, showing low pressure water loss.

Example 7 - Durability and tear resistance

Only durable, flexible and tear-resistant materials in wet and dry environments are suitable as implants in exceptional surgical situations, such as for dura mater replacement materials. For this reason, determining the tear resistance / maximum tensile force of the wetted material provides valuable information regarding structure, strength and the like at the surgical site.

The tear resistance of collagen surgical implants was tested on hydrated samples to mimic the conditions prevailing in the body. The materials were placed for five minutes in isotonic saline.

To measure the tear resistance / maximum tensile force, collagen implant strips were inserted into ^FROMin^andc^{k M}about^de^l 112^{0 All-P}ur^pose Testing ^Machine (^FROMin^andc^{k G}mb^H &amp;

What. KG. Ulm, Germany). Tested collagen implants are shown in Table 6.

Table 6 - Collagen implants

<td>Test strip</td><td>Factor</td><td>Concentration</td><td>Origin</td>

<td>AND</td><td>Sponge collagen "Compress"</td><td>^10,0 m^{g /}cm²</td><td>Cow dermis</td>

<td>B</td><td>Sponge collagen "^FOAM "</td><td>Approximately 2.6 mg / cm2</td><td>horse tendon Achilles</td>

<td>C</td><td>Sponge collagen</td><td>2.8 mg / cm2</td><td>horse tendon Achilles</td>

<td>D</td><td>^Fabout^land collagen</td><td>4.0 mg / cm2</td><td>horse tendon Achilles</td>

<td>E</td><td>Foil from collagen horse</td><td>5.6 mg / cm2</td><td>horse tendon Achilles</td>

Machine control, data collection and testing, including statistical evaluation, were carried out using TestExpert software (Zwick GmbH &amp; Co. KG. Ulm, Germany). Tested samples of sponge A, B and C appear rather fragile. Samples A, B and C were cut into 4 cm wide 1.4 cm wide strips. The results of the experiments with all samples were calculated with reference to a 1.0 cm wide strip. The maximum tensile force was measured in Newtons / cm strip. The test results are shown in Table 7 and Fig. 27.

Table 7 - Tear resistance / maximum voltage

<td>Examined belt</td><td>Tear resistance / maximum voltage (Newtons / cm-strip)</td><td>Deviation standard</td>

<td>AND</td><td> 0^,46</td><td> 0^,19</td>

<td>B</td><td> 0^,45</td><td> 0^,11</td>

<td>C</td><td> 1, 64</td><td> 0^, 69</td>

<td>D</td><td>^3,21</td><td> 0^, 69</td>

<td>E</td><td> 4^,09</td><td> 0^,24</td>

Conclusions

The unique method of producing equine collagen foil increases its tear resistance / maximum tensile strength.

Collagen films showed significantly higher tear resistance / maximum tensile strength compared to collagen sponges.

Tear resistance increases with collagen content per square centimeter (e.g. collagen content 4.0 mg / cm²: 3.21 N / cm-strip; collagen content 5.6 mg / cm²: 4.09 N / cm-strip).

In view of the foregoing, it is clear that several goals have been achieved in the present invention.

With respect to the use of the word "include" or "includes" or "including" throughout this description (including the following claims), the Applicants point out that, unless the context provides otherwise, these words shall be used in their basic and clear meaning and they should be interpreted as open and not exclusive, and this meaning of these words applies throughout the text.