Biocompatible and biodegradable gradient layer system for regenerative medicine and for tissue support

Abstract

The present invention is directed to a biocompatible and preferably biodegradable gradient layer system comprising at least one set of layers comprising a biocompatible and preferably biodegradable cross-linked polymer and at least one biocompatible and preferably biodegradable support layer, wherein a gradient is preferably formed with respect to the mechanical and/or physical properties of one or more layers of the at least one set of layers comprising a biocompatible and biodegradable cross-linked polymer and/or the at least one biocompatible and preferably biodegradable support layer. The at least one support layer preferably comprises a biocompatible and preferably biodegradable meltable polymer and/or a biocompatible and incorporable material. This biocompatible and preferably biodegradable gradient layer system may be used as a biomaterial for regenerative medicine, particularly as a wound dressing or for tissue support. The present invention also provides means utilizing said inventive gradient layer system and methods for producing same.

Claims

1. A biocompatible gradient layer system comprising: at least one biocompatible meltable support layer comprising polycaprolactone (PCL); at least one set of layers disposed on the at least one biocompatible meltable support layer; and a continuous fiber diameter gradient within the at least one set of layers, wherein the at least one set of layers comprises fibers of a biocompatible and biodegradable polymer, wherein the fibers of the biocompatible and biodegradable polymer are formed by electrospinning a solution containing crosslinkers and the biocompatible and biodegradable polymer, wherein the fibers of the biocompatible and biodegradable polymer are selected from gelatin or collagen, wherein the crosslinkers are selected from glyoxal or glyoxal-trimer-dihydrate, wherein the fibers of the biocompatible and biodegradable polymer are internally crosslinked with the crosslinkers glyoxal or glyoxal-trimer-dihydrate, wherein the fibers of the resulting biocompatible and biodegradable crosslinked polymer are loosely packed and not interconnected to each other, wherein the continuous fiber diameter gradient is formed by crosslinking during the electrospinning process, wherein the diameter of the polymeric fibers is increased within the at least one set of layers due to the continuous crosslinking, and wherein the polymeric fibers of the at least one set of layers have a diameter between about 1 nm to about 50 m.

2. The gradient layer system according to claim 1, wherein the fiber diameter increases or decreases from about 0.0001 m to about 1 m per micrometer height of the set of layers either within the at least one set of layers or between several sets of layers of the gradient layer system.

3. The gradient layer system according to claim 1, wherein the at least one biocompatible support layer comprises an incorporable material selected from the group consisting of an incorporable ceramic material, an incorporable ceramic material made from tricalcium phosphate (TCP) and an incorporable ceramic material made from hydroxyl apatite (HA).

4. The gradient layer system according to claim 1, wherein the at least one set of layers comprising the biocompatible and biodegradable cross-linked polymer is present in the form of a fleece, a net or a mesh-like structure.

5. The gradient layer system according to claim 1, wherein the at least one biocompatible support layer is present in the form of bands, strands, fibers, particles, drops, a net or mesh-like structures, a sheet, a film, a foil, or a laminate.

6. The gradient layer system according to claim 1, wherein the gradient layer system is seeded with cells.

7. The gradient layer system according to claim 6, wherein the cells are selected from mammalian, human or non-human cells selected from committed stem cells, differentiated cells, adult stem cells, bone marrow stem cells, umbilical cord stem cells, engineered or non-engineered stem cells, primary or immortalized (cell-line) stem cells, and mesenchymal stem cells, or the cells are selected from a mixture of cells as defined before.

8. The gradient layer system according to claim 1, wherein the gradient layer system further comprises agents selected from cytokines, interleukins, growth factors, immunoglobulins, RGD-peptides, and antibacterial agents.

9. The gradient layer system according to claim 6, wherein the cells are selected from cartilage cells, epithelial cells, endothelial cells, endothelial cells of vascular tissue, endothelial cells of corneal tissue, skin cells, osteocytes, osteoblasts, cementoblasts, bone cells, myoblasts, neuroblasts, fibroblasts cells of all connective tissues including fibroblasts selected from gingival, skin or corneal fibroblasts, and fibroblasts selected from gingival, skin or corneal fibroblasts together with periodontal ligament fibroblasts, keratinocytes, gingival keratinocytes, glioblasts, germ cells, hepatocytes, chondrocytes, cardiac muscle cells, connective tissue cells, glial cells, hormone-secreting cells, cells of the immune system, neurons, cells of the central nervous system, neuronal cells, pericytes, myocytes, adipocytes, astrocytes, melanocytes, tissue cells, tissue cells from autologous tissue sources, tissue cells from allogenic tissue sources, tissue cells from xenogenic tissue sources, autologous cells, allogenic cells, xenogenic cells, tenocytes, cardiomyocytes, hepatocytes, and smooth muscle cells, or the cells are selected from a mixture of cells as defined before.

Description

FIGURES

(1) The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

(2) FIG. 1: illustrates an exemplary inventive biocompatible and preferably biodegradable gradient layer system and its structure. As can be seen, the inventive gradient layer system comprises different sets of layers comprising a biocompatible and preferably biodegradable cross-linked polymer present in a fleece like structure and at least one biocompatible and preferably biodegradable support layer present in a net or mesh-like structure.

(3) FIG. 2: shows a raster electron microscopy (REM) of an inventive biocompatible and preferably biodegradable gradient layer system, wherein fibroblasts of the dermis were cultivated and propagated successfully in vitro. The raster electron microscopy (REM) pictures in FIG. 2 additionally show the pre-culture of fibroblasts from the dermis on the bottom side/undersurface (inlay) of the inventive gradient layer system and establishment of an epidermal keratinocytes compartment on the surface of the substrate.

(4) FIG. 3: depicts a raster electron microscopy (REM) of an inventive gradient layer system, wherein an interactive co-culture of fibroblasts of the dermis and epithelial keratinocytes were cultivated and propagated successfully in vitro on the inventive gradient layer system (see FIG. 3 and inlay).

EXAMPLES

(5) The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto.

Example 1Preparation of Gelatin Layers

(6) Gelatin (type B from bovine skin; Sigma-Aldrich) was solubilized in a solvent mixture of acetic acid (Merck), ethyl acetate (Fluka) and water (5:3:2 vol). The concentration was in a range of about 5 and 20% (weight). Different amounts of cross-linker glyoxal (0.0005-0.1 g/g gelatin solution) were mixed with the gelatin solution. The gelatine/glyoxal mixture was filled into a syringe and mounted in a syringe pump (Model: KDS100 oder KDS101, KD scientific). the flow rate was between about 0.1 und 1.0 ml/h. Between the cannula and the collector an electrical field was established with two high voltage power generators (Heinzinger LNC 30000-2 neg und LNC 30000-2 pos) with field forces between 0.4 und 8 kV/cm.

(7) a) Electrospin/Elektrospray

(8) After depositing a sufficient thick fleece like set of layers of gelatin/glyoxal fibers on the substrate a thin layer of PCL was electrospun or electrosprayed on the fleece like set of layers formed by gelatin/glyoxal fibers. Subsequently further sets of layers comprising gelatin were applied. During the subsequent temperature treatment (60-100 C.; 0.5-24 h) a (further) cross-linking of the gelatin with glyoxal occurred. This was accompanied by a browning of the fleece like sets of layers due to formation to Schiff bases. Furthermore, temperature treatment led to a (partial) melting of the PCL layer. The melt punctually or laminarly surrounds the gelatin fibers and thus provides for significantly improved mechanical properties of the swollen fleece like set of layers of gelatin/glyoxal fibers.
b) 3D-Plotting A plotted PCL layer construct was applied on the fleece like set of layers of gelatin/glyoxal fibers instead of the PCL layers as described under section a).

(9) For both a) and b) it is to be noted that the arrangement of layers or sets of layers may be varied arbitrarily. As an example, it is likewise possible to use a 3D construct as a target for electrospinning. A different arrangement of the layers or sets of layers, the orientation of the different layers or sets of layers and a different number of layers or sets of layers is possible. Optionally, constructs, such as the PCL layers, etc., may be applied onto a support or directly onto the fleece like set of layers of gelatin/glyoxal fibers using a heat gun or hot air blower. A further possibility for improving the mechanical properties of the inventive biocompatible and preferably biodegradable gradient layer system was used based on an additional cross-linking of the gelatin fibers with polyphenol compounds, preferably gallotannine (Sigma-Aldrich). The fleece like set of layers of gelatin/glyoxal fibers was immersed or incubated in an aqueous or alcoholic solution (0.01-10% (weight)) of polyphenol compounds and agitated slightly between 0.5 and 24 h at temperatures of about 20-40 C. Excess polyphenol compounds were removed via several washing steps (destilled water, buffer, alcohol).

Example 2Preparation of Polycaprolactone Layers

(10) Abbreviations:

(11) PCL=polycaprolactone

(12) TCP=tricalciumphosphate

(13) D.sub.inside=bore diameter of nozzle

(14) RPM=rounds per minute

(15) Experimental Part of 3D-Bioplotting

(16) 1. PolycaprolactoneMesh Like Constructs

(17) The mesh like constructs were printed directly with a 3D-plotter (3.sup.rd Generation, Envisiontec, Germany) using CAD data. For this purpose polycaprolactone (PCL, M.sub.n=80.000 g/mol, Aldrich, St. Louis, USA) was filled into the high temperature printer head of the 3D-Bioplotter and molten (90 C.). The molten material was printed in form of rectangles (44 cm). A teflon foil serves as a support for printing. All experimental parameters are listed in table 1.

(18) TABLE-US-00001 TABLE 1 Experimental parameters of the plotting process for mesh-like constructs made from pure PCL. Plotting Nozzle Material medium T.sub.(material) ( C.) T.sub.(plotting medium) ( C.) Nozzle D.sub.inside (mm) PCL Air 90 20 Stahl 0.45 (M.sub.n = 80.000 g/mol) operating applied speed of thickness of pressure printer head distance of strands layer corner delay Material (10.sup.5 Pa) (mm/s) (mm) (mm) (s) PCL 3.6-3.8 100 4.5 0.3 0.1 (M.sub.n = 80.000 g/mol)
2. Polycaprolactone/TricalciumphosphateMesh-Like Constructs: Polycaprolactone (PCL, M.sub.n=80.000 g/mol, Aldrich, St. Louis, USA) and Tricalciumphosphate (10 Gew.-%, Budenheim, Germany) were provided in a lever lid glass container and mixed. The mixture was compounded with a microcompounder (Daca Instruments, Santa Barbara, USA) (100 C., 100 RPM, retention time 2 min). The obtained material was processed as indicated under section a. above. All experimental parameters are listed in table 2.

(19) TABLE-US-00002 TABLE 2 Experimental parameter of the plotting process for mesh-like constructs made from PCL and TCP. Plotting Nozzle Material medium T.sub.(material) ( C.) T.sub.(plotting medium) ( C.) Nozzle D.sub.inside (mm) PCL/TCP Luft 90 20 Stahl 0.45 (M.sub.n = 80.000 g/mol) operating applied speed of thickness of pressure printer head distance of strands layer corner delay Material (10.sup.5 Pa) (mm/s) (mm) (mm) (s) PCL/TCP 3.5 100 4.5 0.3 0.1 (M.sub.n = 80.000 g/mol)

Example 3Preparation of a Biocompatible and Preferably Biodegradable Gradient Layer System

(20) For the preparation of an exemplary inventive gradient layer system gelatin or gelatin blends with other polymers as described above were prepared prior to synthesis of polymeric gelatin fibers and mixed with glyoxal as a crosslinker. In a first step of the inventive method polymeric gelatin fibers was then synthesized via an electrospin procedure as described before leading to a layer of cross-linked polymeric gelatin fibers. The fibers were synthesized using the gelatin blends in the presence of the cross-linker glyoxal to enhance mechanical properties. Furthermore, the fiber strength of the polymeric gelatin fibers was adjusted during the electrospin procedure due to increasing viscosity upon cross-linking the polypeptide with glyoxal. Glyoxal preferably renders the obtained vlies structure water insoluble and thus improves the mechanical properties of the biocompatible and preferably biodegradable gradient layer system. The increasing viscosity led to increasing fiber strength and resulted in a gradient in the layer produced with polymeric gelatin fibers. The gradual fiber strengths had a range as defined above, typically in a range of about 50 to 1500 nm or 100 to 800 nm.

(21) Subsequently after synthesis of a layer of cross-linked polymeric gelatin fibers according to the first step those gradual fiber layers of polymeric gelatin fibers were identified, which exhibit a sufficient stability in aqueous milieu. The pH values were about 5.5 to about 8.5. Excessive alkaline and excessive acidic conditions were avoided to prevent degradation of the gelatin. These gradual fiber layers were then used as a basis in a second step in a 3D-electroplotting procedure. In the 3D-electroplotting procedure the polyester compound polycaprolactone was plotted onto the gradual fiber layer of polymeric gelatin fibers to obtain the inventive polymeric gradient layer system as a three-dimensional biohybrid polymer network structure or scaffold, which significantly stabilized the gradual fiber layer of polymeric gelatin fibers. The resultant scaffold retained the superior properties of the polymers used in the single steps. An exemplary biocompatible and preferably biodegradable gradient layer system and its structure are shown in FIG. 1.

(22) The biocompatible and preferably biodegradable gradient layer system obtained according to step 2, as described before, was then cultured with target cells in a further step 3. Step 3 was carried out by cultivating target cells, preferably fibroblasts of the dermis, on the surface of the inventive polymeric gradient layer system. The target cells were cultivated in cell culture medium. Cultures of primary dermal fibroblasts were maintained for routine cell culture in DME medium (PAA, Pasching, Austria) containing 10% foetal calf serum (Seromed, Biochrom, Berlin, Germany) and 50 g/ml kanamycin (Roche Diagnostics, Mannheim, Germany). Epidermal keratinocytes were maintained in low calcium keratinocyte growth medium (basal keratinocyte medium, KGM, with provided supplements, Promocell, Heidelberg, Germany), containing 50 g/ml kanamycin (Roche Diagnostics, Mannheim, Germany).

(23) Establishment of the respective cell types on the biocompatible and preferably biodegradable gradient layer system was performed by a 24 hours pre-cultivation of dermal fibroblasts at the bottom side of the substrate and subsequently by seeding epidermal keratinocytes at the top side of the device. This co-culture system has been cultivated for further 14 days.

(24) As determined by raster electron microscopy (REM) (see FIG. 2) fibroblasts of the dermis were cultivated and propagated in vitro successfully. The raster electron microscopy (REM) pictures in FIG. 2 additionally show the pre-culture of fibroblasts from the dermis on the bottom side/undersurface (inlay) of the inventive polymeric gradient layer system and establishment of an epidermal keratinocytes compartment on the surface of the substrate. It was furthermore possible to successfully cultivate interactive co-cultures consisting of fibroblasts and epithelial keratinocytes on the inventive polymeric gradient layer system (see FIG. 3 and inlay).