DE102010046218A1 - Device, useful for treatment of injuries and illnesses of e.g. tendons and ligaments, comprises a layer system with at least three layers respectively comprising identical or different, dimensionally stable and biocompatible hydrogel - Google Patents

Abstract

Device comprises a layer system with at least three layers (1, 2, 4), where each layer comprises an identical or different, dimensionally stable and biocompatible hydrogel, and a cell-containing layer (2) is arranged between two cell-free layers (1, 4).

Description

Field of the invention

The invention relates to a cell-gel based injection system for treating musculoskeletal injuries such as cartilage, epithelial layers, bones and the skin to repair or replace damaged human or animal tissue through disease or injury. The novel injection system includes a special mixing and storage container with a new cell-gel suspension.

Background of the invention

Today there are many forms of treatment for tendon defects in humans and animals. There is currently no scientifically validated tendon therapy that leads to a safe restoration of the patient's original (sports) performance [1]. This has the consequence that z. B. Horses despite medical care fail up to a year of riding and bear a considerable risk of injuring themselves at the newly healed place again. Due to the hitherto very limited treatment options for tendon defects, an attempt is increasingly being made to improve the regeneration of the tissue by applying autologous cells to the defect zones. Similar problems also arise from the treatment of other defective tissues, i.a. a. Cartilage, epithelial layers, bones and skin known.

State of the art

In the orthopedic and surgical practice, injuries to the tendon and ligament tissue currently represent a major problem. In addition to traumatic injuries are v. a. also degenerative diseases in the foreground. For the therapy are z. T. autologous transplants available, but often only a primary wound closure is made. It is considered to be particularly problematical that, with previous treatment methods, the functionality of diseased tendon tissue can be restored to a majority of only 60%. This is at least partially an expression of the low regenerative potential of the affected tissue. Tendons consist of collagen-containing extracellular matrix and the interposed tendon cells (tenocytes). These highly specialized cells are of mesenchymal origin. They ensure the structural structure of the tendon through the production of extracellular matrix. However, their small numbers and low mitotic activity in combination with only weak vascularization result in low healing capacity [3]. Another problem is the fact that the regenerated tissue formed is often inferior to healthy tendon tissue. The more it has to be the subject of research to develop therapeutic options that can be used to overcome these deficits.

As part of innovative therapeutic strategies, numerous approaches are being examined to support and positively influence the body's own healing capacity. One approach is to bring about by the application of healing-promoting proteins to improve the therapeutic successes to be achieved so far. Likewise, gene-based therapies are being tested for their applicability, the aim of which is also to support the healing process.

In addition, cell-based therapies provide for improving the rate of healing of substantial tissue defects by introducing metabolically active cells into injured tissue areas. Although no scientific consensus has yet been reached on which cell type is ultimately most appropriate, there is consensus that cell-based methods are particularly promising. In this context, adult mesenchymal stem cells are often the focus of further investigations because they combine a large number of important factors. On the one hand, isolation of these cells is relatively easy and is usually done with bone marrow aspirates [4, 5], adipose tissue [6-12], muscle tissue [13, 14] or peripheral blood [15]. A particular advantage with regard to a later clinical application is also to be seen in the fact that they are highly proliferative cells whose expansion is possible in vitro. In this way, the relatively large amounts of cells needed for a therapeutic application can be generated. Another important advantage of these cells is that they can be differentiated in a defined direction into cells of different tissue types. In the context of tendon injury therapy, this means that defect zones can not be replaced with scar tissue, but with full tendon fibers. This is reflected in a reduced rate of recurrence. In addition, unwanted side effects are not expected as they are cells derived from the body's own tissues and thus can be transmitted in the sense of autologous transplantation.

The use of mesenchymal adult stem cells from bone marrow supernatants for the treatment of tendon, bone or cartilage defects is well known in the art. For example, describes EP 1872789 A1 the use of stem cells for healing of injured tendon tissue.

Another known cell source is the fatty tissue. As in many rapidly developing fields, a variety of names have been used to describe the plastic-adherent cells isolated from adipose tissue by the enzyme collagenase. Each of the following expressions has merit: adipose-derived stromal cells (ASCs), adipose-derived adult cells (ADAS), adipose-derived stromal cells (ADSCs), adipose-derived mesenchymal stem cells (AdMSCs), lipoblast, pericyte, preadipocyte, and processed lipoaspirate cells. This very heterogeneous nomenclature results in a reduced clarity. For this reason, the International Society of Technology has established the term "adipose-derived stem cells" (ASCs) to refer to plastic-adherent multipotent cells. Not least, this reflects the high level of scientific activity in this area of research and is also an indicator of a relatively high level of technology with regard to the isolation of cells from adipose tissue.

The therapeutic utility of ASCs is based on these cells secreting cytokines and growth factors into injured or diseased tissue, thereby stimulating healing. Injected ASCs can also modulate the body's own stem cells by stimulating the migration of endogenous stem cells to the site of the defect [16].

The use of human subcutaneous fatty tissue as a possible source of adult or endogenous stem cells has been widely discussed. This results not least from an increased incidence of obesity, as a result of this phenomenon subcutaneous adipose tissue from now routinely performed interventions for further investigations is available.

Subcutaneous adipose tissue of human patients has been widely used as the starting material for the isolation of the above ASCs. For the cells obtained from this tissue, a stem cell character could be clearly detected. The evidence was provided by a differentiation along the osteogenic, adipogenic, myogenic and chondrogenic lines [17]. In addition, a typical morphological appearance as well as a stem cell-specific surface marker profile were demonstrated [18]. From these characteristics, the applicability of the ASCs for tissue renewal in biotechnology [19].

For the treatment of tendon and ligament tissue injuries, stem cells are to be isolated from subcutaneous fatty tissue. The preparation of the tissue with the aim of cell isolation is analogous to the methods known for the preparation of human tissue. The healing effect, isolation and expansion of adipose tissue-isolated mesenchymal adult stem cells is also known in the art. See the patent application for this WO 2005/035742 , The stem cells described in this invention are isolated from subcutaneous fatty tissue and combined with a carrier material and endogenous serum, resulting in an innovative, ready-to-use product.

The stem cell character of cells derived from canine adipose tissue has already been demonstrated in principle by their differentiability in osteoblasts and in adipocytes. The method for identifying and isolating mesenchymal stem cells from human adipose tissue has been modified for cells of canine adipose tissue. For differentiation, the protocols for human cells were tested and adapted to the requirements of animal cells [20].

In addition to the fundamentally possible recovery of mesenchymal stem cells from the adipose tissue, the survival and growth of tissue precursor cells in hydrogels was examined with regard to the use in the context of regenerative therapeutic strategies (see Patent DE 699 35 786 T2 , In this patent, a liquid hydrogel-cell composition is incorporated into a support structure to repair or replace damaged tissue in living things.). Background of this work is the development of an application-friendly cell-matrix construct that allows a precise introduction of cells into tissue defects and additionally helps to keep the cells at least initially stationary. The following substances may be used as gellable matrix: agarose, agar agar, algina, xanthan, gelatin, polysaccharides, fibrin, polyurethanes, collagen, chitosan.

task

The invention is therefore based on the object to provide a solution which makes it possible to combine a combination of a carrier material, which can be explicitly adapted to the claims of injured tissue, and an expanded to therapeutic amounts of stem cell suspension. A gellable matrix can be used in a three-layered system in combination with a cell mixture for the treatment of injured tissue in humans and animals. The gelable matrix can be dissolved in physiological solution (sodium chloride) and sterilized by steam autoclaving. The cells are preferably centrifuged to a cell pellet (e.g., 500 g, 5 min) and taken up in autologous serum and resuspended. The autologous serum can be directly before the preparation of the gellable matrix are produced individually for each patient. Preferably, it is recovered from the patient's blood by a centrifugation step (eg 1000 g, 10 min) and sterile filtered. The effect of healing is underlined by the longer residence time of the cells at the defect by the biocompatible matrix. In the present application, no additional support framework is used as the matrix. The therapy method can be used for the treatment of defects of the passive and active musculoskeletal system and the skin z. B. in the veterinary field (eg., Horse, dog, cat, camel) are used.

Summary of the invention

• 1. Vorrichtung nach Punkt 1: Vorrichtung zur Injektion, enthaltend ein dreischichtiges, zell-basiertes System, wobei die Zellen in einer flüssigen Gelphase vorliegen, die sich zwischen zwei zellfreien Phasen befindet, dadurch gekennzeichnet, dass: a) die Gelphase ein biokompatibles, injizierbares Hydrogel ist, und b) die Zellen adulten Gewebevorläuferzellen sind, die aus Unterhautfettgewebe isoliert und in vitro expandiert wurden und eine Gewebe bildende Einheit sind, und c) die zellfreie Phase eine höhere Viskosität aufweist als die flüssige Gelphase.
• 2. Vorrichtung nach Punkt 2, dadurch gekennzeichnet, dass die Vorrichtung eine sterile Spritze ist.
• 3. Hdyrogel nach Punkt 3, dadurch gekennzeichnet, dass es aus einer gelierbaren Matrix besteht.
• 4. Gelierbare Matrix nach Punkt 4, ausgewählt aus Agarose, Agar Agar, Alginat, Xanthan, Gelatine, Polysaccharide, Fibrin, Polyurethane, Collagen, Chitosan.
• 5. Gelierbare Matrix nach Punkt 5, dadurch gekennzeichnet, dass sie eine Konzentration von 0,1–0,6% stärker bevorzugt von 0,25–0,5%, am stärksten bevorzugt von 0,4% aufweist.
• 6. Zellen nach Punkt 6, dadurch gekennzeichnet, dass die expandierten adulten Gewebevorläuferzellen mindestens fünf Tage in vitro vermehrt wurden.
• 7. Zellen nach Punkt 7, dadurch gekennzeichnet, dass die Zellen aus Unterhautfettgewebe autologen oder allogenen Ursprung isoliert wurden.
• 8. Zellfreie Phasen nach Punkt 8, dadurch gekennzeichnet, dass sie aus einer gelierbaren Matrix bestehen.
• 9. Geliebare Matrix nach Punkt 9, ausgewählt aus Agarose, Agar Agar, Alginat, Xanthan, Gelatine, Polysaccharide, Fibrin, Polyurethane, Collagen, Chitosan.
• 10. Geliebare Matrix nach Punkt 10, dadurch gekennzeichnet, dass sie eine Konzentration von 0,8–2,5%, stärker bevorzugt von 0,9–1,5%, am stärksten bevorzugt von 1,0% aufweist.
• 11. Verwendung der Vorrichtung nach Punkt 11 zur Behandlung von Verletzungen oder Erkrankungen.
• 12. Verwendung nach Punkt 12, dadurch gekennzeichnet, dass Verletzungen oder Erkrankungen von Sehnen, Bändern, Faszien, Muskel, Haut, Knochen oder Knorpeln behandelt werden können.
• 13. Verwendung nach Punkt 13, dadurch gekennzeichnet, dass alle Säugetiere wie Menschen, Pferde, Hunde und Katzen behandelt werden können.

The inventive embodiments are characterized in the accompanying drawings. The invention describes a novel pharmaceutical composition comprising adipose-derived autologous stem cells for treating injured tendon and ligament tissue. The patient-specific serum produced for each individual therapy, in which the cells are taken up, drastically reduces the risk of rejection of the graft. The "three-layer system" is an ideal composition between carrier material and cells. The vitality of the cells in the carrier material was checked by means of a "living-death-proof". In particular, the present invention relates to the following points:

• Device according to item 1: device for injection containing a three-layer cell-based system, wherein the cells are in a liquid gel phase located between two cell-free phases, characterized in that: a) the gel phase is a biocompatible, injectable Hydrogel; and b) the cells are adult tissue progenitor cells isolated from subcutaneous fatty tissue and expanded in vitro and forming a tissue-forming unit, and c) the cell-free phase has a higher viscosity than the liquid gel phase.
• 2. Device according to item 2, characterized in that the device is a sterile syringe.
• 3. Hdyrogel according to item 3, characterized in that it consists of a gellable matrix.
• 4. Gelable matrix according to item 4 selected from agarose, agar agar, alginate, xanthan, gelatin, polysaccharides, fibrin, polyurethanes, collagen, chitosan.
• 5. Gelable matrix according to item 5, characterized in that it has a concentration of 0.1-0.6%, more preferably of 0.25-0.5%, most preferably of 0.4%.
• 6. cells according to item 6, characterized in that the expanded adult tissue precursor cells were propagated for at least five days in vitro.
• 7. Cells according to item 7, characterized in that the cells have been isolated from subcutaneous fatty tissue of autologous or allogeneic origin.
• 8. Cell-free phases according to item 8, characterized in that they consist of a gellable matrix.
• 9. Gelable matrix according to item 9 selected from agarose, agar agar, alginate, xanthan, gelatin, polysaccharides, fibrin, polyurethanes, collagen, chitosan.
• 10. Gelable matrix according to item 10, characterized in that it has a concentration of 0.8-2.5%, more preferably 0.9-1.5%, most preferably 1.0%.
• 11. Use of the device according to item 11 for the treatment of injuries or illnesses.
• 12. Use according to item 12, characterized in that injuries or diseases of tendons, ligaments, fascia, muscle, skin, bone or cartilage can be treated.
• 13. Use according to item 13, characterized in that all mammals such as humans, horses, dogs and cats can be treated.

Short description of the drawing

Further details, features and advantages of the subject matter of the invention will become apparent from the subclaims and from the following description of the accompanying drawings, in which a preferred embodiment of the invention is shown.

1 is a sketch of the pharmaceutical composition of the stem cell gel suspension. No. 1 represents the first layer which is discarded prior to application in injured tissue. No. 2 represents the layer in which cells (No. 3 ) were introduced into a carrier material. No. 4 represents a top layer of cell-free, gelatinizable Matirx, which is in the application of the composition in the syringe neck, and thereby ensures a 100% application of the cell-carrying hydrogel component. No. 5 represents the storage container used for the application. No. 6 represents the cap of the stem cell gel suspension, which is put on for protection against external influences. No. 7 the piston is the tip.

example 1

Isolating Mesenchymal Stem Cells from Adipose Tissue of the Horse

The sample is taken under local anesthesia. 3-5 g subcutaneous fatty tissue are taken from the tail of the horse. For this, a small linear cut (5 cm) is made at the proximal end of the biceps femoris and semitendinosus muscle. The adipose tissue is removed with scissors and transferred to a phosphate buffered saline (PBS) 50 ml reaction tube. Then the wound is closed with a simple suture.

The isolated adipose tissue is weighed, washed with PBS and cut into small pieces with sterile scissors. All preparation steps are performed under sterile conditions and are performed in a laminar flow hood. This is followed by digestion with 0.1% Collagenase Type 1 (Sigma Aldrich C2674) in a 50 ml Falcon at 37 ° C for 45 min. The digestion is stopped with the same amount of PBS and the cell suspension is filtered off (100 μm mesh). The cell suspension is centrifuged (1000 g for 10 min) and the cell pellet is plated in a culture flask (225 cm 2) and mixed with 20 ml culture medium (UltraCulture (BioWhittaker), 2% UltroserG (CytoGen), L-Glutamine 2 mM, 1% Pen / Strep (PAA)) in the incubator (5% CO2, 37 ° C) expands.

The same procedure can be used to isolate stem cells from adipose tissue of the abdomen which e.g. B. can be obtained in a colic surgery.

Example 2

Increase of adipose-derived mesenchymal stem cells

The adipose-derived mesenchymal stem cells are cultured in the incubator (5% CO ₂ , 37 ° C) for two weeks. The first change of media takes place 4 days after isolation. After about 5-6 days, the cells are split. The cells are washed with PBS and detached with trypsin from the culture flask (1-fold trypsin, 5 min, 37 ° C) to transfer the cells to new culture vessels. In this case, a reduction in the number of cells per area in the ratio of one to three or one to four is achieved. Medium change occurs at intervals of two days. After 10-14 days, the required cell count (about 10 million) is reached. It uses 2-20 million cells per product syringe. With enough large numbers of cells, either multiple syringes can be made or cells frozen at -80 ° C and kept in stock to treat any later occurring injury.

Example 3

Production of the autologous serum for the uptake of the cell pellet for product production

The patient's blood is filled into 50 ml tubes and then centrifuged (1000 g, 10 min). The cell-free supernatant is transferred under sterile conditions to a new vessel and sterile filtered. The serum is stored in the refrigerator (4 ° C) until use.

Example 4

Pharmaceutical kit of mesenchymal stem cells embedded in an agarose hydrogel

1st shift

First, 0.40 g (1%) agarose is dissolved in 40 ml sodium chloride in a 100 ml glass bottle. The agarose solution is autoclaved in a steam autoclave at 121 ° C for 20 minutes, dissolving completely. It is then transferred to a syringe (eg 2 ml) under a sterile bench during cooling. The consistency of the carrier material with the subsequently added cell suspension is adjusted so that an injectable gel can be produced reproducibly.

Next, 100 μl of the liquid hot agarose solution is poured into the product syringe. The viscous carrier material introduced at about 95 ° C. is cooled to room temperature under sterile conditions (eg in a sterile bench) (about 5 minutes) and solidifies. This layer forms the conclusion or underlays the later to be injected into a defect cell gel suspension ("final layer"). This layer is schematically in 1 shown under No. 4.

2 layer:

After cell isolation and proliferation, the cells are washed with PBS and incubated with 1% trypsin in a 37 ° C CO2 incubator for 5 min. The cells detach from the surface of the culture vessels. The trypsin is inactivated by adding fresh culture medium. The cells are transferred to a 50 ml Falcon and an aliquot taken to determine the number of cells using a Neubauer counting chamber. It is a cell suspension with min. 5 x 10 ⁶ cells at 500 g for 5 min centrifuged to a cell pellet. The cell pellet is resuspended in 300 μl of fluid (eg PBS, autologous serum, FBS, NaCl). The cell suspension is submerged at the final agarose layer Using a 200 μl pipette (200 μl + 100 μl) added. This step can be performed manually, but also standardized, automated.

The cell suspension develops by adding 200 μl of the cooled, autoclaved carrier material to a viscous mixture of cells and gel. To the cell suspension, the 1% agarose is added and homogeneously mixed without destroying the "finishing layer". To generate a homogeneous distribution of the cells in the gel, the solution is mixed with a fixed number (10-12 times) of stirring cycles (eg with a cannula). By mixing the cell suspension with the 1% agarose, the injectable 0.4% cell-hydrogel mixture is formed. This layer is schematically in 1 under no. 2 represented, wherein the cells by no. 3 Marked are.

3 layer:

The conclusion of this process step is to cover the stem cell suspension in the syringe with a thin layer of 1% agarose (eg 100 μl) so that the cells are not affected by external influences (eg fluctuations in temperature or oxygen concentration ) to be influenced. This layer is schematically in 1 under no. 1 shown.

Example 5

Application of the injection system of stem cells embedded in an agarose hydrogel

Before the application of the stem cell gel suspension in a tissue defect, the cap (see 1 under no. 6 ) and the first drop (cover layer of the viscous carrier material 1 under no. 1 ) discarded, leaving only the fresh cell gel (see 1 No. 2 ) is injected into the defect. Thereafter, the stem cells are applied under ultrasound control in the tendon defect site.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 1872789 A1 [0006]
• WO 2005/035742 [0011]
• DE 69935786 T2 [0013]

Claims (10)

Device comprising a layer system with at least three layers ( 1 . 2 . 4 ), each containing an identical or different, dimensionally stable and biocompatible hydrogel, wherein a cell-containing layer ( 2 ) between two cell-free layers ( 1 . 4 ) is arranged. The device of claim 1, wherein the solid component of the hydrogel of each layer is in a concentration of 0.1% (w / v) to 2.5% (w / v). Device according to claim 1 or 2, wherein the solid component concentrations of the hydrogels in the layers ( 1 . 2 . 4 ) are the same or different. Device according to one of claims 1 to 3, wherein the solid component concentrations in the cell-containing layer ( 2 ) in the range of 0.1 (w / v) to 0.6 (w / v), more preferably from 0.25% (w / v) to 0.5% (w / v). Device according to one of claims 1 to 4, wherein the solid component concentrations in the cell-free layer ( 1 . 4 ) ranges from 0.8 (w / v) to 2.5 (w / v), more preferably from 0.9% (w / v) to 1.5% (w / v). Device according to one of claims 1 to 5, wherein the solids component concentration of the hydrogel in the cell-containing layer ( 2 ) lower than that of the cell-free layers ( 1 . 4 ). Device according to one of claims 1 to 6, wherein the solid component is selected from the group consisting of agarose, agar-agar, alginate, xanthan, gelatin, polysaccharides, fibrin, ployurethane, collagen, chitosan and mixtures. Device according to one of claims 1 to 7, wherein the cells ( 3 ) in the cell-containing layer ( 2 ) are adult tissue progenitor cells. Device according to one of claims 1 to 8, which is a sterile syringe, wherein the layer system between pistons ( 7 ) and opening ( 6 ) is arranged. Device according to one of claims 1 to 9 for the treatment of injuries and diseases, in particular of tendons, ligaments, fasciae, muscles, skin, cooking or cartilage in mammals, in particular humans, horses, dogs and cats.

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