Transdifferentiated tissue graft

Abstract

The invention provides a method of producing a connective tissue graft suitable for correcting a connective tissue defect, comprising determining the size and shape of a tissue defect, obtaining a fat tissue from a patient modelled to fit the size and shape of the tissue defect, contacting the fat tissue with one or more connective tissue specific growth or differentiation factors; and kits for such a method.

Claims

1-17. (canceled)

18. A method of producing a bioengineered connective tissue graft by direct transdifferentiation of a donor connective tissue, preferably fat tissue, into another connective tissue type comprising contacting the donor connective tissue in vitro or in vivo by one or more administered connective tissue specific growth or differentiation factor.

19. A method of producing a connective tissue graft suitable for correcting a connective tissue defect, comprising deter-mining the size and shape of a tissue defect, treating a donor connective tissue, preferably fat tissue, obtained from a patient by, in any order: modelling donor connective tissue, preferably fat tissue, to fit the size and shape of the tissue defect and contacting the donor connective tissue, preferably fat tissue, with one or more connective tissue specific growth or differentiation factors, thereby initiating differentiation of the tissue graft into another connective tissue.

20. The method of claim 18 further comprising placing the differentiated tissue into the tissue defect of a patient, preferably wherein the patient with the tissue defect and the patient providing the donor tissue is the same patient (autologous tissue).

21. The method of claim 18 wherein the step of culturing the donor connective tissue or the contacting step is for at least 1 hour, at least 1 day, preferably at least 2 or at least 3 days, especially preferred at least 4 days.

22. The method of claim 18, wherein the another connective tissue is bone, cartilage, muscle, tendon, ligament or nerve, preferably bone or cartilage.

23. The method of claim 18, wherein the connective tissue is cartilage and differentiation comprises the differentiation into chondrocytes and/or chondroblasts, with the differentiation factor being a chondrocyte differentiation factor, preferably wherein said factor includes TGF-beta.

24. The method of claim 18, wherein the connective tissue is bone and differentiation comprises the differentiation into osteocytes and/or osteoblasts, with the differentiation factor being an osteogenic differentiation factor, preferably wherein said factor includes beta-glycerophosphate.

25. The method of claim 18, wherein connective tissue specific growth or differentiation factors comprise ascorbic acid or an ascorbic acid ester, preferably ascorbate-2-phosphate, or any pharmaceutically acceptable salt thereof.

26. The method of claim 18, wherein the fat tissue comprises stromal cells and adipocytes, such as white and/or brown adipocytes.

27. The method of claim 18, wherein the differentiated tissue graft is inserted into a tissue defect and/or wherein the differentiated tissue graft is inserted into an intervertebral disc compartment or into a cage designed for insertion into the intervertebral disc compartment for spinal fusion, and further preferably wherein the insertion is fixed by a tissue sealant, preferably a fibrin glue.

28. The method of claim 18 further defined as an ex vivo method for preparing a donor, preferably fat, tissue into a differentiated graft suitable for connective tissue repair, comprising contacting the donor, preferably fat, tissue with one or more connective tissue specific growth or differentiation factors, thereby initiating differentiation of the tissue graft, wherein said contacting is for a time period of between 1 hour to 6 weeks at a temperature of between 30 to 40° C., 0.01% to 10% (w/v) CO.sub.2 and between 70% to 98% humidity, wherein the connective tissue specific growth or differentiation factors is not a nucleic acid or consist of proteins, peptides and small molecules with a size of at most 10 kDa.

29. The method of claim 18 further comprising contacting the tissue with a tissue sealant, preferably after treatment with said connective tissue specific growth or differentiation factors.

30. A connective tissue specific growth or differentiation factor for use in a method of claim 18.

31. A kit suitable for performing a method of claim 27 comprising a connective tissue specific growth or differentiation factor and a tissue sealant, preferably a fibrin glue.

32. The kit of claim 31 further comprising a cartilage or bone tissue label or marker.

33. Use of the kit of claim 31 comprising the insertion is fixed by the tissue sealant.

Description

FIGURES

[0071] FIG. 1: Smooth surface transformation of chondrogenic transdifferentiated fat graft (a) compared to corresponding control (b) showing irregular, uneven surface formation

[0072] FIG. 2: Alcian Blue Glycosaminoglycan staining of a chondrogenic transdifferentiated fat graft (a) and the corresponding control (d); Bismarckbrown staining of fat graft (b) and control (e); Safranin O staining of fat graft (c) and control (f).

[0073] FIG. 3: Glycosaminoglycan content of a chondrogenic transdifferentiated fat graft.

[0074] FIG. 4: Mineralisation of an osteogenic transdifferentiated fat graft indicated by positive von Kossa staining (a) and Alizarin red staining (b). The corresponding controls (d,e) show no signs of mineralization. Azan staining demonstrates an increase in collagenous tissue in the transdifferentiated fat graft (c) compared to the undifferentiated control (f).

[0075] FIG. 5: Quantification of mineralisation in 5 μm sections of an osteogenic transdifferentiated graft

[0076] FIG. 6: Evaluation of angiogenesis and tissue integration of an osteogenic transdifferentiated graft using the HET-CAM angiogenesis assay. The graft demonstrates good tissue integration and is connected to the vessel system of the recipient after 5 days in vivo (a,b). Numerous blood vessels are visible within the graft (c; arrows). Osteogenic differentiation is maintained as demonstrated by positive von Kossa staining (d).

[0077] FIG. 7: Hyalinous cartilage of the knee joint 14 days after implantation of a chondrogenic transdifferentiated fat tissue graft. The graft is well integrated into the recipient tissue.

[0078] FIG. 8: Isolated, neurogenic differentiated mesenchymal stem cells exhibit typical neuron and axon formation (FIG. 8a, arrows). Axon and neuron formation is not observed in the control group (FIG. 8b). The neurogenic transdiffereniated fat graft demonstrates positive Nissl body staining (FIG. 8c, arrows), which is not observed in the control group (8d).

[0079] FIG. 9: a) Control fat tissue demonstrating the typical adipose tissue phenotype. b) Neurogenic transdifferentiated fat pad after 6 weeks: The fat vacuoles have been replaced by neurogenic tissue indicating positive cresylviolett staining. c) The zonal differentiation of peripheral nervous tissue, containing perineurium (black arrow) and epineurium (grey arrows) as well as concomitant blood vessels (yellow arrows) can be observed. d) Nissl bodies within the perineurium are visible in the neurogenic transdifferentiated sample (arrows).

[0080] FIG. 10: a) Differentiation of isolated mesenchymal stem cells towards a tenocytic phenotype two weeks after initiation of differentiation. b) unaltered phenotype of the control group. c) Islands of tenocytic differentiated cells showing circular orientation were present in the transdifferentiated fat pads, but not in the control tissues (d).

[0081] FIG. 11: Myogenic differentiation: a) Early differentiation towards oriented myocytes; b) no differentiation in the control group. c) Fat vacuoles were partially replaced by muscle tissue demonstrating longitudinal orientation and positive Goldner staining; d) muscle tissue formation was absent in the control sample.

EXAMPLES

Example 1

Transdifferentiation of Fat Tissue into a Hyaline Cartilage Graft

Fat Graft Preparation:

[0082] A small fat biopsy obtained during spinal decompression surgery was placed in a sterile container for transportation to the tissue culture laboratory. The sample was washed in sterile saline solution to remove contaminating erythrocytes. After passing the contamination check, the sample was divided into two parts. Part A (Transdifferentiation sample) was incubated in a commercially available chondrogenic differentiation medium (Promocell, Heidelberg/Germany) intended to use for mesenchymal stem cell differentiation. To obtain mesenchymal stem cell differentiation, cells are usually placed in aggregate or pellet cultures in a defined medium containing dexamethasone, ascorbate-2-phosphate, insulin, selenious acid, transferrin, sodium pyruvate and transforming growth factor β (TGF-β).sup.15. Part B (control) was incubated in a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 pg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity for 2-3 weeks. Medium was exchanged twice a week.

Histological Evaluation:

[0083] At the end of the incubation period, samples were fixed in 4% formaldehyde, washed in phosphate buffered saline and drained in ethanol in ascending concentrations. Tissues were embedded in paraplast and 5 μm sections were prepared. Chondrogenic differentiation was evaluated via Alcian Blue, Bismarck Brown and Safranine O staining.

Evaluation of Glycosaminoglycan Syntheses:

[0084] After 2 weeks of transdifferentiation, samples were digested over night in 1 mg/ml Proteinase K dissolved in 50 mM Tris containing 1 mM EDTA. Glycosaminoglcan content was measured using the Dimethyl-Methylenblue assay and absorbance was read at 525 nm. Shark chondroitin sulphate was used for generation of the standard curve.

Morphological Results:

[0085] After 3 weeks of transdifferentiation in vitro, the fat tissue showed a compact, spherical morphology with smooth surface remodelling (FIG. 1).

Histological Results:

[0086] Histological staining for chondrogenic differentiation was positive in the transdifferentiated fat tissue: Glycosaminoglycan synthesis could be detected via Alcian Blue staining. Proteoglycans were further visualized via positive Safranine O staining, and Positive Bismarck-Brown staining indicated the presence of an extracellular matrix typical for cartilaginous tissue (FIG. 2).

Evaluation of Glycosaminoglycan Syntheses:

[0087] Two weeks after inition of the transdifferentiation process, transdifferentiated fat grafts contained 16.56 μg glycosaminoglycans/mg tissue while the controls only showed an average glycosaminoglycan content of 1.92 μg/mg (p<0.0001; FIG. 3).

Example 2

Transdifferentiation of Fat Tissue into a Bone Graft

Fat Graft Preparation:

[0088] A small fat biopsy obtained during spinal decompression surgery was placed in a sterile container for transportation to the tissue culture laboratory. The sample was washed in sterile saline solution to remove contaminating erythrocytes. After passing the contamination check, the sample was divided into two parts. Part A (Transdifferentiation sample) was subjected to repeated mechanical stimulation followed by incubation in osteogenic differentiation medium. Osteogenic differentiation medium consists of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, 0.05 mg/ml ascorbic acid, 2 mM L-glutamine, 1 μM dexamethasone, 10 mM Na-β-glycerolphosphate and 1 μg/ml leptin. Part B (control) was incubated in a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 μg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 95% humidity for 3 weeks. Medium was exchanged twice a week.

Histological Evaluation:

[0089] At the end of the incubation period, samples were fixed in 4% formaldehyde, washed in phosphate buffered saline and drained in ethanol in ascending concentrations. Tissues were embedded in paraplast and 5 μm sections were prepared. Samples were stained with Azan, von Kossa and Alizarin Red.

Histological Results:

[0090] The transdifferentiated fat graft shows an increase in collagen content and signs of mineralization as indicated by positive von Kossa and Alizarin red staining (FIG. 4).

Quantification of Mineralization:

[0091] The degree of mineralization was quantified from 5 μm sections by determining the optical density (OD) of alizarin red staining after 3 weeks of osteogenic transdifferention.

Alizarin Red Staining Results:

[0092] Average OD of the osteogenic transdifferentiated graft was 0.25 per 5 μm section. OD of the corresponding control section was 0.12 (p<0.005; FIG. 5) Evaluation of angiogenesis and tissue integration:

[0093] Angiogenesis and tissue integration were evaluated using the HET-CAM (Hen Egg Test—Chorionallantoic Membrane) assay. The osteogenic transdifferentiated grafts were heterotopically implanted onto the exposed chorionallantoic membrane of fertilized, specific pathogen free chicken eggs. 5 days after implantation, the graft bearing area of the CAM was excised and processed for histological analysis.

HET-CAM Testing Results:

[0094] The implant was well integrated and connected to the recipient's vascular system after 5 days in vivo (FIG. 6a,b). Numerous small blood vessels were visible within the graft (FIG. 6c; arrows). Despite the deprivation of differentiation factors, osteogenic differentiation was maintained (FIG. 6d).

Example 3

Treatment of Cartilage Lesions

Graft Harvest and Preparation:

[0095] A subcutaneous fat biopsy is harvested under local anaesthesia. This can be done in an outpatient setting approximately 14 days prior to the planned surgical procedure. The fat tissue is aseptically placed in a sterile container containing tissue culture medium and e.g. treated as described above, i.e. the graft is subjected to chondrogenic transdifferentiation for 2 weeks at 37° C., 5% CO.sub.2 and 90% humidity. On the day of the planned procedure, the graft is sent to the operating room. An in vitro transdifferentiated cartilage graft implant is shown in FIG. 7.

Defect Preparation:

[0096] A mini-arthrotomy is performed and the defect is carefully debrided. Using a stencil (e.g. sterile tin foil), an exact mould of the defect is fabricated.

Graft Preparation and Implantation:

[0097] Using the stencil, the graft is fitted to the size of the defect. The graft is then implanted into the defect using fibrin glue. After 5 minutes of hardening time, excessive glue is removed with a scalpel and the joint is flexed and extended completely for 10 times. Stability and position of the graft is inspected during joint movement. Subsequently, the wound is closed.

Post-Surgical Procedure:

[0098] The patients undergo partial weight bearing (10 kg) treatment of the joint for 14 days, afterwards progressive weight bearing depending on swelling. The graft is full weight bearing after about 8 weeks.

Example 4

Treatment of a Vertebral Bone Fracture

Graft Harvest and Preparation:

[0099] A subcutaneous fat biopsy is harvested under local anaesthesia. This can be done in an outpatient setting approximately 1-2 weeks prior to the planned surgical procedure. The fat is aseptically dissected into slices of 2 mm.sup.2 length of edge, placed in a sterile container containing tissue culture medium and e.g. treated as described above, i.e. the graft is subjected to osteogenic transdifferentiation for 1-2 weeks at 37° C., 5% CO.sub.2 and 90% humidity. On the day of the planned procedure, the graft is sent to the operating room.

Surgical Procure:

[0100] The patient is placed in a prone position on a radiolucent table. After determining the location of the incision under fluoroscopy, a stab incision is made. The access instrumentation is inserted and moved forward until pedicle contact is reached. After confirmation of proper trajectory, the instrument is advanced into the vertebral body. Access to the vertebral body can be obtained via guide wire or trocar. Vertebral height can be restored performing a balloon Kyphoplasty procedure if desired.

Graft Preparation and Implantation:

[0101] The graft is delivered in a sterile application device. The application device is connected to the access device. Graft and fibrin are injected simultaneously into the vertebral body under fluoroscopic guidance. After having inserted the desired amount of graft in the vertebral body, the access instruments are removed and the wound is closed.

Post-Surgical Procedure:

[0102] Mobilisation can be started o the day of the procedure. Bracing is recommended until the absence of pain, analgesics should be prescribed as adequate. An exercise program focusing on lumbar stabilisation should be started as soon as permitted by the pain situation.

Example 5

Initiation of Neurogenic Transdifferentiation

Proof of Concept:

[0103] Mesenchymal stem cells were isolated by collagenase digestion from a fat tissue biopsy. Cells were expanded in monolayer culture. After a sufficient amount cells was obtained, cells were plated at a density of 3×10.sup.4 cells into two wells of a 48 well plate. Neurogenic differentiation was initiated in one well by addition of a commercially available neurogenic differentiation medium. The remaining cells were cultivated in control medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 μg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity. Medium was exchanged twice a week. After 3 days, a formation of dendrites and axons typical for neuron-like cells was observed (FIG. 8a). Cells cultured in control medium maintained their polygonal shape typical for mesenchymal stem cells (FIG. 8b).

Fat Graft Preparation:

[0104] A small fat biopsy was placed in a sterile container for transportation to the tissue culture laboratory. The sample was washed in sterile saline solution to remove contaminating erythrocytes. After passing the contamination check, the sample was divided into two parts. Part A (Transdifferentiation sample) was incubated in a commercially available neurogenic differentiation medium (Promocell, Heidelberg/Germany) intended to use for mesenchymal stem cell differentiation. Part B (control) was incubated in a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 pg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity for 6 weeks. Medium was exchanged twice a week.

Histological Evaluation:

[0105] At the end of the incubation period, samples were fixed in 4% formaldehyde, washed in phosphate buffered saline and drained in ethanol in ascending concentrations. Tissues were embedded in paraplast and 5 μm sections were prepared. Neurogenic differentiation was evaluated via histochemical stain of Nissl Bodies using Cresyl violet.

Result:

[0106] No morphological changes were observed in the control tissue (FIG. 9a). The neurogenic transdifferentiated graft demonstrated positive Cresyl violet staining, indicated by the presence of black-violet Nissl bodies within thy cytoplasm (FIG. 8c, arrows). In the neurogenic transdifferentiated sample, the fat vacuoles were gradually replaced by neurogenic tissue containing round cells with large pericaryons demonstrating positive Cresyl violet staining typical for neural cells (FIG. 9b). The zonal differentiation of peripheral nervous tissue, containing a clearly distinguishable peri- and epineurium surrounding the neural cells as well as concomitant blood vessels, could be observed in the transdifferentiated samples (FIG. 9c). The formation of Nissl bodies was not observed in the control sample (FIG. 8d).

Example 6

Induction of Tenogenic Differentiation

Initial Proof of Concept:

[0107] As an initial evaluation of the tenogenic differentiation medium, mesenchymal stem cells were isolated by collagenase digestion from a fat tissue biopsy. Cells were expanded in monolayer culture. After a sufficient amount cells was obtained, cells were plated at a density of 5×10.sup.4 cells into two wells of a 48 well plate. Tenogenic differentiation was initiated in one well by addition of a tenogenic differentiation medium consisting of DMEM-F12 supplemented with 1% FCS and 10 ng/ml BMP-12. The remaining cells were cultivated in control medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 μg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity. Medium was exchanged twice a week. Differentiation towards spindle shaped tenocytes was visible in the differentiation group after two weeks (FIG. 10a). No morphological changes were observed in the control group (FIG. 10b).

Fat Graft Preparation:

[0108] A small fat biopsy was placed in a sterile container for transportation to the tissue culture laboratory. The sample was washed in sterile saline solution to remove contaminating erythrocytes. After passing the contamination check, the sample was divided into two parts. Part A (Transdifferentiation sample) was incubated in a tenogenic differentiation medium. Part B (control) was incubated in a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 μg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity for 6 weeks. Medium was exchanged twice a week.

Histological Evaluation:

[0109] At the end of the incubation period, samples were fixed in 4% formaldehyde, washed in phosphate buffered saline and drained in ethanol in ascending concentrations. Tissues were embedded in paraplast and 5 μm sections were prepared. Tenogenic differentiation was evaluated using H/E staining.

Result:

[0110] Islands of tenocytic differentiated tissue showing circular orientation were present in the transdifferentiated fat pads (FIG. 10c), but not in the control group (d).

Example 7

Induction of Myogenic Differentiation

Initial Proof of Concept:

[0111] As an initial proof of concept, mesenchymal stem cells were isolated by collagenase digestion from a fat tissue biopsy. Cells were expanded in monolayer culture. After a sufficient amount cells was obtained, cells were plated at a density of 5×10.sup.4 cells into two wells of a 48 well plate. Myogenic differentiation was initiated in one well by addition of a commercially available myogenic differentiation medium. The remaining cells were cultivated in control medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 μg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity. Medium was exchanged twice a week. Differentiation towards oriented myocytes were visible after two weeks in the differentiation group (FIG. 11a), but not in the control group (FIG. 11b).

Fat Graft Preparation:

[0112] A small fat biopsy was placed in a sterile container for transportation to the tissue culture laboratory. The sample was washed in sterile saline solution to remove contaminating erythrocytes. After passing the contamination check, the sample was divided into two parts. Part A (Transdifferentiation sample) was incubated in a myogenic differentiation medium. Part B (control) was incubated in a 1:1 mixture of Dulbecco's modified Eagle's Medium and Ham's nutrient mix F12 supplemented with 10% Fetal Calf Serum and 2 mM L-glutamine. To prevent bacterial contamination, 50 μg/ml Gentamycin was added to both culture media. Incubation took place at 37° C., 5% CO.sub.2 and 90% humidity for 6 weeks. Medium was exchanged twice a week.

Histological Evaluation:

[0113] At the end of the incubation period, samples were fixed in 4% formaldehyde, washed in phosphate buffered saline and drained in ethanol in ascending concentrations. Tissues were embedded in paraplast and 5 μm sections were prepared. Myogenic differentiation was evaluated using Masson Goldner staining.

Result:

[0114] After 6 weeks of differentiation, fat vacuoles were partially replaced by muscle tissue demonstrating longitudinal orientation and positive Goldner staining (FIG. 11c). Muscle tissue formation was absent in the control sample (FIG. 11d).

REFERENCES

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