PRODUCTS FOR THERAPY OF A MUSCULOSKELETAL CONDITION AND METHODS FOR THEIR PRODUCTION

Abstract

A method for obtaining a fraction of a fetal cell culture supernatant, including the steps of obtaining a cell-containing sample of tissue (such as cartilage) or (cord-)blood or bone marrow from a non-human mammalian fetus, culturing the sample in a liquid cell culture medium, thereby obtaining a cell culture with a liquid supernatant, and isolating a fraction from the supernatant. Furthermore, a fraction obtainable by this method is provided. A pharmaceutical composition including this fraction is also provided, preferably for use in therapy, such as for use in a prevention or treatment of osteoarthritis, arthritis, tendinitis, tendinopathy, cartilage injury, tendon injury, rheumatoid arthritis, discospondylitis, meniscus injury, desmitis, desmopathy, intervertebral disc injuries, degenerative disease of intervertebral discs, reperfusion injury, wounds or inflammatory disease.

Claims

1. A method for obtaining a fraction of a fetal cell culture supernatant, comprising the steps of: obtaining a cell-containing sample of tissue from a non-human mammalian fetus; culturing the sample in a liquid cell culture medium, thereby obtaining a cell culture with a liquid supernatant; and isolating a fraction from the supernatant.

2. The method of claim 1, wherein the fetus is within the first two trimesters of gestation, preferably within the first half of gestation.

3. The method of claim 1, wherein the sample comprises chondrocytes, chondroblasts, chondroprogenitor cells, tenocytes, tenoblasts, tendon progenitor cells, fibrocytes, fibroblasts, fibrochondrocytes, fibrochondroblasts, synoviocytes, synovioblasts, osteocytes, osteoblasts, osteoclasts, hepatocytes, monocyte, macrophage, mesenchymal stem cells, mesenchymal progenitor cells and/or interzone cells.

4. The method of claim 1, wherein the tissue is selected from cartilage, tendon, ligament, bone, bone marrow and blood, in particular cord-blood.

5. The method of claim 1, wherein the tissue is articular cartilage.

6. The method of claim 1, wherein the culturing step comprises injuring, preferably chemically or mechanically injuring, the cells.

7. The method of claim 1, wherein the fraction comprises proteins, lipids, metabolites, extracellular vesicles and/or RNA, in particular miRNA.

8. The method of claim 1, wherein the liquid cell culture medium is a serum-free cell culture medium, a protein-free cell culture medium or a chemically defined cell culture medium.

9. The method of claim 1, wherein the isolating step comprises a preservation step, preferably a freezing or drying step, especially lyophilisation.

10. A fraction, obtainable by the method of claim 1.

11. A cell supernatant fraction from non-human fetal cells, preferably wherein the cells are defined as in claim 3, wherein the fraction comprises proteins, lipids, metabolites, extracellular vesicles and/or RNA, in particular miRNA.

12. The fraction of claim 10, wherein the fraction is dry, preferably lyophilised.

13. A pharmaceutical composition, comprising the fraction of claim 10.

14. The pharmaceutical composition of claim 13 for use in therapy.

15. The pharmaceutical composition of claim 14 for use in a prevention or treatment of osteoarthritis, arthritis, tendinitis, tendinopathy, cartilage injury, tendon injury, rheumatoid arthritis, discospondylitis, meniscus injury, desmitis, desmopathy, intervertebral disc injuries, degenerative disease of intervertebral discs, reperfusion injury, wounds or inflammatory disease.

Description

[0040] The present invention is further illustrated by the following figures and examples, without being limited thereto.

[0041] FIG. 1: Diagram of the distal femur with the medial and lateral trochlea ridge and the medial and lateral condyle identified as landmarks. Cartilage lesion location and size is indicated in blue in in adult and in green in fetal sheep. The lesion was centred 15 mm (adult) resp. 3 mm (fetus) distant from the medial trochlear-condylar junction on a line, which virtually extended the medial trochlear ridge.

[0042] FIG. 2: Healing of adult (5 months post injury) and fetal (28 days post injury) cartilage defects: Haematoxylin and Eosin (H&E) stained sections. Adult (A and B): no repair except in areas of micro fracture, tissue mixture of fibrocartilage with partial hyalinisation, no integration with surrounding cartilage (see insert). Fetal (C and D): defect filled to 80-90% with differentiating hyaline cartilage and the superficial 10-20% with repair tissue with progressing hyalinisation, good integration with surrounding cartilage, processing artefact (*).

[0043] FIG. 3: Morphology and extracellular matrix composition of healthy and injured adult and fetal cartilage: Haematoxylin and Eosin (H&E), Safranin 0, and collagen type 2 (Col2) staining. Arrows mark the transition from healthy cartilage to the lesion site; asterisks indicate sites of microfracture penetrating the subchondral bone plate in D, E, F. Fibrous tissue partly covering the surface of the lesion (arrows in J, K, L) was found in fetal injured condyles.

[0044] FIG. 4: Distribution of proliferation marker Ki67 (A, D, G, J) and matrix metalloproteinases (MMPs, B, C, E, F, H, I, K, L) in healthy and injured adult and fetal cartilage. Inserts in fetal injured condyles indicate transition from healthy cartilage to the lesion site. Scale bar in adult samples=100 μm, scale bar in fetal samples=200 μm and scale bar in inserts=20 μm.

[0045] FIG. 5: The Venn diagram gives an overview of genes implicated by a range of differential screening tests (n=3 biological replicates/group, 2 technical replicates/biological replicate). Specifically, we examine the average Total Response to injury (magenta), the Fetal Response (blue), the Adult Response (red), and significant Response Differences (green). Separately assessing significances for each of the four tests improves sensitivity and specificity by avoiding an accumulation of thresholding artefacts. Comparing cartilage on day 3 after injury with matching control tissues yielded 385 genes implicated in the total response incorporating the average evidence from all sample types (7+9+0+35+261+2+56+15). Analogously, 74 genes were implicated in the fetal response (9+3+35+8+2+2+15), of which 13 were newly identified (3+8+2+0). Conversely, 445 genes were implicated in the adult response (45+261+64+2+56+2+15), of which 111 were newly identified (45+64+2+0). Response differences are shown by 356 genes with an injury response in fetal samples that was significantly different to that in adult samples (3+0+45+35+261+2+8+2), including 3 previously not implicated genes, 8 genes so far only implicated in the fetal response, 45 genes so far only implicated in the adult response, 2 genes already implicated in both, 35 genes already implicated in the total and the fetal responses, 261 genes already implicated in the total and the adult responses, and 2 genes implicated in all, reflecting that response strength and direction of expression change can be affected differently in the injury response of fetal and adult samples.

[0046] FIG. 6: Sample correlation structure. This figure compares pairwise sample correlations, with Spearman rank correlation coefficients given in the boxes below the diagonal, which are visualized above the diagonal, with darker and narrower ellipses indicating higher correlations. Rows and columns show sample labels, where A/F=adult/fetal, c/i=control/injured, and #.# show biological and technical replicate numbers (n=3 biological replicates/group, 2 technical replicates/biological replicate).

[0047] FIG. 7: Diagram of the superficial digital flexor tendon of the gastrocnemius tendon bundle with the calcaneus identified as landmark. Tendon lesion location and size is indicated in blue in in adult and in green in fetal sheep. The distal lesion was placed 9 mm (adult) resp. 4 mm (fetus) proximal to the calcaneus, the proximal lesion at a distance of 9 mm (adult) resp. 4 mm (fetus) proximal to the distal lesion.

[0048] FIG. 8: Proinflammatory factors S100A8, S100A9 and S100A12 were clearly upregulated in the supernatant of adult injured tendon whereas they remained essentially unchanged in the supernatant of fetal tendon upon injury (ctrl: control, inj: injured).

[0049] FIG. 9: Of the growth factors SERPINH1, TGFBR3 and EIF3I, the factor SERPIN1 was clearly upregulated in the supernatant of fetal tendon. (ctrl: control, inj: injured).

[0050] FIG. 10: Of the extracellular matrix components collagen 1 A1 (Col1A1, versican (VCAN) and decorin (DCN), Col1A1 was clearly upregulated in the supernatant of fetal tendon, VCAN was upregulated in the supernatant of fetal tendon and even increased upon injury, whereas DCN was lower in the supernatant of fetal tendon. (ctrl: control, inj: injured).

[0051] FIG. 11: The extracellular matrix components biglycan (BGN) and tenascin-C (TNC) exhibited a mixed pattern. (ctrl: control, inj: injured).

[0052] FIG. 12: From the abundance of inflammatory factors TIMP1, ADAM12, MMP2 and MMP3 in supernatants, several trends become apparent. (ctrl: control, inj: injured).

[0053] FIG. 13: Gene expression in inflamed chondrocytes following treatment with supernatants from adult mesenchymal stem cells (MSCs; “aMSC SN”), fetal MSCs (“fMSCs”) or fetal chondrocytes (“fChondro”). Uninjured chondrocytes (“control healthy”) and injured but not treated chondrocytes (“control inflamed”) served as controls. The individual panels show gene expression levels of collagen type II alpha 1 (Col2), aggrecan and telomerase reverse transcriptase (TERT). The expression of all three genes was reduced in injured compared to healthy chondrocytes. Treatment with supernatants from fetal cells increased the expression close to or even above normal levels.

[0054] FIG. 14: Gene expression in inflamed tenocytes following treatment with supernatants from adult MSCs (“aMSC SN”), fetal MSCs (“fMSC SN”) or fetal tenocytes (“fTeno SN”). Uninjured tenocytes (“control healthy”) and injured but not treated tenocytes (“control inf”) served as controls. The individual panels show gene expression levels of Decorin and Tenascin C. The expression of both genes was reduced in injured compared to healthy tenocytes. Treatment with supernatants from fetal cells increased the expression levels.

[0055] FIG. 15: Effects of supernatants from fetal chondrocytes or fetal MSCs on the senescence of inflamed adult chondrocytes as determined using a beta galactosidase senescence assay. Following 48 h treatment with supernatants from injured fetal cells, senescence of adult chondrocytes decreased.

[0056] FIG. 16: Effects of the secretome of fetal and adult MSCs on injured tenocytes. Inflamed ovine tenocytes were either treated with supernatants from adult MSCs (“aMSC SN”) or fetal MSCs (“fMSC SN”). Uninjured tenocytes (“Control Healthy”) and injured but not treated tenocytes (“Control Inflamed”) served as controls. Exposure to the secretome of MSCs led to decreased expression levels of inflammatory genes IL6 and MMP1 compared to untreated control. The expression of tendon extracellular matrix (ECM) gene collagen III (Col3a) was decreased in response to inflammation (control inflamed) but returned to almost normal levels (healthy control) following treatment with the secretome of MSCs.

[0057] FIG. 17: Wound healing assay (chondrocyte migration). Inflamed ovine chondrocytes were scratched and subsequently treated with supernatants from either fetal MSCs (fMSCs) or fetal chondrocytes (fChondro). Uninjured chondrocytes (control healthy) and injured but not treated chondrocytes (control inflamed) served as controls. Supernatants from fetal cells significantly improved wound healing with supernatants of fetal MSCs showing an even stronger effect than supernatants of fetal chondrocytes.

[0058] FIG. 18: Wound healing assay (tenocyte migration). Injured ovine tenocytes were scratched and subsequently treated with the supernatant of fetal MSCs (fMSCs) or fetal tenocytes (fTeno). Uninjured tenocytes (control healthy) and injured but not treated tenocytes (control inflamed) served as controls. It was found that the supernatants of fetal cells significantly improved wound healing with supernatants of fetal MSCs showing an even stronger effect than supernatants of fetal tenocytes.

EXAMPLE 1—FETAL ARTICULAR CARTILAGE REGENERATION VERSUS ADULT FIBROCARTILAGINOUS REPAIR

[0059] This study aimed to 1) establish a standardized cartilage lesion model allowing comparison of cartilage healing in adult and fetal sheep (ovis aries); 2) establish the feasibility, repeatability and relevance of proteomic analysis of minute fetal and adult cartilage samples; and 3) compare fetal and adult protein regulation in response to cartilage injury.

[0060] The proteomic analysis of the differential response of fetal and adult cartilage to injury will have a major impact on our understanding of cartilage biology and of the molecular mechanisms underlying OA and cartilage regeneration, could help identify and target factors that are crucial to promote a regenerative response and allows the development of disease-modifying treatment strategies to induce cartilage regeneration in adult mammals. A major challenge to the proteomic characterization of the complex protein mixture in cartilage extract is the wide dynamic range of protein abundance, making the detection of low-abundant proteins very difficult (Stenberg et al., 2013; Wilson and Bateman, 2008). However, while technically demanding, studying the functional proteome gives a more comprehensive picture of disease aetiopathogenesis than gene expression analysis alone, as it can capture post-transcriptional regulation of protein expression levels as well as post-translational modifications (Ritter et al., 2013b).

Materials and Methods

Animal Model

[0061] Standardized cartilage lesions in musculoskeletally mature (2-5 years, body weight 95±12 kg), female, healthy, non-gravid Merino-cross ewes (ovis aries) without orthopaedic disease and fetal lambs (gestational day 80, term=150 days) were created with approval of the national (Federal Ministry of Science, BMWFW) and institutional animal welfare committee. For the fetal subjects, only twin pregnancies were included to provide a twin lamb as uninjured control on a background of low genetic variation to allow differentiation between protein secretion of regular fetal development and fetal response to cartilage injury. Fetal hind limbs were exteriorized from the uterus via a standard ventral-midline laparotomy followed by an uterotomy over a randomly chosen uterine horn.

[0062] For the purpose of lesion induction, a minimally invasive medial parapatellar arthrotomy (Orth and Madry, 2013) was performed in both knees in adult and fetal sheep. A bilateral full-thickness cartilage lesion with a diameter of 7 mm (adult sheep) resp. 1 mm (fetal lamb) was created in the medial femoral condyle region (FIG. 1) using a custom-made precision surgical instrument (trocar with sleeve) for adult sheep and for fetal lambs a Versi-handle (Ellis Instruments, Madison, N.J., USA) with adjustable depth control, which was set at a lesion depth of 1 mm.

[0063] To ensure that differences in the healing response between fetal and adult articular cartilage were not caused by differences in blood supply, which in fetal sheep is starting at an articular cartilage depth of 400 μm (Namba et al., 1998), we created 3 microfractures through the subchondral bone plate in adult cartilage defects to gain access to the bone marrow vasculature (Dorotka et al., 2005a). The arthrotomy was closed in 1 dermal layer using 6-0 monofilament nylon (Monosof, Covidien, Minneapolis, USA) in fetal lambs and in 3 layers using 2-0 monofilament absorbable polyester (Biosyn, Covidien) for the joint capsule and subcutaneous tissue and 2-0 monofilament nylon for the skin in adult sheep. Adult animals were allowed full weight-bearing immediately after surgery. All adult sheep were treated with antibiotics peri-operatively and received pain management. Pain management was provided with morphine to avoid anti-inflammatory drugs, which would influence the result of the study.

Pilot Study

[0064] In a pilot study, as a proof of principle of fetal regeneration versus adult fibrocartilaginous repair, 3 adult and 3 fetal injured sheep were euthanized at 5 months (adult) respectively 28 days (fetal) postoperatively for macroscopic and histologic evaluation of the defect repair. At the time of euthanasia, digital photographs were taken and the articular cartilage surface and the cartilage defect healing response was macroscopically evaluated using the Osteoarthritis Research Society International (OARSI) recommendations for macroscopic scoring of cartilage damage in sheep, taking into account surface roughening, fibrillation, fissures as well as presence and size of osteophytes and erosions (Little et al., 2010). For fetal sheep the OARSI macroscopic score was size-adjusted by multiplying the adult lesion size cut-off values with 3.4/36.4, the ratio of the reported tibia length of fetal versus adult sheep (Mufti et al., 2000; Salami et al., 2011).

Tissue Harvest

[0065] At day 3 after injury, samples were collected from 3 biological replicates per comparison group (adult injured, fetal injured, fetal uninjured twin control). In adult sheep, samples were also harvested from uninjured controls (n=3).

[0066] After macroscopic scoring, the medial femoral condyles were surgically excised and left and right knees were randomly assigned to mass spectrometry and histology. For mass spectrometry the (cartilage)-tissue remnants contained in the defect area and the cartilage rim surrounding the lesion (3 mm width: adults, 1 mm width in fetal sheep) were excised.

Histology and Immunohistochemistry

[0067] For histological analysis the femoral condyles were fixed in 4% buffered formalin. Condyles from adult sheep were decalcified in 8% neutral EDTA. After embedding in paraffin, 4 μm-thick sections were cut and mounted on APES-glutaraldehyde-coated slides (3-aminopropyltriethoxysilane; Sigma-Aldrich, Vienna, Austria). Consecutive sections were stained with Haematoxylin and Eosin (H&E), and Safranin 0.

[0068] For immunohistochemistry, sections were deparaffinised, rehydrated and endogenous peroxidase was blocked with 0.6% hydrogen peroxide in methanol (15 min at room temperature). Nonspecific binding of antibodies was prevented by incubation with 1.5% normal goat serum (Dako Cytomation, Glostrup, Denmark) in phosphate-buffered saline (PBS; 30 min at room temperature). Primary antibodies (anti-collagen type 2, anti-Ki67, anti-MMP9, and anti-MMP13; table 2) were incubated overnight at 4° C. An appropriate BrightVision Peroxidase system (Immunologic, Duiven, The Netherlands) was used and peroxidase activities were localized with diaminobenzidine (DAB; Sigma-Aldrich). Cell nuclei were counterstained with Mayer's haematoxylin.

[0069] Tissue from adult sheep mammary glands and human placenta served as positive controls. For negative controls, the primary antibody was omitted.

Mass Spectrometry

[0070] The cartilage rim and the tissue remnants obtained from the lesion site were cultivated in serum-free RPMI medium (Gibco, Life Technologies, Austria) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (ATCC, LGC Standards GmbH, Germany) for 6 h under standard cell culture conditions (37° C. and 5% CO2). Afterwards, medium was sterile-filtered through a 0.2 μm filter and precipitated overnight with ice-cold ethanol at −20° C. After precipitation, proteins were dissolved in sample buffer (7.5 M urea, 1.5 M thiourea, 4% CHAPS, 0.05% SDS, 100 mM dithiothreitol (DDT)) and protein concentrations were determined using Bradford assay (Bio-Rad-Laboratories, Munich, Germany).

[0071] Twenty microgram of each protein sample was used for a filter-aided digestion as described previously (Aukland, 1991; Aukland et al., 1997a; Aukland et al., 1997b; Bileck et al., 2014; Wiśniewski et al., 2009). Briefly, 3 kD molecular weight cut-off filters (Pall Austria Filter GmbH) were conditioned with MS grade water (Millipore GmbH). Protein samples were concentrated on the pre-washed filter by centrifugation at 15000 g for 15 min. After reduction with DTT (5 mg/ml dissolved in 8 M guanidinium hydrochloride in 50 mM ammonium bicarbonate buffer (ABC buffer), pH 8) and alkylation with iodoacetamide (10 mg/ml in 8 M guanidinium hydrochloride in 50 mM ABC buffer), samples were washed and 1 μg trypsin was added prior to incubation at 37° C. for 18 h. After enzymatic digestion, peptide samples were cleaned with C-18 spin columns (Pierce, Thermo Scientific, Germany), dried and stored at −20° C. until analysis.

[0072] For mass spectrometric analyses, dried samples were reconstituted in 5 μl 30% formic acid (FA) containing 10 fmol of four synthetic standard peptides each and diluted with 40 μl mobile phase A (H2O:ACN:FA=98:2:0.1). Ten microliter of the peptide solution were loaded onto a 2 cm×75 μm C18 Pepmap100 pre-column (Thermo Fisher Scientific, Austria) at a flow rate of 10 μl/min using mobile phase A. Afterwards, peptides were eluted from the pre-column to a 50 cm×75 μm Pepmap100 analytical column (Thermo Fisher Scientific, Austria) at a flow rate of 300 nl/min and separation was achieved using a gradient of 8% to 40% mobile phase B (ACN:H2O:FA=80:20:0.1) over 95 min. For mass spectrometric analyses, MS scans were performed in the range of m/z 400-1400 at a resolution of 70000 (at m/z=200). MS/MS scans of the 8 most abundant ions were achieved through HCD fragmentation at 30% normalized collision energy and analysed in the orbitrap at a resolution of 17500 (at m/z=200). All samples were analysed in duplicates.

Data Analysis and Statistics of Mass Spectrometry Experiments

[0073] Protein identification as well as label-free quantitative (LFQ) data analysis was performed using the open source software MaxQuant 1.3.0.5 including the Andromeda search engine (Cox and Mann, 2008). Protein identification was achieved searching against Ovis aries in the Uniprot Database (version 09/2014 with 26 864 entries) allowing a mass tolerance of 5 ppm for MS spectra and 20 ppm for MS/MS spectra as well as a maximum of 2 missed cleavages. In addition, carbamidomethylation on cysteins was included as fixed modification whereas methionine oxidation as well as N-terminal protein acetylation were included as variable modifications. Furthermore, search criteria included a minimum of two peptide identifications per protein, at least one of them unique, and the calculation based on q-values performed for both, peptide identification as well as protein identification, less than 0.01. Prior to statistical analyses, proteins were filtered for reversed sequences, contaminants and we required a minimum of three independent identifications per protein.

[0074] Missing values were replaced by a global s, set to the minimum intensity observed in the entire data set divided by 4. This sensitivity based pseudo-count reflects the prior belief of non-observed protein expression, maintaining a lower bound of a 4-fold change for differences to proteins not observed in one sample group, thus maintaining sensitivity, while improving specificity by mitigating the effects of random non-observations. The Spearman rank correlations between samples shown in FIG. 6 are not affected by this transform. For the visualization of the sample correlation structure in that figure, ellipses were plotted as (x, y)=(cos(θ+d/2), cos(θ−d/2)), where θ in [0,2π) and cos(d)=ρ, with ρ the Spearman rank correlation coefficient (Murdoch and Chow, 1996).

[0075] Protein expression profile analysis was performed in the statistical environment R (www.r-project.org). Differential expression contrasts were computed with Bioconductor libraries (www.bioconductor.org). Data were normalized by a mean log-shift, standardizing mean expression levels per sample. Linear models were fit separately for each protein, computing second-level contrasts for a direct test of differences between fetal and adult responses to injury. Conservative Benjamini-Yekutieli correction was used to adjust for multiple testing to give strong control of the false discovery rate (FDR). We call significant features for q-values <0.05. Linear models were adjusted for the nested correlation structure of technical and biological replicates (cf. FIG. 6). Significance was assessed by regularized t-tests. For these, group variances are shrunk by an Empirical Bayes procedure to mitigate the high uncertainty of variance estimates for the available sample sizes (Sun et al., 2009). The employed algorithms are implemented in the package limma (Smyth, 2005), which is available from Bioconductor.

Results

The Ovine Model Supports Complex Surgical Manipulations Required for the Investigation of Cartilage Regeneration

[0076] Ewes and fetal sheep tolerated the laparotomy, uterotomy and fetal manipulation well. No postoperative complications or abortions were encountered. Fetal sheep at 80 days of gestation (gd) had age-appropriate crown-anus lengths within the reported range of 10.1±1.3 cm (Mufti et al., 2000). The landmarks for standardized induction of medial femoral condylar cartilage lesions (FIG. 1) were easily identified and allowed placement of the lesion in the centre of the condyle.

Long-Term Evaluation Confirmed Fetal Regenerative Versus Adult Scarring Cartilage Repair

[0077] In the pilot study designed as a proof of principle of fetal regeneration at 28 days postoperatively versus adult scarring repair at 5 months post injury, the defect was macroscopically not detectable in fetal sheep resulting in an OARSI (Osteoarthritis Research Society International) macroscopic score (Little et al., 2010) of 0 for cartilage, osteophytes and synovium, while in adult sheep the defect was clearly evident and only partially filled with fibrocartilaginous tissue resulting in an OARSI macroscopic score of 5/16 for cartilage (surface roughening plus defect), 0/5 for osteophytes and 2/5 for synovium.

[0078] Histologically (FIG. 2), no defect repair and only minimal fibrocartilaginous regeneration adjacent to microfractures without integration with the surrounding cartilage was achieved in adult sheep 5 months postoperatively, whereas in fetal sheep, 28 days after surgery, the defect was filled with differentiating hyaline cartilage in about 80-90% of the repair tissue and 10-20% with progressing hyalinisation and full integration with the surrounding cartilage.

Injury- and Repair-Associated Macroscopic and Histologic Changes in Adult and Fetal Sheep 3 Days Post Injury

[0079] Upon harvest at 3 days postoperatively, the OARSI macroscopic score was 4/16 for cartilage due to the 7 mm (adults) resp. 1 mm (fetal) size defect in the medial femoral condyle and 0 for osteophytes (due to the short time since surgery, no OARSI score was assigned to the macroscopic appearance of the synovium). Within the first 3 days after injury, no histologically visible cartilage repair could be detected. Therefore, none of the established repair scoring systems could be applied. Thus, the description of the structural conditions was based on the evaluation criteria of the ICRS assessment including the cartilage surface and matrix, cell distribution, cell viability, and subchondral bone but without scores (Mainil-Varlet et al., 2003).

[0080] Adult control condyles showed healthy articular cartilage with a smooth surface, physiologic matrix composition, and typical distribution of chondrocytes (FIG. 3A, B). Col2 staining was homogeneous and distinct throughout the whole articular cartilage (FIG. 3C).

[0081] Creation of the cartilage lesion in adults resulted in matrix depletion at the site of injury as well as in the superficial zone (FIG. 3 D, E). Next to the cartilage lesion an acellular area of about 100 μm thickness was found with either empty lacunae or homogenous matrix lacking apparent lacunae. No cell clustering was observed. The microfractures penetrating the subchondral bone plate were visible (FIG. 3 D, E). One sample showed a focal accumulation of granulocytes in the bone marrow cavity below the cartilage lesion. In the immediate vicinity (˜10 μm) of the cartilage lesion, Col2 staining intensity was decreased (FIG. 3 F).

[0082] Similar to the adults, fetal uninjured control samples showed a smooth cartilage surface, homogenous matrix composition, and distinct Col2 staining throughout the whole condyles (FIG. 3 G-I).

[0083] Although matrix depletion was also detected around the cartilage lesions in the fetal samples it was less marked compared to the adults (FIG. 3 J, K). An almost acellular area of 50 μm surrounded the cartilage lesion. The lesion surface was partly covered with fibrous tissue originating either from cartilage canals or connective tissue flanking the articular surface. The pattern of the Col2 staining around the fetal cartilage lesions was similar to the adults with a 10 μm thick zone of decreased staining intensity (FIG. 3 L).

[0084] In adult control animals, no proliferating cells (demonstrated by expression of Ki67) were found in the articular cartilage or the subchondral bone (FIG. 4 A). Few Ki67-positive cells were detected in the bone marrow cavities. Chondrocytes in all cartilage zones expressed MMP9 and MMP13 with a stronger staining intensity for MMP9 (FIG. 4 B, C), however no MMP-positive osteocytes were observed.

[0085] In the injured adult cartilage samples also no Ki67 positive cells were observed (FIG. 4 D). However, in one sample, an accumulation of Ki67-positive cells was found in a microfracture gap, which was filled with bone marrow. Both, MMP9 and MMP13-expression was reduced within and adjacent to the cartilage lesions (FIG. 4 E, F) as compared to the intact cartilage of the respective sample.

[0086] In fetal healthy cartilage, evenly distributed Ki67-positive cells (FIG. 4 G) and MMP-expressing cells (FIG. 4 H, I) were detected throughout the whole cartilage. The staining pattern of MMP9 appeared identical to MMP13.

[0087] Although, in the fetal injured cartilage an almost cell free zone of 50 μm was found to surround the lesions, single Ki67-expressing cells as well as MMP-positive cells could still be detected in this area (FIG. 4 J-L). More MMP-expressing cells were located adjacent to the cell free zone as well as in the cartilage canals of the injured condyle.

The Ovine Model Supports Comprehensive Molecular Profiling by High Resolution Mass Spectrometry

[0088] Secretome analysis of control and injured (3 days postoperative) cartilage tissue samples derived from adult and fetal sheep, respectively, using high-resolution mass spectrometry (MS) enabled the identification of a total number of 2106 distinct proteins. Thereof, 445 proteins were found significantly regulated (q-value <0.05) in response to cartilage injury in adult animals, in contrast to 74 proteins in fetal animals (FIG. 5). Comparing protein baseline expression, 1288 proteins were found significantly differentially regulated between fetal and adult control animals. The injury response of fetal and adult sheep was significantly differently regulated in 356 proteins. A comparison of protein regulation in adult and fetal animals (FIG. 5) revealed differences concerning the following groups of proteins: (i) proteins associated with immune response and inflammation, (ii) proteins specific for cartilage tissue and cartilage development and (iii) proteins involved in cell growth and proliferation (table 1). Multiple well-known actors in inflammatory processes, such as S100A8, S100A9, S100A12 and Ccdc88A were found significantly up-regulated following injury in adult (q<0.001) but not in fetal animals (table 1). In contrast, several proteins with anti-inflammatory and growth-supporting effects, such as protein phosphatase, Mg2+/Mn2+ dependent 1A (Ppm1A) and cell division cycle 42 (Cdc42) showed a significant increase in response to injury in fetal sheep (q=0.005 and 0.006) compared to adults (table 1). Cartilage-specific proteins, such as aggrecan (Acan), cartilage oligomeric protein (Comp), chondroadherin (Chad) and proteoglycan-4 (Prg4) had a significantly higher baseline expression in adults (q<0.001) and showed little injury response in either age group with the exception of Prg4, which was significantly up-regulated in fetal injured sheep (q=0.01). Other proteins related to cell growth and proliferation, such as mitogen-activated protein kinase 3 (Mapk3/Erk1) and GA binding protein transcription factor alpha subunit (Gabpa), also displayed differences in abundance (q=0.02 and 0.04) as well as regulation between adult and fetal sheep (q=0.003 and 0.0001).

[0089] Our results demonstrate the biological relevance and reproducibility of our new ovine cartilage defect model and MS analysis (FIG. 6). Technical measurement reproducibility was excellent, with variation clearly lower than variation between biological replicates, indicating a high sensitivity of the proteomics profiling workflow (FIG. 6). The robustness of our new cartilage defect model is reflected in the variance across biological replicates being small in relation to the examined biological effects, whether injury versus control, or differences between adult and fetal samples (FIG. 6). For both adult and fetal samples, low variance across replicates indicates good reproducibility of our experimental setup, confirming that biologically meaningful signals can sensitively be obtained already from moderate sample size. Furthermore, it confirms good standardization of our articular cartilage defects between individuals of both the adult and fetal age group.

Discussion

[0090] The results illustrate the biological relevance, the technical feasibility and repeatability of the new ovine cartilage defect model (FIG. 1) and analytical approaches and confirm regeneration in fetal versus scarring repair in adult sheep (FIG. 2). Specific characteristics that make sheep particularly well-suited for OA, regenerative medicine and fetal regeneration research to obtain results of high clinical relevance are: 1) large size facilitating repeated sampling from individual animals and harvest of adequate sample sizes; 2) comparable bodyweight to humans; 3) similar mechanical exertion to humans (Bruns et al., 2000; Russo et al., 2015); 4) relatively long life expectancy (lifespan 8-12 years) allowing longitudinal analysis as well as evaluation of long-term efficacy and safety of treatments; 5) long gestational period (150 days) provides sufficient temporal resolution to translate findings obtained in sheep into human parameters (Jeanblanc et al., 2014); 6) extremely well characterized immune development analogous to humans; 7) bone marrow ontogeny and niche development closely paralleling humans.

[0091] Furthermore, for the sheep model, a quite acceptable annotation status and representative subsets of identified proteins are available on sources such as the NCBI (e.g. 30584 genes and 69889 proteins) allowing good applicability and translation of the results.

[0092] In this study we compared the adult and fetal response to cartilage injury 3 days after lesion induction as this time point is established to be within the time window of inflammation for both adult and fetal individuals, one of the injury responses hypothesized to crucially contribute to the difference between adult and fetal healing. For the fetal injury response, it is only known that cartilage regeneration occurs within 4 weeks, which is in stark contrast to the adult injury response with an inflammatory phase of 3-5 days, a proliferative phase of 3-6 weeks and a remodeling phase of up to one year duration resulting in a fibrocartilaginous scar. As the timeline of the fetal injury response is not yet established, choosing a later date would have made data interpretation and correlation of adult and fetal data much harder. Three days is within the peak period of the adult inflammatory response, allows for recruitment of monocytes/macrophages to the injury site and has been shown to be associated with signs of inflammation also in fetal injuries in other tissues.

[0093] The main factors identified within the secretome were extracellular matrix proteins, growth factors and inflammatory mediators such as cytokines and chemokines. Considering the key chondrocyte signalling pathways regulating processes of inflammation, cell proliferation, differentiation and matrix remodelling, which include the p38, Jnk and Erk Map kinases, the PI-3 kinase-Akt pathway, the Jak-Stat pathway, Rho GTPases and Wnt-β-catenin and Smad pathways (Beier and Loeser, 2010), the data provide an indication of differences in the inflammatory response to injury between adult and fetal cartilage and suggest the active production of anti-inflammatory and growth factors, such as Ppm1A and Cdc42 in the fetal environment.

[0094] Ppm1A is a bona fide phosphatase, which is involved in the regulation of many developmental processes such as skeletal and cardiovascular development. Through its role as phosphatase of many signalling molecules such as p38 kinase, Cdk2, phosphatidylinositol 3-kinase (PI3K), Axin and Smad, up-regulation of Ppm1A abolishes for example TGF-β-induced antiproliferative and transcriptional responses (Wang et al., 2014) as well as BMP signalling (Duan et al., 2006). Furthermore, Ppm1A by dephosphorylating IκB kinase-β and thus terminating TNFα-induced NF-κB activation, partakes in the regulation of inflammation, immune-response and apoptosis (Sun et al., 2009).

[0095] Cdc42 belongs to the family of Rho GTPases and controls a broad variety of signal transduction pathways regulating cell migration, polarization, adhesion proliferation, differentiation, and apoptosis in a variety of cell types (Sun et al., 2009). Cdc42 is required in successive steps of chondrogenesis by promoting mesenchymal condensation via the BMP2/Cdc42/Pak/p38/Smad signalling cascade and chondrogenic differentiation via the TGF-β/Cdc42/Pak/Akt/Sox9 signalling pathway (Wang et al., 2016). Another essential Cdc42 function relevant to the current study is its involvement in wound healing by attenuating MMP1 expression (Rohani et al., 2014) and regulating spatially distinct aspects of the cytoskeleton machinery, especially actin mobilization toward the wound (Benink and Bement, 2005) which, given the increase of actin-containing articular chondrocytes in response to cartilage injury, could also play a role in the healing of cartilage defects (Wang et al., 2000).

[0096] In contrast to the anti-inflammatory factors up-regulated in fetal sheep in response to injury, adult sheep displayed a significant increase of inflammatory mediators such as alarmins S100A8, S100A9, S100A12 and coiled-coil domain containing 88A (Ccdc88A). The alarmin 5100 proteins are markers of destructive processes such as those occurring in OA (Liu-Bryan and Terkeltaub, 2015; Nefla et al., 2016; van den Bosch et al., 2015). Accordingly, in OA articular S100A8 and S100A9 protein secretion is increased, recruiting immune cells to inflamed synovia, initiating the adaptive immune response, inducing canonical Wnt signalling and promoting cartilage matrix catabolism, osteophyte formation, angiogenesis and hypertrophic differentiation (Liu-Bryan and Terkeltaub, 2015; Nefla et al., 2016; van den Bosch et al., 2015). S100A8/A9 up-regulate markers characteristic for ECM degradation (MMPs 1, 3, 9, and 13, interleukin-6 (IL-6), IL-8) and down-regulate growth promotion markers (aggrecan and Col2) and thus have a destructive effect on chondrocytes, causing proteoglycan depletion and cartilage breakdown (Schelbergen et al., 2012). Also S100A12 is up-regulated in OA cartilage and has been shown to increase the production of MMP-13 and Vegf in OA chondrocytes via p38 Mapk and NF-κB pathways (Nakashima et al., 2012). Another relevant protein, which was significantly down-regulated upon injury in fetal sheep but significantly up-regulated in injured adult sheep is Ccdc88A. Ccdc88A is a multimodular signal transducer, which modulates growth factor signalling during diverse biological and disease processes including cell migration, chemotaxis, development, self-renewal, apoptosis and autophagy by integrating signals downstream of a variety of growth factors, such as Efg, Igf, Vegf, Insulin, Stat3, Pdgfr and trimeric G protein Gi (Dunkel et al., 2012; Ghosh et al., 2008). In addition, Ccdc88A, which is expressed at high level in immune cells of the lymphoid lineage, plays an important role in T cell maturation, activation and cytokine production during pathological inflammation and its inhibition could help treat inflammatory conditions as shown in in-vitro and mouse studies (Kennedy et al., 2014). Furthermore Ccdc88A, via activation of Gαi, simultaneously enhances the profibrotic (Pi3k-Akt-FoxO1 and TGF-β-Smad) and inhibits the antifibrotic (cAMP-PKA-pCREB) pathways, shifting the fibrogenic signalling network toward a profibrotic programme (Lopez-Sanchez et al., 2014). Interestingly, in the liver, sustained up-regulation of Ccdc88A occurs only in all forms of chronic fibrogenic injuries but not in acute injuries that heal without fibrosis, indicating that increased expression of Ccdc88A during acute injuries may enhance progression to chronicity and fibrosis (Lopez-Sanchez et al., 2014). Ccdc88A also regulates the Pi3 kinase-Akt pathway, which exhibits pleiotropic functions in chondrogenesis, cartilage homeostasis and inflammation. It may further induce an increase in MMP production by chondrocytes leading to subsequent cartilage degeneration, via its multiple downstream target proteins (Chen et al., 2013; Fujita et al., 2004; Greene and Loeser, 2015; Kita et al., 2008; Litherland et al., 2008; Starkman et al., 2005; Xu et al., 2015).

[0097] Remarkably, in this study, the cartilage matrix proteins Prg4, Acan, Comp and Chad had a significantly higher baseline expression in adult sheep and showed little injury response in either age group with the exception of Prg4, which was significantly up-regulated in fetal injured sheep. Prg4, in response to injury increased 3.2 fold (q=0.01) in fetal sheep, which is a 4.6 fold higher increase compared to adults (q=0.002), indicating a stronger and more rapid cartilage matrix production. Since Prg4 expressing cells constitute a cartilage progenitor cell population, the higher baseline expression in adults is particularly surprising but can be explained by its restriction to the most superficial cell layer in fetal joints compared to a distribution throughout the entire cartilage depth in older individuals (Kozhemyakina et al., 2015).

[0098] In contrast to the cartilage matrix glycoproteins, many growth factors, such as Gabpa and Mapk3 showed differential regulation following injury between adult and fetal sheep. Gabpa, a member of the ets protein family, which is ubiquitously expressed and plays an essential role in cellular functions such as cell cycle regulation, cellular growth, apoptosis, and differentiation (Rosmarin, 2004) showed a further significant up-regulation in fetal injury and no response to adult injury (q=0.0001). Gabpa activates the transcriptional co-activator Yes-associated protein (Yap), which is essential for cellular and tissue defences against oxidative stress, cell survival and proliferation and can induce the expression of growth-promoting genes important for tissue regeneration after injury (Wen Chun Juan, 2016). The cellular importance of Gabpa is further highlighted by the observation that in Gabpa conditional knockout embryonic stem cells (ESCs), disruption of Gabpa drastically repressed ESC proliferation and cells started to die within 2 days (Ueda et al., 2017).

[0099] The growth-regulator Mapk3 had a higher baseline expression in adult sheep (log FC=7.8, q=0>02) but significantly decreased (13.7 log FC, q<0.0001) after injury, while fetal Mapk3 remained essentially unchanged. Mapk3 acts as an essential component of the MAP kinase signal transduction pathway and as such contributes to cell growth, adhesion, survival and differentiation through the regulation of transcription, translation and cytoskeletal rearrangements. Mapk3 also fulfils an essential role in the control of chondrogenesis and osteogenesis of MSCs under TGF-β or mechanical induction and positively regulates chondrogenesis of MSCs (Bobick et al., 2010).

[0100] The different inflammatory response as demonstrated herein in the fetus may be a major contributor to fetal scarless cartilage healing. This is especially surprising as fetal sheep have a normally functioning immune system by 75 gd (Almeida-Porada et al., 2004; Emmert et al., 2013). Leukocytes have been shown to be present and increase rapidly at the end of the first trimester (Mackay et al., 1986; Maddox et al., 1987d). Fetal sheep are able to form large amounts of specific antibodies in response to antigen stimuli by 70 gd (Silverstein et al., 1963) and reject orthotopic skin grafts and stem cell xenotransplants administered after 75-77 gd with the same competence and rapidity as adult (Silverstein et al., 1964). Furthermore, fetal sheep have an inflammatory response to injury before 80 gd (Kumta et al., 1994; Moss et al., 2008; Nitsos et al., 2016). The first evidence of inflammation, the presence of TNF and IL-1 has even been shown as early as 30-40 gd (Dziegielewska et al., 2000).

[0101] In conclusion, the results demonstrate the power of the new ovine fetal cartilage regeneration model and of the analytical approach. Both positive and negative regulators of inflammatory events were found to be differentially regulated, which holds promise for therapeutic interventions based on cell culture supernatants, in particular as the presence of a negative regulator is more easily mimicked than the absence of a positive regulator.

Tables

[0102]

TABLE-US-00001 TABLE 1 Selected relevant proteins, logFC represents the fold change in a logarithmic scale to the basis 2 based on label-free quantification (LFQ) intensities. Fetal ctrl Fetal D 3 inj. Adult D 3 inj. Fetal vs adult vs Adult ctrl vs ctrl vs ctrl inj. response Name logFC q logFC q logFC q logFC q Acan −10.74 5.54E−11 1.77 1.00E+00 −1.36 9.15E−01 3.13 1.01E−01 Ccdc88A 4.32 5.29E−01 −10.45 5.62E−03 10.05 8.04E−04 −20.50 1.90E−05 Ccdc42 −3.58 5.97E−01 8.68 4.27E−02 −3.45 9.39E−01 12.13 5.47E−03 Chad −10.94 2.14E−09 1.48 1.00E+00 −1.22 1.00E+00 2.70 6.95E−01 Col2a1 −2.07 3.69E−01 1.14 1.00E+00 −1.01 1.00E+00 2.15 1.00E+00 Comp −9.64 1.13E−10 1.67 1.00E+00 −0.05 1.00E+00 1.72 1.00E+00 Gabpa −3.01 3.50E−02 8.23 1.52E−05 −0.29 1.00E+00 8.52 1.09E−04 Mapk3 −7.81 1.50E−02 1.11 1.00E+00 −13.73 1.23E−05 14.84 2.55E−03 Ppm1A −1.62 1.00E+00 8.91 1.36E−03 −0.21 1.00E+00 9.12 4.69E−03 Prg4 −11.56 3.94E−12 3.18 1.27E−02 −1.43 5.29E−01 4.61 1.63E−03 S100A12 −2.71 1.86E−01 8.39 4.99E−05 13.54 7.37E−10 −5.15 7.15E−02 S100A8 −2.78 3.48E−01 7.49 1.29E−03 15.80 7.15E−10 −8.32 3.53E−03 S100A9 0.08 1.00E+00 6.34 4.25E−01 15.45 9.32E−08 −9.11 4.15E−02

TABLE-US-00002 TABLE 2 Sources, pre-treatments and dilutions of the antibodies used for histology. Concen- Anti- tration body Clone (v/v) Pre-treatment Source Col2 2B1.5 1/100 0.04% hyaluronidase Thermo (Sigma Aldrich) in PBS.sup.#, 4 h Fisher at 37° C.; followed by 1% Scientific, protease (Sigma Aldrich) in Waltham, PBS, 30 min at 37° C. MA Ki67 SP6 1/400 0.01M citrate buffer pH 6.0, Thermo 2 h at 85° C. Fisher Scientific Mmp9 poly 1/100 0.01M citrate buffer pH 6.0, Abnova, 30 min at 90° C.-95° C. Heidelberg, Germany Mmp13 poly 1/50 0.01M citrate buffer pH 6.0, Thermo 30 min at 90° C.-95° C. Fisher Scientific

EXAMPLE 2—CELL CULTURE SUPERNATANT OF TENDON CELLS OBTAINED FROM ADULT AND FETAL SHEEP

[0103] In adult (2-4 years of age) and fetal (day 80 of gestation) sheep, surgical lesions were induced at a tendon (see FIG. 7). Tendon samples were collected before lesion induction and on day 3 after lesion induction (leading to four sample groups: adult control, adult injured, fetal control, fetal injured). Cell culture, supernatant collection and fractionation, and mass-spectrometric secretome analysis was performed essentially as in Example 1. Many proteins in the secretome were diffentially secreted into the supernatant between the sample groups. A selection of relevant examples is shown in FIGS. 8 to 12. Further examples can be found in Table 3 below.

TABLE-US-00003 TABLE 3 Selected relevant proteins, which are differentially regulated between the adult and fetal injury response. LogFC represents the fold change in a logarithmic scale to the basis 2 based on label-free quantification (LFQ) intensities. Protein FDR adj. p- Abbreviation Accession Name logFC value (q-value) FHL1 W5PP66 Four and a half LIM domains protein 1 14.51 8.15E−12 UBE2M W5P1I7 Ubiquitin conjugating enzyme E2 M −10.37 8.28E−11 RPL7A W5P0H3 ribosomal protein L7a −10.15 2.65E−10 MARCKS W5PIF5 Myristoylated alanine-rich protein kinase C substrate −12.36 2.77E−09 DUT W5QIN9 deoxyuridine triphosphatase −10.28 4.20E−09 H2AFJ W5QGA9 Histone H2A −17.45 6.05E−09 H1FX W5PY64 H1 histone family member X −11.07 6.05E−09 NUCKS1 W5P2B2 Nuclear casein kinase and cyclin dependent kinase substrate 1 −10.35 7.77E−09 CARHSP1 W5P5P6 calcium regulated heat stable protein 1 −11.97 1.06E−08 PAPSS1 W5PG10 3′-phosphoadenosine 5′-phosphosulfate synthase 1 −10.54 1.08E−08 Palm W5PHI5 Paralemmin 16.22 1.26E−08 CTRB1 W5P9F4 chymotrypsinogen B1 13.77 1.31E−08 Stam 2 W5PEU9 signal transducing adaptor molecule 2 −12.73 2.52E−08 COLGALT1 W5Q3J3 Collagen beta(1-O)galactosyltransferase 1 −12.56 2.52E−08 NUTF2 W5NZ10 nuclear transport factor 2 14.26 2.52E−08 GOLGA1 W5PUI3 golgin A1 14.81 2.52E−08 ENSA W5QI48 endosulfine alpha −11.40 2.80E−08 RPL4 W5Q9Z9 ribosomal protein L4 −9.90 2.80E−08 C11orf68 W5NPL7 chromosome 11 open reading frame 68 11.96 2.80E−08 TLN2 W5QI12 talin 2 13.68 3.37E−08

[0104] In this study, comparative proteomics of the fetal and adult response to acute tendon injury (3 days after injury) demonstrated differential regulation of a range of proteins between the adult and fetal response to injury. A protein with significantly higher upregulation in the fetal compared to the adult response to injury, CTRB1, participates in the “activation of matrix metalloproteinases”, “degradation of extracellular matrix” and “extracellular matrix organisation” pathway. Other proteins, which showed stronger regulation in adult sheep in response to injury, have a role in the “mitotic cell cycle”, “developmental biology”, “extracellular matrix organization”, “gene expression”, “metabolism of proteins” and “immune system” including “signaling by interleukins” and the “TNFR2 non-canonical NF-kappaB pathway. The majority of regulated proteins were upregulated in response to injury, including CARHSP1 (TNF signaling), COLGAT1, DUT, ENSA, H1FX, HSAFJ, MARCKS, NUCKS1, PAPSS1, RPL4, RPL7a, STAM2, UBE2M, while downregulated proteins included C11ORF68, FHL1, Golgal, NUTF2, PALM, TLN2.

[0105] The results demonstrated large differences between the fetal and adult response to tendon injury, implying therapeutic relevance of fetal supernatants due to the much higher regeneration potential of injured fetal tendon.

EXAMPLE 3—TREATMENT OF INFLAMED CHONDROCYTES WITH SUPERNATANTS FROM FETAL CELLS

[0106] Ovine chondrocytes were injured (inflamed for 24 h with TNF-α and IL-1β—each 10 ng/ml) and subsequently treated with the supernatant (SN) of adult mesenchymal stem cells (MSCs; “aMSCs”), fetal MSCs (“fMSCs”) or fetal chondrocytes (“fChondro”). Uninjured chondrocytes (“control healthy”) and injured but not treated chondrocytes (“control inflamed”) served as controls. As readout, gene expression analysis by real-time quantitative PCR (q-PCR) was performed in order to determine the expression levels of collagen type II alpha 1 (Col2), aggrecan and telomerase reverse transcriptase (TERT).

[0107] The expression of all three genes was reduced in injured compared to healthy chondrocytes. Treatment with supernatants from fetal cells were found to increase the expression close to or even above normal levels (FIG. 13).

EXAMPLE 4—TREATMENT OF INFLAMED TENOCYTES WITH SUPERNATANTS FROM FETAL CELLS

[0108] Ovine adult tenocytes were injured (inflamed for 24 h with TNF-α and IL-1β—each 10 ng/ml) and subsequently treated with the supernatant (SN) of adult MSCs (“aMSC SN”), fetal MSCs (“fMSC SN”) or fetal tenocytes (“fTeno SN”). Uninjured tenocytes (“control healthy”) and injured but not treated tenocytes (“control inf”) served as controls. As readout, gene expression analysis by real-time quantitative PCR (q-PCR) was performed in order to determine the expression levels of Decorin and Tenascin C.

[0109] The expression of both genes was reduced in injured compared to healthy tenocytes. Treatment with supernatants from fetal cells were found to increase the expression levels (FIG. 14).

EXAMPLE 5—EFFECTS OF SUPERNATANTS FROM FETAL CELLS ON THE SENESCENCE OF ADULT CHONDROCYTES

[0110] Supernatants of the fetal chondrocytes and fetal MSCs were collected in serum free medium (secretion time 6 hours). This supernatant was then used as a “treatment” for inflamed adult chondrocytes (again inflamed with 10 ng/ml IL1B+10 ng/ml TNF-alpha for 24 h). Commercial serum-free medium served as control. After 48 h a beta-galactosidase staining was performed using a commercially available kit (96 well Cellular senescence assay Beta Gal Activity from BioCat).

[0111] More precisely, on day one fetal chondrocytes, fetal MSCs and adult chondrocytes were seeded. On day two, adult articular chondrocytes (“patient”-cells) were inflamed with 10 ng/ml IL1B+10 ng/ml TNF-alpha for 24 h. On day three medium of the fetal chondrocytes and fetal MSCs was changed to fresh serum free medium. After 6 hours the so produced supernatant was used as a “treatment” on the adult “patient” cells which had been inflamed the day before. As control, adult “patient cells” received fresh commercial serum free medium. On day 5 (so after 48 h) the beta-galactosidase staining was performed.

[0112] It was found that treatment with the supernatants from injured fetal chondrocytes and injured fetal MSCs significantly reduced senescence in adult chondrocytes (FIG. 15).

EXAMPLE 6—EFFECTS OF THE SECRETOME OF FETAL AND ADULT CELLS ON INJURED TENOCYTES

[0113] In a further test of the beneficial effect of the fetal secretome on adult healing the bioactivity of both fetal and adult MSC-derived trophic factors with respect to their tendon regeneration potential were evaluated. For that purpose injured (inflamed for 24 h with TNF-α and IL-1β—each 10 ng/ml) ovine tenocytes were “treated” with the supernatant (SN) of adult or fetal MSCs. Uninjured tenocytes and injured but not treated tenocytes served as controls. As readouts gene expression analysis by q-PCR was performed. It could be shown that trophic factors secreted by MSCs decreased inflammation and increased expression of ECM genes as well as migration activity of injured tenocytes (see Example 8), indicating an inherent regenerative potential. The SN of fetal MSCs induced a faster “healing” compared to adult MSCs SN. Gene expression analysis confirmed the anti-inflammatory effect of MSCs shown by downregulation of IL6 and MMP1 expression in all “treated” samples with a slightly stronger effect achieved by fetal MSCs (FIG. 16). Intriguingly, collagen 3a1 expression was restored quicker and at a higher level in samples “treated” with fetal MSCs SN.

[0114] The results confirm that the supernatant of fetal MSCs has beneficial effects on adult tenocyte healing, supernatants from adult MSCs have significantly weaker effects.

EXAMPLE 7—WOUND HEALING ASSAY (CHONDROCYTE MIGRATION)

[0115] Methods to study cell migration in vitro are essential to simulate and explore critical mechanisms of action involved in the process and to investigate therapeutics. The “wound healing” assay is a commonly used in vitro model to study cellular response to injury by evaluating cell migration into a cell-free area within a confluent monolayer. This cell-free area is created either by removing the cells post adherence by mechanical, electrical, chemical, optical or thermal means, or through physical exclusion of cells during seeding.

[0116] In this experiment injured (inflamed for 24 h with TNF-α and IL-1β—each 10 ng/ml) ovine chondrocytes were scratched and subsequently “treated” with the supernatant (SN) of fetal MSCs or fetal chondrocytes. Uninjured chondrocytes and injured but not treated chondrocytes served as controls.

[0117] It was found that the supernatants of fetal cells significantly improved wound healing (FIG. 17) with SN of fetal MSCs showing a stronger effect than SN of fetal chondrocytes.

EXAMPLE 8—WOUND HEALING ASSAY (TENOCYTE MIGRATION)

[0118] In this experiment injured (inflamed for 24 h with TNF-α and IL-1β—each 10 ng/ml) ovine tenocytes were scratched and subsequently “treated” with the supernatant (SN) of fetal MSCs or fetal tenocytes. Uninjured tenocytes and injured but not treated tenocytes served as controls.

[0119] It was found that the supernatants of fetal cells significantly improved wound healing (FIG. 18) with SN of fetal MSCs showing a stronger effect than SN of fetal tenocytes.

[0120] Accordingly, the present invention discloses the following preferred embodiments:

1. A method for obtaining a fraction of a fetal cell culture supernatant, comprising the steps of [0121] obtaining a cell-containing sample of tissue from a non-human mammalian fetus, [0122] culturing the sample in a liquid cell culture medium, thereby obtaining a cell culture with a liquid supernatant, and [0123] isolating a fraction from the supernatant.
2. The method of embodiment 1, wherein the fetus is within the first two trimesters of gestation, preferably within the first half of gestation.
3. The method of any one of embodiments 1 to 2, wherein the sample comprises chondrocytes, chondroblasts, chondroprogenitor cells, tenocytes, tenoblasts, tendon progenitor cells, fibrocytes, fibroblasts, fibrochondrocytes, fibrochondroblasts, synoviocytes, synovioblasts, osteocytes, osteoblasts, osteoclasts, hepatocytes, monocyte, macrophage, mesenchymal stem cells, mesenchymal progenitor cells and/or interzone cells.
4. The method of any one of embodiments 1 to 3, wherein the tissue is selected from cartilage, tendon, ligament, bone, bone marrow and blood, in particular cord-blood.
5. The method of any one of embodiments 1 to 4, wherein the tissue is articular cartilage.
6. The method of any one of embodiments 1 to 5, wherein the culturing step comprises injuring, preferably chemically or mechanically injuring, the cells.
7. The method of any one of embodiments 1 to 6, wherein the fraction comprises proteins, lipids, metabolites, extracellular vesicles and/or RNA, in particular miRNA.
8. The method of any one of embodiments 1 to 7, wherein the liquid cell culture medium is a serum-free cell culture medium, a protein-free cell culture medium or a chemically defined cell culture medium.
9. The method of any one of embodiments 1 to 8, wherein the isolating step comprises a sterile filtration.
10. The method of any one of embodiments 1 to 9, wherein the isolating step comprises adding a protein-precipitating agent.
11. The method of any one of embodiments 1 to 10, wherein the isolating step comprises a centrifugation step.
12. The method of any one of embodiments 1 to 11, wherein the isolating step comprises a preservation step, preferably a freezing or drying step, especially lyophilisation.
13. The method according to any one of embodiments 1 to 12, wherein the cell culture medium comprises fetal calf serum (FCS) or other nutrient additives.
14. A fraction, obtainable by the method of any one of embodiments 1 to 13.
15. A cell supernatant fraction from non-human fetal cells, preferably wherein the cells are defined as in embodiment 3, wherein the fraction comprises proteins, lipids, metabolites, extracellular vesicles and/or RNA, in particular miRNA.
16. The fraction of embodiment 14 or 15, wherein the fraction is dry, preferably lyophilised.
17. A pharmaceutical composition, comprising the fraction of any one of embodiments 14 to 16.
18. The pharmaceutical composition of embodiment 17 for use in therapy.
19. The pharmaceutical composition of embodiment 18 for use in a prevention or treatment of osteoarthritis, arthritis, tendinitis, tendinopathy, cartilage injury, tendon injury, rheumatoid arthritis, discospondylitis, meniscus injury, desmitis, desmopathy, intervertebral disc injuries, degenerative disease of intervertebral discs, reperfusion injury, wounds or inflammatory disease.

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