MESENCHYMAL STROMAL CELL BONE GRAFT MATERIAL

Abstract

The invention pertains to the use of mesenchymal stromal cells (MSC) in the treatment of bone disorders or injuries. The invention provides MSC and preparations of specifically pooled MSC for use in the manufacturing of bone graft material for implanting into or attaching to bones in order to enhance bone regeneration after surgery or injury, or to treat various bone disorders, such as osteonecrosis. The invention provides bone graft material, a method for its production, bone graft implants, and medical methods and uses of the inventive products.

Claims

1. A bone graft material for use in the treatment of a subject, comprising a scaffold material and a biological cell material, wherein the biological cell material comprises cell material genetically allogenic to the subject.

2. The bone graft material for use according to claim 1, wherein the biological cell material comprises mesenchymal stromal cells (MSC) derived from any source, or comprises bone marrow mononuclear cells (BMC).

3. The bone graft material for use according to claim 1 or 2, wherein the biological cell material comprises living cells of one donor subject, preferably of more than two genetically distinct donor subjects, preferably from at least 3, 4, 5, 6, 7, 8. 9 or 10 or more genetically distinct donor subjects.

4. The bone graft material for use according to any one of claims 1 to 3, wherein the treatment comprises surgical insertion or attachment of the bone replacement material to a bone of the subject.

5. The bone graft material for use according to any one of claims 1 to 4, wherein the treatment is for promoting bone growth or healing, such as for example to treat a bone injury or disorder selected from bone fracture, bone trauma, arthrodesis, a bone deficit condition associated with post-traumatic bone surgery, post-prosthetic joint surgery, post-plastic bone surgery, post-dental surgery, bone chemotherapy treatment, congenital bone loss, post traumatic bone loss, post-surgical bone loss, post infectious bone loss, allograft incorporation or bone radiotherapy treatment, and preferably in the treatment of osteonecrosis (avascular necrosis (AVN)).

6. The bone graft material for use according to claim 5, wherein the osteonecrosis is a secondary disease caused by corticosteroid treatment, trauma, alcohol, sickle cell disease, leukaemia, or is idiopathic osteonecrosis.

7. The bone graft material for use according to any one of claims 1 to 6, wherein the scaffold material is synthetic or natural, preferably wherein the scaffold material comprises calcium phosphate, preferably α- or β-tricalciumphosphate (TCP), or comprises polylactic acid or polycarpolactone (or other biodegradable polymer), or a mixture of these materials.

8. The bone graft material for use according to any one of claims 1 to 7, wherein the bone graft material is in the form of a tablet, and the tablet has a height of 1 to 10 mm, preferably 3 to 5 mm, and/or a diameter of 1 to 15 mm, preferably 5 to 8 mm.

9. A bone graft implant, comprising a scaffold material and a biological cell material, wherein the bone graft implant is in the form of an implantable tablet having a height of 1 to 10 mm, preferably 3 to 5 mm, and/or a diameter of 1 to 15 mm, preferably 5 to 8 mm.

10. The bone graft implant according to claim 10, wherein the biological cell material comprises mesenchymal stromal cells (MSC) derived from any source, or comprises bone marrow mononuclear cells (BMC).

11. The bone graft implant according to any one of claims 10 to 12, for a use recited in any one of claims 1 to 9.

12. A method for producing cryopreserved bone graft material, comprising the steps of (i) Seeding biological cell material as recited in any one of claims 1 to 8 on a scaffold material as recited in any one of claims 1 to 8 to obtain a bone graft material, and (ii) Immediately cryopreserving the bone graft material obtained in (a).

13. The method according to any one of claims 23 to 26, wherein the biological cell material is seeded on the scaffold material in a density of at least 0.1×10.sup.6 cells per mL scaffold material, preferably of at least 0.5×10.sup.6 cells per mL scaffold material, more preferably of about 1×10.sup.6 cells per mL scaffold material.

14. A therapeutic kit, comprising (i) A first container comprising a scaffold material recited in any one of the preceding claims, (ii) A second container comprising a biological cell material recited in any one of the preceding claims; wherein the (i) and (ii) are provided in the therapeutic kit in separate containers for combining immediately before surgical insertion.

15. A mesenchymal stromal cell (MSC), or a MSC preparation, for use in the treatment of a disease, wherein the treatment comprises the administration of the MSC, or the MSC preparation, to a subject in need of the treatment by adhering the MSC or MSC preparation to a bone graft material to obtain an MSC-bone graft material, and implanting or attaching the MSC-bone graft material into/to one or more bone(s) of the patient.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0068] The figures show:

[0069] FIG. 1: shows the metabolic activity of MSC seeded on β-TCP, frozen and thawed (second bars) and of MSC cultured on β-TCP permanently at 37° C. (first bars). Mean values and standard deviation are shown. MSC were from the same donor, measurement was done in triplicate. Metabolic activity was assessed by means of MTT assay.

[0070] FIG. 2: shows a photograph of the tablet of the invention

[0071] FIG. 3: shows human MSC (cell pool of n=8 donors) were placed for 3 h on β-TCP granules, which were modelled into the approximate dimensions of the planned MSC tablet (radius 4 mm, height 5 mm) by using a permeable form. MSC were applied at a density of 1×106 cells per cm.sup.3 β-TCP according to established seeding protocols and cultivated for 3 h at 37° C. and then first frozen at −80° C. (overnight) and then −196° C. (7 days) (experimental group). Metabolic activity was determined by MTT test in parallel cultures 24 h and 48 h after thawing. The control group consisted of MSC seeded at the same density and in the same manner on β-TCP, but incubated for 24 h at 37° C. only. Subsequently, the metabolic activity was also determined.

EXAMPLES

[0072] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0073] The examples show:

Comparative Example 1: Collection of Bone Marrow from 8 Healthy Third-Party Donors and Isolation of BM-MNCs

[0074] After obtaining a written informed consent, from each bone marrow donor were collected up to 250 ml additional bone marrow aspirate for the purpose of MSC banking with approval by the local Ethics Committee in full agreement with the Declaration of Helsinki. In total from 8 donors the inventors obtained 1.66 liters bone marrow. For isolation of bone marrow mono-nuclear cells by Ficoll-gradient the inventors used the Sepax machine as shown in FIG. 1. The absolute number of BM-MNCs per 1 ml of bone marrow after this isolation procedure was 3.3×10.sup.6±6.3×10.sup.5 cells. Total number of BM-MNCs which was obtained from eight donors after two washing steps was 9.86×10.sup.9. These cells were resuspended in cryomedium and distributed in the bags, that were frozen using a rate-controlled freezer and then stored in the vapour phase of liquid nitrogen until use.

Comparative Example 2: Generation of Mesenchymal Stromal Cells from Bone Marrow Mononuclear Cells and Establishment of a MSC-Bank

[0075] To generate the MSC-Bank, bone marrow mononuclear cells from 8 donors were thawed, washed and pooled in DMEM supplemented with 5% PL. To find out the optimal concentration of platelet lysate for the adherence of progenitor cells of MSCs the inventors cultured BM-MNCs with both concentrations of PL: 5% and 10%. The obtained results have demonstrated that the 5% concentration of platelet lysate is much more efficacious in promotion of BM-MNCs and generation of MSCs than 10% concentration of PL. In addition, the inventors asked which of these two concentrations of PLs is better for clinical-scale expansion of MSCs. The inventors found that that the 10% concentration of PLs is significantly more efficient in expanding the MSCs than the 5% PL. Moreover, in both cases the unfiltered platelet lysates were more effective for generation and expansion of MSCs than the filtered ones. These preliminary experiments paved the way for establishing the master MSC-bank. Therefore, the inventors thawed the BM-MNCs from each donor and after washing twice they were pooled together and thereafter cultured for 14 days, as described in the section of methods. The inventors were able to generate from 9.89×10.sup.9 BM-MNCs 3.2×10.sup.8 MSCs of passage 1. These MSCs expressed the typical markers for MSCs, such as CD73, CD90 and CD105 but were negative for hematopoietic cell markers e.g. CD14, CD34, CD45. According to trypan blue staining the viability of these MSCs before freezing was 95±5%.

[0076] The total number of MSCs was distributed in 210 cryovials each containing 1.5×10.sup.6 MSC P1 and finally frozen in the gaseous phase of liquid nitrogen until use. The inventors referred to this set of vials as MSC-bank. The MSC as isolated according to comparative example 2 have improved allo suppressive potential as can be derived from WO 2016/008895.

Example 1: Scaffold Materials

[0077] Dimensions and shape: Cylindrical, height 3-5 mm, diameter of 5-8 mm, depending on the size of the bone defect. A number of scaffolds can be combined to fill larger defects as they occur in the treatment of femoral head osteonecrosis.

[0078] The scaffold should consist of natural or synthetic matrices e.g. α-TCP, β-TCP, demineralized bone matrix, polylactic acid or polycaprolactone.

[0079] The scaffold should preferably be mechanically stable in order to prevent collapsing during surgical procedures and implantation. The scaffold should offer communicating macropores of the size range 100 μm to 500 μm. Pore sizes from 100 μm improve angiogenesis, while pore diameters of 300-400 μm have a positive effect on osteoconductivity. Micropores enabling surface enlargement, cellular adhesion and improvement of nutritional support should range from 1 μm to 10 μm. Overall porosity of the scaffold should be 60-70%, thus ensuring highest possible degree of porosity without compromising the mechanical strength.

[0080] The surface of the material should be rather smooth, rough microstructures in size range 1-10 μm should be prevented. Preferably, the surface consists of smooth slightly convex structures with a diameter ranging from 8-15 μm that are arranged in a honey comb similar matter.

Example 2: Manufacture of a Bone Implant Graft

[0081] MSC derived from the cell bank of comparative example 2 were seeded to the scaffolds as described in Henrich et al, 2009, 2013, 2014; Seebach et al 2010, 2012, 2015. In brief, cells were harvested and preferably adjusted to a density of 1*10.sup.6 cells/mL (Range 1-1*10.sup.7 cells/mL). The cell suspension is carefully dripped on an equal volume of scaffold.

[0082] Non adsorbed cell suspension is carefully distributed once again evenly on the scaffold followed by 10 min incubation at 37° C., 5% CO2, 100% humidity. This procedure (Wetting of scaffold with non-adsorbed cell suspension followed by incubation) will be repeated two more times.

[0083] Such obtained functionalized graft material is ready for use or can be cryopreserved.

Example 3: Cryopreservation of Bone Graft Implants

[0084] Cryopreservation will be performed preferably immediately after the seeding procedure but time span between seeding and cryopreservation may vary in a cell type dependent manner from 0 min to 24 hrs. In the latter case the scaffold will be stored in medium (DMEM+5% platelet lysate) at 37° C., 5% CO2, 100% humidity until cryopreservation procedure takes place.

[0085] The cell populated scaffolds were subjected to cryopreservation medium preferably consisting of physiologic NaCl solution (65% v/v, final NaCl concentration 0.7%), human serum albumin solution (HSA, 25% v/v, final HSA concentration 5%) and DMSO (10% v/v).

[0086] The cryopreservation medium is provided in sterile plastic bags. After addition of cell seeded scaffolds (range 1 to 20 scaffolds per bag), the bags are closed by heat sealing. The sealed bags were immediately subjected to a controlled rate freezer. Long term storage will be performed in liquid nitrogen vapor phase. The possibility for short term storage at −30° C. to −20° C. will be analysed.

Example 4: Thawing and Use of Bone Graft Implants

[0087] Opening of sterile envelope and immediate subjection of the scaffold to a 50 mL vial filled with room temperature or prewarmed saline+HSA (0.5-5%, cleansing solution) followed by 5 min incubation in order to remove DMSO from the scaffold. The scaffold can be aseptically removed from the cleansing solution and should be placed immediately into the bone defect.

Example 5: Medical Use of Bone Graft Implants

[0088] MSC were seeded on approximately 200 μL densely compressed β-TCP granules (size 1.4-2.8 mm) in a density of 1*10.sup.6 cells/mL β-TCP-scaffold. The seeding procedure consists of repeated dripping of the cell suspension over the scaffolds during a period of 30 minutes. Subsequently scaffolds were immediately frozen at −80° C. overnight in a medium containing 10% DMSO and 90% FCS. As control, MSC on β-TCP (same donor) were cultivated at 37° C. in parallel. All procedures described in the following were also performed with the control approach. Next day the well plate containing the scaffolds was placed for 3 min in a water bath (37° C.) in order to enable recovery of scaffolds. Scaffolds were then immediately dropped into 30 mL prewarmed (room temperature) medium (RPMI+10% FCS) for 1 min and recovered by filtering with cell strainer (100 μm mesh). Scaffolds were equally distributed into a 96-well plate using sterile forceps and 100 μL medium (Mesencult+supplements) were added to each well. In order to assess metabolic activity of the MSC on the scaffold an MTT assay was performed in triplicate following the instructions of the manufacturer. Incubation time with MTT reagent was 4 hrs. In order to assess long term survival of the MSC a portion of cells was cultured for additional 24 hrs after thawing followed by an MTT assay.

[0089] Metabolic cell equivalent (MCE) of frozen and thawed MSC was 2.7*10.sup.4 and around 25% of MSC cultured at 37° C. on β-TCP (5.8*104 MCE). If MSC were cultured additional 24 hrs after thawing, the metabolic activity remained approximately constant (2.6*10.sup.4 MCE) compared to cell activity directly measured after thawing (2.7*10.sup.4 MCE) whereas the metabolic activity of MSC cultured permanently at 37° C. increased further (1.7*10.sup.5 MCE) compared to the initial value (1.2*10.sup.5 MCE, FIG. 1). These results indicate that MSC seeded on a β-TCP scaffold can be stored frozen at −80° C. and remain in part vital. Furthermore, it can be concluded that frozen/thawed MSC did not further deteriorate 24 hrs after thawing, though they did not improve either. One might assume that the freeze/thaw procedure induced a lag phase of MSC proliferation.

Example 6: Optimization of Cell Seeding as Well as Cryopreservation, Thawing and Reconstitution of the Cell-Populated Scaffolds

[0090] Human MSC-Pool (8 donors, passage 2) was obtained from the Clinic for Pediatrics and Adolescent Medicine. The cells were further cultivated up to a maximum of the 5th passage and used in experiments, or cryopreserved for later experiments. MSC phenotype was activated by FACS analysis (CD90+, CD105+, CD34−, CD45−) and the ability for osteogenic differentiation.

[0091] Using the MSC pool, our previous results were reproduced in a series of pilot experiments (n=3), which showed that hMSC, seeded on β-TCP scaffold and cultivated for a short time (3 h) at 37° C., are reduced metabolically active after freezing and thawing.

[0092] The metabolic activity of the cells was measured on the day after sowing for the control group and 1 and 2 days after rethawing using the MTT test. The mean metabolic activity of the cells of the experimental group increased significantly with increasing cultivation time after thawing (see FIG. 3: 36.3% of the control on day 1 and 56.2% of the control on day 2 after thawing; p<0.05, n=3).