WO2014033325A1

WO2014033325A1 - Cell-based activation of devitalized engineered grafts for tissue repair

Info

Publication number: WO2014033325A1
Application number: PCT/EP2013/068194
Authority: WO
Inventors: Adam Papadimitropoulos; Ivan Martin; Atanas TODOROV
Original assignee: Universitätsspital Basel Rechtsdienst
Priority date: 2012-09-03
Filing date: 2013-09-03
Publication date: 2014-03-06

Abstract

The invention relates to a method for production of a graft for use in a method of connective tissue repair or connective tissue regeneration or connective tissue replacement, characterized by (a) providing a hypertrophic cartilaginous template (HCT), (b) providing a preparation of osteoclast precursor cells, (c) seeding said preparation onto said HCT under conditions of cell culture in the presence of MCSF and RANKL, yielding seeded HCT, and (d) maintaining said seeded HCT under conditions of cell culture for a period of at least 12 hrs. The invention further relates to grafts provided by the method of the invention for repair, regeneration and replacement of connective tissue, particularly bone.

Description

Cell-based activation of devitalized engineered grafts for tissue repair

Field of the Invention

The present invention relates to a method of providing a graft for tissue repair by in vitro seeding of osteoclast. The invention further relates to graft tissue provided by the method of the invention.

Background of the invention

Bone loss from trauma, neoplasia, reconstructive surgery and congenital defects remain major health problems, making the development of effective bone regeneration therapies a primary priority. Existing treatments such as the use of autologous or allogenic bone grafts are often successful. Complications, however, due to the additional autograft surgery, limited quantity of material available for harvesting, and limited availability of transplant tissue from bone banks constitute serious shortcomings.

The efficacy of non-viable bone obtained from cadavers, or made of synthetic materials, is still under debate. The combination of osteoconductive substrates with osteoinductive factors (e.g., bone morphogenetic proteins) capable to recruit the appropriate endogenous cells holds a great promise, though optimal combinations, doses and release kinetics for such factors are still far from being identified and implemented in safe and clinically effective products. Therefore, an alternative approach that can improve the outcome of bone defects is highly desirable.

Implants based on three-dimensional (3D) porous biomaterials combined with autologous Mesenchymal Stem/Stromal Cells (MSC) provide promising approaches for the repair of the aforementioned defects, but so far have not been considered convincingly effective for routine clinical applications (Quarto et al., N Engl J Med. 2001 Feb 1 ;344(5):385-6; Meijer et al., PLoS Med. 2007;4(2):e9). One of the most critical issues yet to be addressed is the efficient engraftment and vascularization of the implants, particularly for large scale constructs for the treatment of critically sized defects.

Human bone marrow-derived Mesenchymal Stem/Stromal Cells (hMSCs) are defined as a cellular fraction positive for CD73, CD90, CD105, and negative for hematopoietic markers, while being able to stably differentiate in vitro into osteoblasts, adipocytes and

chondrocytes [Pittenger et al., Science 1999; 284:143-147]. The multi-differentiation capacity of hMSCs makes them promising candidates for regenerative medicine. The chondrogenic and osteogenic potential of hMSCs is used for the repair of damaged articular cartilage but also for bone tissue engineering by recapitulating intramembranous or endochondral ossification processes.

Osteoclast precursor cells are hematopoietic cells of myeloid origin that under specific stimuli can give rise to osteoclasts (see Xing et al., Immunol Rev. 2005 Dec;208:19-29).

MSC have typically been used to generate bone tissue by a process resembling intramembranous ossification, i.e. by direct osteoblastic differentiation. However, most bones develop by endochondral ossification, i.e. via remodelling of hypertrophic cartilaginous templates. Moreover, this process is conserved in adult vertebrates and present during fracture healing. Due to the intrinsic capability of hypertrophic chondrocytes to efficiently survive in hypoxic conditions and to produce potent angiogenic factors, endochondral bone formation could solve the issue of limited initial vascularisation, which typically leads to cell-death and formation of necrotic cores.

Recently, the inventors demonstrated that hypertrophic cartilage tissues engineered in vitro using human MSC and ectopically implanted in nude mice can recapitulate the events of endochondral ossification and efficiently generate functional bone tissue (Scotti et al., Proc Natl Acad Sci U S A, 2010 Apr 20;107(16):7251 -6). The study showed that bone formation critically depended on the template's stage of maturity, i.e. hypertrophic stage of cartilagineous templates.

The extracellular matrix (ECM) not only provides structural stability but also works as a reservoir of chemoattractants and growth factors, including bone inductive proteins, such as bone morphogenetic proteins (BMPs) and transforming growth factor beta (TGF-β) (Pfeilschifter et al., J Bone Miner Res. 1990 Aug;5(8):825-30) as well as angiogenic factors (VEGFs), which are released in response to specific endogenous or exogenous stimuli. In this context, an important aspect is that all implants generated in vitro required ECM remodelling in vivo, which was mediated mainly by host osteoclast/chondroclasts. Recent studies showed the importance of ECM remodelling by osteoclasts in

endochondral ossification process (Ota et al., Endocrinology. 2009 Nov; 150(1 1 ):4823-34; Vu et al., Cell. 1998 May 1 ;93(3):41 1 -22.), while impairment of remodelling process resulted in delayed fracture healing (Kosaki et al., Biochem Biophys Res Commun. 2007 Mar 23;354(4):846-51 ). Importantly, Tang et al. (Nat Med. 2009 Jul; 15(7):757-65) demonstrated that TGF-b1 activation and release due to osteoclastic-mediated resorption during remodelling of bone tissue is responsible to guide the migration of marrow derived bone progenitor cells (BMSC) to the resorption sites and form new bone. The objective of the present invention is to improve on the above state of the art, to provide safe and efficacious means for the generation of grafts for tissue repair, regeneration and replacement in a variety of clinical approaches. This objective is attained by the subject matter of the independent claims.

Brief summary of the invention

The invention provides devitalized engineered tissues with the capacity to induce bone formation. The inventors surprisingly found that devitalized hyperthropic cartilage tissues (HCT) produced in vitro by adult human mesenchymal stromal cells can be activated by peripheral blood-derived osteoclast progenitors prior to implantation to induce ectopic bone tissue formation.

According to a first aspect of the invention, a method for producing a graft is provided, characterized by

a. providing a cartilaginous template,

b. providing a preparation of osteoclast precursor cells,

c. seeding the preparation of osteoclast precursor cells onto the cartilaginous

template under conditions of cell culture in the presence of MCSF and RANKL, yielding seeded cartilaginous template, and

d. maintaining said seeded cartilaginous template under conditions of cell culture for a period of at least 12 hrs.

In general, any cartilaginous template amenable to rearrangement by osteoclast activity is contemplated. In addition to the hypertrophic cartilaginous template described in the examples, a cartilaginous template derived, by way of non-limiting example, from demineralized bone, cartilage tissue or any other suitable source of material providing an extracellular matrix, may be employed.

In some embodiments, the cartilaginous template is a hypertrophic cartilaginous template (HCT).

In some embodiments, the seeded cartilaginous template is maintained under conditions of cell culture for 1 , 2, 3, 4, 5, 6, or 7 days.

The graft is suitable for use in a method of connective tissue repair or connective tissue regeneration or connective tissue replacement, particularly for repair, regeneration or replacement of bone. In some embodiments, the HCT is provided in according to the protocol provided by Scotti et al., PNAS 2010, 7251 -7256.

In some embodiments, the HCT is provided by a method wherein

a. an adherent cell population derived from a stem cell preparation is propagated under conditions of cell culture (ex vivo);

b. said adherent cell population is maintained under conditions of cell culture in the presence of ΤΘΡβ1 for 15 to 25 days in a chondrogenic step, and c. subsequently, said adherent cell population is maintained under conditions of cell culture in the absence of ΤΘΡβ1 for 10 to 20 days in a hypertrophic step. In some embodiments, the stem cell preparation is a bone marrow stem cell preparation, for example obtained by selecting stem cells from a bone marrow aspirate.

In some embodiments, the stem cell preparation is a human stem cell preparation.

In some embodiments, fibroblast growth factor-2 is present in the chondrogenic step or the hypertrophic step.

In some embodiments, the concentration of said MCSF is between 20 and 30 ng/ml. In some embodiments, the concentration of said MCSF is about 25 ng/ml. In some embodiments, the concentration of said RANKL is between 40 and 60 ng/ml. In some embodiments, the concentration of said RANKL is 50 ng/ml.

In some embodiments, HCT is obtained by growing said adherent cell population on a 3D porous scaffold.

In some embodiments, said preparation of osteoclast precursor cells is derived by selecting peripheral blood cells that express CD14 on their surface. Cell selection can be achieved, by way of non-limiting example, by magnetic separation (MACS) or

fluorescence-based separation (FACS) based on the attachment of cells to labelled antibodies directed to CD14.

In some embodiments, said HCT is devitalized. The devitalization method is a key step in the generation of cell-free grafts. It aims to remove all cellular material without adversely affecting the composition, mechanical integrity but also the biologic activity of the remaining ECM that carries specific properties. Physical treatment (freeze & thaw cycles, sonication, pressure, mechanical agitation), enzymatic (Trypsin) or chemical treatments (Sodium deoxycholate, Triton X solutions) are used to eliminate the living fraction from the generated graft. Those methods must maintain a tight balance of having an optimal preservation of the ECM properties while obtaining an efficient removal of the cellular component.

In some embodiments, the HCT is devitalized by two, three, four or more freeze-thaw cycles after the hypertrophic step.

In some embodiments, the graft provided by the current invention is for use in a human patient. Accordingly, the cells employed are of human origin, autologous or allogenic.

In some embodiments, the preparation of osteoclast precursor cells is an autologous preparation, i.e. it is derived from the same patient who is the intended recipient of the graft provided herein.

In some embodiments, the stem cell preparation employed to generate the HCT is an autologous preparation.

According to a second aspect of the invention, a graft is provided for use in a method of connective tissue repair or connective tissue regeneration or connective tissue

replacement, comprising

a. a devitalized hypertrophic cartilaginous template (HCT) and

b. autologous osteoclasts or osteoclast precursor cells.

The graft can be obtained by the method of the present invention. In some embodiments, the HCT is derived from MSC cells passaged at least one week in the presence of JGF^ . In some embodiments, the HCT is derived from MSC cells passaged at least two weeks in the presence of JGF^ , and subsequently for one to two weeks in the absence of TGFB1.

RANKL in the present context refers to the protein known as "Receptor activator of nuclear factor kappa-B ligand", also known as tumor necrosis factor ligand superfamily member 1 1 (TNFSF1 1 ), TNF-related activation-induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF) (UniProt ID Nr. 014788).

M-CSF in the present context refers to the protein known as "Macrophage Colony stimulating factor" (UniProt ID Nr. P09603).

In some embodiments, RANKL and/or M-CSF are employed as recombinant proteins.

ΤΘΡβ1 in the present context refers to the protein known as "Transforming growth factor beta 1 " (UniProt ID No. P01 137). FGF2 in the present context refers to the protein known as„Basic fibroblast growth factor", also known as bFGF, FGF2 or FGF-β, UNiProt ID No. P09038.

The in vitro seeding of osteoclast precursor cells on devitalized hyperthrophic

cartilaginous templates induces and accelerates bone formation through endochondral ossification by anticipating the host osteoclast-mediated remodelling.

From a clinical and commercial standpoint, devitalized engineered constructs with the capacity to induce bone formation are highly attractive since they allow for using specific cell lines capable of generating a proper ECM, and therefore facilitate the development of standardized "off the shelf products. Reactivation/stimulation of these products with autologous cells (osteoclast precursor cells from peripheral blood) improves/accelerates the healing process and thus reduces hospitalization time and health costs.

The addition of osteoclastic cells on devitalized HCT likely primed the onset of the remodeling process, which is a critical trigger of the endochondral ossification process. The activation/stimulation of off-the-shelf, devitalized engineered tissues using easily available autologous cells represents a novel paradigm in regenerative medicine.

The invention is further illustrated by the following figures and examples, from which further advantages and embodiments can be drawn. The figures and examples are not intended to limit the scope of the claimed invention.

Figure Legends

Figure 1 : shows mineral volume quantification (through quantitative microtomography) of the devitalised grafts co-cultured with osteoclast precursor cells (1 ) or without osteoclast precursor cells (2) after 8 weeks of ectopic implantation in nude mice, x-axis represents the experimental groups and y-axis represents the volume in mm³ (cubic millimetres).

Figure 2: shows mineral thickness quantification (through quantitative microtomography) of the devitalised grafts co-cultured with osteoclast precursor cells (1 ) or without osteoclast precursor cells (2) after 8 weeks of ectopic implantation in nude mice, x-axis represents the experimental groups and y-axis represents the length in micrometres.

Figure 3: shows histologic staining (Masson Trichrome) of the devitalised grafts co- cultured with osteoclast precursor cells (1 ) or without osteoclast precursor cells (2) after 8 weeks of ectopic implantation in nude mice. Examples

In overview, buffy coat derived from peripheral blood is processed by sorting out the CD14+ cell population that contains osteoclast precursor cell population. Devitalized engineered grafts are then co-cultured in the presence of osteoclastogenic stimulating factors with the osteoclast precursor cells in order to stimulate the differentiation of osteoclast precursor cells to osteoclasts on the devitalized engineered graft. 3D

visualisation (through quantitative micro-tomography) of the devitalised grafts co-cultured with osteoclast precursor cells after 8 weeks of ectopic implantation in nude mice shows frank bone formation in comparison to absence of frank bone formation in samples without osteoclast precursor cells present.

HCT were devitalized by successive cycles of freeze/thaw and either co-cultured with freshly isolated and sorted CD14+ osteoclast progenitors from human peripheral blood, or cultured alone, in the presence of osteoclastogenic factors (MCSF; RANK-ligand). Samples were implanted ectopically in nude mice for up to 8 weeks. Only the co-cultured constructs generated frank bone tissue through endochondral ossification, with 3.5-fold higher mineralized volume and less remnants of cartilaginous tissue as compared to cell- free devitalized HCT.

Analysis consisted of biochemistry, protein assays, histology and microtomography.

In vitro matrix degradation through significant loss of glycosaminoglycans was detected only if HCT was cultured with osteoclastic cells. Supernatants of co-cultures contained significantly higher amounts of chemoattractant (MCP-1 192-fold; SDF-1 4-fold), angiogenic (IL-8 556-fold; VEGF-A 5.4-fold) and matrix degrading (MMP9 13534-fold; MMP13 8.5-fold) factors as compared to controls. In vivo only co-cultured HCT generated frank bone through endochondral ossification with a 3.5-fold higher mineralized volume as compared to controls.

Example 1

Hyperthropic cartilaginous templates were prepared in vitro using expanded BMSCs (bone marrow derived stem cells) with the already established protocols (Scotti et al. 2010):

MSC isolation, in vitro culture, and in vivo implantation:

Human mesenchymal stem cells (MSC) were isolated from ten bone marrow aspirates and processed as previously described (Braccini et al. (2005) Stem Cells, 23:1066-1072). The two donors with the best chondrogenic potential, screened by means of micromass culture system (Muraglia et al. (2003) J Cell Sci, 1 16:2949-2955), were chosen for the following experiments. MSC were expanded for two passages (referred to as

postexpanded MSC) and differentiated into chondrogenic lineages in transwell culture (Murdoch et al. (2007) Stem Cells, 25:2786-2796) for 1 or 2 weeks in a serum-free chondrogenic medium (Barbero et al., (2003) Arthritis Rheum, 48:1315-1325 ), or for 3 weeks in chondrogenic medium followed by 2 weeks in a serum-free hypertrophic medium, supplemented with 50 nmol/l thyroxine (Mackay et al. (1998) Tissue Eng, 4:415- 428 ), 7.0 x 10^~3 mol/l β-glycerophosphate, 10-8 M dexamethasone, and 2.5 * 10^~4 mol/l ascorbic acid (Muraglia, ibid.). Samples were implanted in subcutaneous pouches of nude mice (4 samples/mouse) and retrieved after 4, 8, or 1 1 weeks. During in vitro culture, selected transwells were supplemented with 5 pmol/l 3-keto-N-(aminoethyl-aminocaproyl- dihydro- cinnamoyl)-cyclopamine (Calbiochem), a potent derivative of cyclopamine, and cultured for 5 weeks.

In short, human bone marrow stem cells were isolated from human bone marrow mononuclear cells by adherence over 24 hours to tissue culture plastic and were expanded in monolayer culture in Mesenchymal Stem Cell Growth Medium (Lonza Biosciences, http://www.lonza.com) supplemented with 5 ng/ml fibroblast growth factor-2 (R&D Systems Europe, http://www.rndsystems.com). Cultures were maintained in a humid atmosphere of 5% C0₂/95% air at 37°C. Once cells had reached confluence (passage 1 [P1]), they were passaged using Trypsin/EDTA at a split ratio of 1 :3.

MSC were expanded for two passages and cultured in transwell (5 ^χ 10⁵ cells/insert) for 3 weeks in chondrogenic medium (with TGF31 ) followed by 2 weeks in a hypertrophic medium (without TGF31 and with beta-glycerophosphate and thyroxine; late

hypertrophic). These templates were devitalized using 3 cycles of freeze and thaw and kept in -80°C until use.

Mononuclear cells were isolated from human peripheral blood buffy coats from healthy donors by gradient centrifugation (Ficoll, Histopaque 1077, Sigma-Aldrich, St. Louis, MO, USA). Subsequently, Osteoclast precursor cells were sorted using anti-CD 14-coated magnetic beads (Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer's instructions. Frozen devitalized hyperthropic cartilaginous templates were then kept under the hood at room temperature to thaw again. Subsequently, osteoclast precursor cells were seeded on the top of the templates at the density of 1.25 Mio cells /cm² in the presence of osteoclastogenic factors, namely Macrophage Colony Stimulating Factor (25ng/ml MCSF) and Receptor Activator of Nuclear factor Kappa-B Ligand (50ng/ml RANKL) both purchased from RnD (Switzerland) and cultured in serum-free alpha-MEM medium for 24 hours. Devitalized hyperthropic cartilaginous templates were also cultured in the above defined osteoclastogenic medium for 24 hours but without osteoclasts and used as controls. After 24 hours of culture, grafts from both experimental groups were ectopically implanted for 8 weeks in nude mice (CD1 - Foxnl nu obtained from Charles River, Germany). After 8 weeks, explants from both experimental groups were analyzed by means of histology (Figure 3; Masson Trichrome) and quantitative microtomography (Figure 1 , 2; using Skyscan 1074 device, Belgium) to assess and characterize bone formation.

Claims

1. A method for production of a graft, said method being characterized by the steps of a. providing a cartilaginous template,

b. providing a preparation of osteoclast precursor cells,

c. seeding said preparation of osteoclast precursor cells onto said cartilaginous template under conditions of cell culture in the presence of MCSF and/or RANKL, yielding a seeded cartilaginous template, and

2. The method according to claim 1 , wherein said cartilaginous template is a

hypertrophic cartilaginous template (HCT).

3. The method of claim 2, wherein said HCT is provided by a method wherein

a. an adherent cell population derived from a stem cell preparation is propagated under conditions of cell culture;

b. said adherent cell population is maintained under conditions of cell culture in the presence of ΤΘΡβ1 for 15 to 25 days in a chondrogenic step, and

c. subsequently, said adherent cell population is maintained under conditions of cell culture in the absence of ΤΘΡβ1 for 10 to 20 days in a hypertrophic step.

4. The method according to claim 3, wherein fibroblast growth factor-2 is present in the chondrogenic step or the hypertrophic step.

5. The method according to any one of the above claims, wherein the concentration of said MCSF is between 20 and 30 ng/ml, and/or the concentration of said RANKL is between 40 and 60 ng/ml.

6. The method according to any one of the above claims, wherein said preparation of osteoclast precursor cells is derived by selecting peripheral blood cells that express CD14 on their surface.

7. The method according to any one of the above claims, wherein said cartilaginous template is devitalized.

8. The method according to any one of claims 3 to 7, wherein subsequent to said

hypertrophic step, said adherent cell population is devitalized.

9. The method according to any one of the above claims, wherein said preparation of osteoclast precursor cells is an autologous preparation.

10. The method according to any one of claims 2 to 8, wherein said stem cell preparation is an autologous preparation.

1 1 . A graft for use in a method of connective tissue repair or connective tissue

regeneration or connective tissue replacement, comprising

a. a devitalized hypertrophic cartilaginous template (HCT) and

b. autologous osteoclasts or osteoclast precursor cells.

12. The graft according to claim 1 1 , characterized in that said HCT comprises, or is

essentially composed of, devitalized autologous MSC passaged in the presence of TGFB1 .

13. The graft according to claim 1 1 or 12, obtainable by a method according to any one of claims 1 to 10.

14. A method of use of a graft obtained by a method according to any one of claims 1 to 10, or of a graft according to claim 1 1 to 13, in a method of connective tissue repair or connective tissue regeneration or connective tissue replacement.

15. The method of use according to claim 14, in a human patient.

16. A method of use according to any one of claims 14 or 15, wherein said connective tissue is bone.