In vitro method for creating a viable connective tissue and/or osseous tissue

Abstract

The present invention relates to an in vitro method for creating a viable connective tissue and/or osseous tissue obtained by tribological solicitations of a biological culture. It further relates to a viable connective tissue and/or osseous tissue susceptible to be obtained by said method as well as to the use of said method or viable connective tissue and/or osseous tissue to prepare a biological implant.

Claims

1. An in vitro method for creating a viable connective tissue and/or osseous tissue obtained by tribological solicitations of a biological culture to repair a patient's bone defect, the method comprising: (a) culturing a blood sample or biopsy from the patient on a pyrocarbon (Pyc) applicator in the presence of a tissue culture media in a bioreactor for at least about 4 days thus resulting in tissue that is growing, wherein the Pyc applicator has a matched convex/concave shape that mimics a shape of the patient's bone defect to be repaired, and concomitantly, afterwards, or with an overlap of; (b) submitting the growing tissue obtained at step (a) to tribological solicitations for at least about 4 days; wherein the growing tissue obtained at step (a) is submitted to a perpendicular pressure force to obtain an osseous tissue; and/or is submitted to a perpendicular pressure force and a movement parallel to a surface of the pyrocarbon applicator to obtain a connective tissue; and (c) preparing a biological implant.

2. The method of claim 1, wherein the step (b) is conducted using a material selected from the group consisting of polymers, ceramics, and combinations thereof, the growing tissue being positioned between the material and the pyrocarbon applicator, the pyrocarbon applicator and the material applying the perpendicular pressure force on the tissue.

3. The method of claim 1, wherein step (b) comprises: a step (b0) in which the growing tissue is submitted to a perpendicular pressure force for at least 4 days; and concomitantly, after or with an overlap, a step (b1) in which said growing tissue is submitted to a perpendicular pressure force and a movement parallel to the surface of said pyrocarbon applicator for at least 4 days.

4. The method of claim 1, wherein the step (b) comprises: a step (b0) in which the growing tissue is positioned between a material selected from the group consisting of polymers, ceramics and combinations thereof, and the pyrocarbon applicator, the pyrocarbon applicator and the material applying the perpendicular pressure force on the tissue; and concomitantly, afterwards, or with an overlap of, a step (b1) in which the growing tissue is submitted to the perpendicular pressure force and the movement parallel to the surface of the pyrocarbon applicator.

5. The method of claim 1, wherein each step of the method is conducted for between 4 and 10 days.

6. The method of claim 2, wherein the material selected from the group consisting of polymers, ceramics, and combinations thereof is a pyrocarbon material.

7. The method of claim 1, wherein the blood sample or biopsy of the patient includes stem cells.

8. The method of claim 7, comprising a step (a0) consisting in isolating the stem cells from the blood sample or biopsy, and further using said isolated stem cells in step (a).

9. The method of claim 1, wherein the tissue culture media comprises synovial fluid components.

10. The method of claim 1, wherein the biological implant is a resurfacing cap, a sheet of cartilage, or an entire articular end of a bone.

11. The method of claim 1, wherein the biological implant is configured to repair a localized cartilage defect on an articular surface.

12. The method of claim 11, wherein the defect has been abraded and covered by the biological implant.

Description

FIGURES

(1) The figure is a diagram of a bioreactor according to an embodiment of the present disclosure.

EXAMPLES

(2) Cartilage tissue has been grown in vitro from stem cells (extracted from the patient's blood) and is intended to be implanted.

(3) Obtaining cartilage tissue has required several steps: Isolation of stem cells from the extracellular matrix contained in the patient's blood. Multiplication (amplification) of the cells making to obtain a sufficient number of cells Forming a stem cell aggregate, or developing a biological support (like constructions of hyaluronan-gelatin for example) which will be seeded by the stem cells Mechanical (and biochemical) stimulation of the biological culture containing the cells (or the stem cell aggregate) in shear and compression against a Pyrocarbon disk in a mechanical simulator, in an incubator allowing the cultivation of cells for several weeks. Many parameters had to be controlled: mechanical constraints (shaking, mechanical solicitation), intake of growth factors and nutrients, oxygen and CO.sub.2 levels as shown in Table 1 below.

(4) TABLE-US-00001 TABLE 1 Criteria Level Normal Force 1 N < F < 70 N applied Normal force 0.1 Hz < f < 10 Hz frequency Tangential force 0.1 Hz < f < 10 Hz frequency Torque applied 0.001 N .Math. m < C < 1 N .Math. m to cells Physical/chemical 37° environment 5% CO.sub.2 Pyrocarbon Graphite disks (1 mm thick, diam 20 mm) covered applicator with 200 μm of PyC, polished at Ra 0.03 μm. Cells culture Dulbecco's Modified Eagle Medium DMEM, 4.5 g/L media for cells glucose, 100 mM HEPES and 10% FBS (still culture step containing L-glutamine, penicillin and streptomycin) (step (a)) Cells culture Dulbecco's Modified Eagle Medium (DMEM) media during containing 20 mM L-glutamine, 1 g/L glucose, mechanical step 100 U/mL penicillin and 100 μg/mL streptomycin, (step (b)) 10% Foetal Bovine Serum (FBS) + synovial components

(5) Results obtained by this setting have provided a cortical graft covered with articular cartilage layer, proven by specific histological and immunohistological staining.

(6) These analyses showed the existence of biological tissue adherent to the bone.

(7) Specific histological and immunohistological staining indicate that the surface layer of this tissue has the characteristics of cartilaginous tissue.