Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Implant or insert
Reexamination Certificate
2001-04-04
2003-11-25
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Implant or insert
C424S093700, C435S177000, C435S395000, C623S011110
Reexamination Certificate
active
06652872
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns scaffold matrices for supporting three-dimensional tissues and systems for maintaining three-dimensional viable tissues.
BACKGROUND OF THE INVENTION
The following publications are believed to be relevant as background of the invention.
1. WO 98/22573
2. U.S. Pat. No. 4,880,429
3. U.S. Pat. No. 4,108,438
4. U.S. Pat. No. 5,843,182
5. Mikos, A. G., Sarakinos, G., Leite, S. M., Vacanti, J. P., and Langer, R., “
Laminated three
-
dimensional biodegradable foams for use in tissue ingineering”, Biomaterials,
14:323-330, 1993.
6. Hutmacher, D., Kirsch, A., Ackeman, K. L., and Huerzeler, M. B., “
Matrix and carrier materials for bone growth factors—state of the art and future prospectives in:
Stark, G. B., Horch, T, Tancos, E. (eds).
Biological Matrices and Tissue Reconstruction.
Springer Verlag, 1998, pp. 197-203.
7. Kandel, R. A., Chen, H., Clark, J. and Renlund, R.,
Transplantation of cartilaginous tissue generated in vitro into articular joint defects. Biotechnol,
23:565-577, 1995.
Cartilage is a specialized form of connective tissue composed of cells and matrix. The cartilage cells synthesize matrix and become encased in cavities (lacunae) within it. The matrix is composed of fibers embedded in ground substance and endows cartilage with its specialized physico-chemical properties.
Trauma, single or repetitive, and minute imbalance in joint stability are the most known causes of damage and degeneration of articular cartilage, that leads to pain, chronic disability and ultimately to joint failure. The current options for treatment provide temporary improvement of symptoms and function, however, there is no full restoration of joint performance. Prosthetic joint replacement is currently the ultimate and the most commonly employed treatment. Modem biological grafting is the other alternative for resurfacing the damaged joint, but is still imperfect.
A large number of candidate grafts have been studied for enhancing the repair of cartilage defects which include: (i) Osteochondral graft (autografts or allografts); (ii) Intact cartilage grafts; (iii) Growth plate; (iv) Isolated allogeneic chondrocytes; (v) Cultured autologous chondrocytes (dedifferentiated) (vi) Periosteum; (vii) Perichondrium; (viii) Bone marrow mesenchymal derived cells and (ix) Synovial membrane derived cells.
Another approach was the attempt to use natural occurring or synthetic biodegradable scaffolds which support three-dimensional growth of cartilage cells. The scaffolds may be impregnated with cells, which together with the scaffold form the graft. Alternatively, the scaffold may initially be devoid of impregnated cell, and endogenous cells from the patient are expected to migrate into the scaffold after its implantation.
Examples of such scaffolds are: (a) Fibrin polymers; (b) Collagen Type I; (c) Natural hyaluronic acid (HA) and chemically modified HA and (d) Synthetic biopolymers either biodegradable e.g. polylactic acid, polyglycolic acid or non-biodegradable (e.g. alginic acid). However, none of the above scaffolds can induce regeneration of hyaline-like cartilage. Fibrin adhesive polymers tend to induce dedifferentiation and thus do not permit production of functional tissue. Collagen Type I has no inherent chemotactic ability for chondrocytes, but stimulates proliferation of fibroblasts. Thus, instead of encouraging migration of chondrocytes, the tissue formed in this scaffold tends to be fibrous. Hyaluronic acid can stimulate chondrogenic differentiation, but does not stimulate chondrocytes proliferation. Alginic acid is a foreign sea weed derived carbohydrate and thus might induce an antigenic reaction, and furthermore it is not biodegradable. Polyglycolic and polylactic acid scaffolds do not support good hyaline cartilage regeneration due to acidic conditions formed during their degradation.
Damaged or missing hyaline cartilage is frequently repaired by transplantation of homografts. Homografts are immunologically privileged since the matrix acts as a barrier that permits only limited diffusion of low-molecular weight substances and contains an anti-angiogenesis factor to prevent invasion of host blood vessels and fibroblasts.
Various culturing systems have been developed for maintaining the viability and growth of tissues in culture. Generally, these are divided into static and perfusion bioreactors. Perfusion bioreactors are reactors which essentially keep constant, growth permissible conditions (such as nutrition, gas composition, temperature, pH, etc.) in which the growth fluid medium is constantly perfused in and out of the system. Typically, perfusion is carried out by utilizing a constant velocity flow of the medium.
SUMMARY OF THE INVENTION
By a first aspect, the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising a naturally derived connective or skeletal tissue which has been treated for elimination of cellular and cytosolic elements, and which has been modified by cross-linking with an agent selected from the group consisting of: hyaluronic acid, proteoglycans, glycosaminoglycan, chondroitin sulfates, heparan sulfates, heparins and dextran sulfates.
By a second aspect, the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising a naturally derived connective or skeletal tissue which has been treated for elimination of cellular and cytosolic elements, and which is formed of flakes having a size below 1000&mgr;, which are attached to each other, the scaffold having a porosity of at least 85%, most preferably a porosity of 95%-98%.
By a third aspect the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising an aggregate of at least 3, preferably 7-8 embryonal epiphyses, obtained from a third to midterm old fetuses (11 days old chick embryo, or 17-22 weeks human embryos). The aggregates are formed spontaneously, when several individual epiphyses are attached to each other, due to the presence of mesenchymal progenitor cells in the periphery of the epiphysis, cells which feature high expression of adhesive molecules such as integrins, cadherins and CAMs.
It has been found that such scaffolds according to the first, second and third aspects of the invention, have the properties of encouraging cells' adherence both to the matrix and to other cells as well, and enablement of propagation of cells. It was further found that the scaffolds of the invention supports chondrocyte proliferation at the expense of fibroblasts, resulting in a hyaline-like reparative repair tissue. Cross-linking with the agents specified above in connection with the first aspect gives the scaffold an additional mechanical strength and produces a substance which is less brittle with prolonged biodegradation time.
The term “scaffold” in the context of the first and second aspects of the present invention refers to the connective/skeletal tissue which has been treated for elimination of cellular and cytosolic elements. In accordance with the first aspect of the invention, the scaffold has been modified by cross-linking as described above. In accordance with the second aspect, the scaffold is composed of attached flakes having a size of less than 1000&mgr; and having porosity of at least 85%. This term also refers in accordance with the first and second aspect to such a construct containing additional agents such as adhesive molecules or growth factors.
The term “flakes” refers to particles or chips produced as a result of crushing the connective tissue, to particles of a size less than 1000&mgr;.
In accordance with the third aspect of the invention the term “scaffold” concerns freshly aborted human epiphyses from around midterm (18±4 weeks), which are composed of mesenchymal progenitor stem cells and committed chondrocytes. At least 3 and preferably 7-8 epiphyses have been fused to form aggregates which
Nevo Zvi
Robinson Dror
Naff David M.
Ramat at Tel Aviv University Ltd.
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