Chemistry: molecular biology and microbiology – Apparatus – Including condition or time responsive control means
Reexamination Certificate
1999-07-06
2003-10-14
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Apparatus
Including condition or time responsive control means
C435S177000, C435S395000, C435S283100, C435S284100, C435S286100, C435S289100, C435S001100
Reexamination Certificate
active
06632651
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns scaffold matrixes 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.
WO 98/22573
U.S. Pat. No. 4,880,429
U.S. Pat. No. 4,108,438
U.S. Pat. No. 5,843,182.
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, is the most known cause 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.
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) Collagent Type I; (c) Natural hyaluronic acid (HA) and chemically modified HA and (d) Synthetic bipolymers either biodegradable or non-biodegradable (e.g. alginic acid) and (e) Polylactic acid, polyglycolic acid. However, none of the above scaffolds can induce generation of hyaline-like cartilage. Fibrin 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 stimulate proliferation of fibroblast. 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 cabohydrate and thus might induce an antigenic reaction, and furthermore is not biodegradable. Polyglycolic and polylactic acid scaffolds do not support good hyaline cartilage regeneration due to acidic conditions during degradation.
Damaged or missing hyaline cartilage is frequently repaired by transplantation of homografts. Homografts are immunologically privileged the matrix acts as a barrier that permits only limited diffusion of high-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 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 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.
It has been found that such a scaffold has the properties of encouraging cells adherence thereto and enablement of propagation of cells on the one hand, while the cross-linking with the agents specified above gives the scaffold mechanical strength, produces a substance which is less brittle with prolonged degradation time on the other hand. It was further found that the scaffold of the invention supports chondrocyte proliferation at the expense of fibroblasts, resulting in a hyaline-like repair tissue.
The term “scaffold” in the context of the present invention refers to the connective/skeletal tissue which has been treated for elimination of cellular and cytosolic agents and modified by cross-linking as described above, as well as to such a construct containing additional agents such as adhesive molecules or growth factors.
The term “three-dimensional tissue” (3D tissue) refers to any type of tissue which has an orderly three-dimensional structure, i.e., is not naturally present in the body in the form limited to a single layer of cells or lamina, but has a stucture which is spatially ordered. Examples of three-dimensional tissue are: mesenchymal tissue, cartilage and bone tissue, liver tissue, kidney tissue, neuronal tissue, fibrous tissue, dermis tissue etc. Another three-dimensional tissue is the whole embryonal epiphyseal organ derived from embryos at a post limb-bud stage.
The naturally derived connective or skeletal tissue is, in general, tissue that was derived from mesenchymal tissues that express, temporarily or continuously fibroblast growth factor receptor 3 (FGFR3). Examples of such tissue are mainly members of chondrogenic and osteogenic anlagen, as well as the residual mesenchymal stem cell reservoirs found in tissues all along life, ready to carry wound healing, repair and regeneration tasks. Another example of connective or skeletal tissue is epiphyseal tissue.
The tissue should be treated for elimination of cellular and cytosolic elements such as: DNA, RNA, proteins, lipids, proteoglycans and in general most elements of the cells which are immunogenic, as well as treated for removal of calcification-mineralization centers. Methods for elimination of the above cellular and cytosolic elements are in general known in the art.
The naturally derived connective or skeletal tissue treated as described above for elimination of cellular and cytosolic components is preferably further treated for producing porosity by the production of pores in a controlled manner. The treatment may be mechanical, for example, by hammering the tissue on a scraper device.
Alternatively, the treatment for producing porosity may be a chemical extraction process carried out by exposing the tissue, for a controlled amount of time, in a controlled environment to chemical agents capable of degradation of the tissue. In addition or alternatively, the treatment for producing porosity may be by exposing the tissue to enzymatic agents such as proteolytic enzymes, capable of partial degradation of the tissue. Example of such chemical agents which can produce pores in the tissue are guanidium chloride. The pores should have preferably a size of 10-500&mgr;, most preferably 20-100&mgr;.
The agents either specified in above (i.e. hyaluronic acid, proteoglycans, glycosaminoglycan, chondrotin sulfates, heparan sulfates, heparin and dextran sulfates) or additional agents such as adhesive molecules or growth factor moieties may be linked to the residual scaffold either by sugar cross-linking, (for example using ribose and xylose) or by carbodiimideor 1, 1 carbonyl di-imidazole. Cross-linking with the above agents i
Nevo Zvi
Robinson Dror
Ladas & Parry
Naff David M.
Ramot at Tel Aviv University Ltd.
LandOfFree
Tissue maintenance system that applies rhythmic pulses of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Tissue maintenance system that applies rhythmic pulses of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Tissue maintenance system that applies rhythmic pulses of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3134714