Cell-culture and polymer constructs

Details Cell-culture and polymer constructs Cell-culture and polymer constructs

2000-11-14

2003-10-28

McDermott, Corrine (Department: 3738)

Surgery

Miscellaneous

Methods

C623S023720

Reexamination Certificate

active

06637437

ABSTRACT:

FIELD OF THE INVENTION
The herein disclosed invention finds applicability in the field of cell culture, as well as in the field of tissue substitutes for tissue replacement and for prosthesis.
BACKGROUND
Attempts at replacing or rebuilding diseased or damaged structures in the human body go back to 3000 B.C. It was not until the middle of the 1900's, however, that the use of synthetic materials for rebuilding body structures met with widespread and reproducible success. Advances in material science and biomaterials and science have afforded much of the success. The need for new and better implants exists in every field of medicine in which disease or trauma can be treated surgically.
As technology advances continue to improve the state of the art, the standards for successful implants continue to improve including strength, biocompatibility and elasticity. The new research being conducted today on growth factors and controlled drug release tell of the day when implant material will be expected to promote healing, dissipate disease and stimulate tissue regeneration.
The inventors have continued to make improvements to more efficiently produce good quality cells and of sufficient quantity which are able to be transplanted effectively.
Beta-1 Integrin Immunolocalization on Human Chondrocytes Attached to Collagen Microcarriers
Introduction: Interactions between the extracellular and the intracellular environment are known to be mediated by transmembrane glycoprotein receptors called integrins. Consisting of alpha and beta chains, integrins are non-covalently bonded protein complexes. These proteins are present in a whole array of cells, including human chondrocytes. Integrins are known to mediate cell attachment and are also involved in cell signaling pathways (Boudreau
1
). It has been documented that cartilage homeostasis and metabolism is highly influenced by the interaction between the chondrocytes and the extracellular matrix. The role of integrins as mediators of this interaction on chondrocytes has recently been reported (Lapadula
2
). It has also been documented that the expression of integrin chains may be inversely correlated to the degree of damage in pathological conditions such as osteoarthritis (Lapadula
2
).
It has been previously reported that human articular chondrocytes propagated in microcarrier spinner culture produce the extracellular matrix components collagen type II and proteoglycans more actively than cells in monolayer culture (Frondoza
3
). The mechanism by which the microcarrier spinner culture promotes the chondrocytic phenotype is not clear. The present study tests the hypothesis that maintenance of chondrocytic phenotype in microcarrier spinner culture may involve integrin &bgr;1. The inventors have immunolocalized integrin &bgr;-1 while the cells are still attached to the collagen beads. The main goal was to visualize the expression of integrin &bgr;-1 on chondrocytes without disrupting the cell-material interaction.
Methods: Cartilage was obtained from two different tissue sites: (a) knee cartilage from osteoarthritic patients at the time of total knee replacement, (b) nasal cartilage from patients during nasal septum reconstruction. Chondrocytes isolated by collagenase digestion were directly seeded at 4×10
3
cells/cm
2
onto collagen microcarriers (Cellagen™ 100-400 &mgr;m derived from bovine corium, ICN, Cleveland, Ohio) previously described (Frondoza
3
). Microcarrier spinner cultures were incubated at 37° C., 5% CO
2
for fourteen days. Chondrocytes were sedimented in conical tubes and then aliquoted onto microscope slides. Viability of chondrocytes was determined using trypan blue vital dye. Chondrocytes were transferred to microscope slides, fixed with 2% paraformaldehyde, and air-dried. Chondrocytes on microcarriers were then immunostained using immunoperoxidase with monospecific antibodies for &bgr;1 (Chemicon International, Inc.), collagen types I and II (Fisher Scientific, Pittsburgh, Pa.); and keratan sulfate (ICN Biomedicals, Inc., Aurora, Ohio).
Total RNA was isolated by the TRIzol Reagent method (Life Technologies, Rockville, Md.). A total cDNA library was synthesized using the Advantage RT-PCR Kit (Clontech Laboratories, Palo Alto, Calif.) with the Oligo (dT
18
) primer. The resulting reverse transcriptase product was expanded using the SuperTaq Plus (Ambion, Austin, Tex.) PCR Kit and specific primers for collagen type II, type I, aggrecan and the housekeeping genes GADPH and ribosomal RNA S14 subunit. The PCR products were analyzed by agarose gel electrophoresis.
Results: Cells attached to the surface of microcarriers remained viable after two weeks in culture. Chondrocytes isolated from knee or nasal cartilage displayed similar growth patterns and immunostaining characteristics. Microcarriers are seen covered by cells with their surrounding dense extracellular matrix. Many microcarriers exhibited halo-like outgrowths of cells with their matrix-like material. Cells on microcarriers stained more intensely for integrin &bgr;, collagen type II, and keratan sulfate. Microcarriers were seen forming clusters of up to 8 beads. There was insignificant staining for collagen type I, and also when the primary antibodies were omitted. Expression of &bgr;1 integrin, collagens type I and II and proteoglycans was verified with the RT-PCR semi-quantitative analysis.
The inventors have found that &bgr;1 integrin is strongly co-expressed with collagen type II and proteoglycans by chondrocytes on microcarrier spinner culture. Cell inside-out and outside in signaling, as well as regulation of extracellular matrix metabolism, have been documented to be integrin-mediated (Lapadula
2
). Microcarrier spinner culture, a biomechanically active environment, may promote and enhance integrin &bgr;1 expression on chondrocytes, as well as their participation in the maintenance of the original chondrocyte phenotype. The inventors intend to employ integrin expression as a measure of viability of chondroctyes.
Collagen Microcarriers Support the Phenotypic Expression of Chondrocytes from Human Knee, Nasal, and Ankle Cartilage
Introduction: A novel approach to repair articular cartilage currently being investigated, is cell therapy. Cells are transplanted by themselves or transplanted in a delivery vehicle such as resorbable polymers. As an avascular and alymphatic tissue, articular cartilage does not adequately heal and the repair tissue is frequently fibrocartilage. The repair consists primarily of collagen type I, rather than collagen type II which is characteristic of hyaline articular cartilage. The proteoglycan content is altered from high (aggrecan) to low molecular weight. The inability of cartilage to heal is also attributed to the limited capacity of chondrocytes, the only cellular constituent of cartilage to proliferate and produce components of the surrounding extracellular matrix (Buckwater
4
). Alteration in the chemical composition of articular cartilage leads to physical changes that compromise the biomechanical function of the joint.
A major problem in the use of cell-based therapy is the limited number of cells capable of producing the appropriate extracellular matrix that constitute hyaline cartilage. The inventors have propagated chondrocytes from three distinct cartilaginous tissues: the knee, nose, and ankle. Propagation has taken place on collagen microcarriers using the suspension spinner culture technique. Collagen microcarrier spinner culture promotes the chondrocytic phenotype of cells retrieved from different cartilage sites. It is obvious that additional tissue sources of chondrocytic cells are desirable and provide additional donor pool of cells for articular cartilage repair. This is a major object of this invention.
Cartilage was obtained from three different tissue sites: (a) knee cartilage from 5 osteoarthritic patients at the time of total knee replacement, (b) ankle cartilage from 5 patients with vascular disease undergoing below the knee amputation, and (c) nasal cartilage from 5 patients during nasal septum reconstru

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