Surgery – Miscellaneous – Methods
Reexamination Certificate
1996-09-06
2001-02-20
Yu, Mickey (Department: 3738)
Surgery
Miscellaneous
Methods
C623S016110, C623S011110, C623S066100, C623S901000
Reexamination Certificate
active
06189537
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns a process and apparatus for producing osteoinductive bone and osteoinductive bone produced thereby. The present inventor has surprisingly discovered that ground bone can be optimally demineralized to produce maximally osteoinductive bone and that bone is optimally demineralized when it is demineralized to the point of containing approximately 2 wt % residual calcium is maximally osteoinductive. The invention is also directed to a controlled-flow apparatus for extracting undesirable constituents from tissue. The present controlled-flow apparatus is used with the present process to produce osteoinductive bone.
BACKGROUND OF THE INVENTION
It is known that implantation of acid demineralized bone (DMB) in the form of a powder in extraskeletal sites stimulates new bone formation. Various research groups (Syftestad, 1982; Urist et al., 1967; Urist and Strates, 1979; Urist and Strates, 1971; Urist et al., 1983) have suggested that a noncollagenous protein(s) present in demineralized bone has the ability to induce new bone formation when present within the implanted bone matrix.
Procedures presently utilized to demineralize ground bone fragments involve the use of ethanol to remove lipids and hydrochloric acid to remove the mineral components of bone.
It is also known to treat bones and bone particles to render them biocompatible so that they can be implanted in living animal and human bodies without being rejected. Included among the known methods for treating bone is the dilapidation of bone using ethanol or chloroform. It is further known to demineralize bone matrix with an inorganic acid such as hydrochloric acid.
Although it is well known to defat and demineralize bone for implantation purposes, known methods of demineralizing and removing lipids have been extremely tedious, labor intensive, and slow. Furthermore, an excessive amount of handling and/or exposure of the bone to non-sterile conditions has been necessary during the various phases of processing.
SUMMARY OF THE INVENTION
The present invention is based on the present inventors surprising discovery that bone can be over and under demineralized and that this alters the osteoinductivity, as one function of the demineralized bone material, of the demineralized bone. It has been presently shown that a linear relationship exists between the pH of the eluent demineralization solution and the wt % residual calcium. Thus, it is one object of the present invention to provide controlled-flow apparatus for extracting undesirable constituents from tissue, including use with the present dilapidation and demineralization process for producing osteoinductive bone.
Included in this object is the method to restore overly demineralized bone to a state of being maximally osteoinductive.
It is further object of the present invention to provide an apparatus and method for the simultaneous dilapidation and demineralization of multiple bone samples.
It is a further object of the present invention to provide an apparatus and process for dilapidation and demineralization of bone samples which is less tedious and labor intensive than prior art methods and which is less likely to produce bone which is microbiologically contaminated following the dilapidation and demineralization process.
It is a further object of the present invention to provide a method of reprecipitating calcium phosphate in overly demineralized bone to restore it to a state of being maximally osteoinductive.
Other objects and advantages of the present invention and advantages of other features thereof will become apparent as the description proceeds herein.
Included in the description are in vivo and in vitro assays used in assessing the osteoinductive potential of demineralized bone and that demineralized bone particles of particular particle size ranges and wt % residual calcium are maximally osteoinductive. The desired wt % residual calcium levels are readily achieved using the apparatus and process described herein.
The apparatus for processing tissue comprises at least one vessel for containing the solid matter where each vessel has an upper end and lower end. Each lower end has an inlet port and each upper end has an eluent drain. Each vessel is adapted to contain solid matter during the extent of processing. The apparatus may further include a driving mechanism for conducting the solvent flow of through each vessel, which is preferably at least one peristaltic pump and solvent reservoirs for holding the solvents which are to be conducted through the vessels. Eluent solution can be monitored for change in pH, calcium ion concentration, or conductivity, through use of a pH meter or calcium specific electrode attached to an ion meter.
Preferred solid materials to be processed in the apparatus include any body tissue preferably particulate bone, cancellous bone, and/or strips or cubes of cortical bone. The entire apparatus can be contained within a controlled environment chamber which is capable of maintaining the apparatus at a processing temperature range of 4° C. to 42° C., preferably 15° C. to 30° C., and most preferably 20° C. to 25° C.
The invention further includes a method involving the soaking of overly demineralized bone in concentrated solutions of calcium phosphate made sufficiently acid to dissolve the calcium phosphate followed by reprecipitation of calcium phosphate onto and within the collagen and noncollagen matrix of the bone by increasing the pH of the solution to the point that the calcium phosphate is no longer soluble.
The invention also includes in vitro and in vivo assays for assessing the osteoinductive potential of the produced osteoinductive bone. The in vivo assay includes an athymic “nude” mouse system where the 5 to 10 mg of ground osteoinductive bone is implanted in muscle pouches in the gluteal region. The implanted material(s) are explanted, optimally after 4 weeks, cleaned of excess soft tissue and solubilized in dilute hydrochloric acid prior to determination of calcium content using any available assay, for example atomic absorption. The in vitro assay includes a tissue culture based assay where periosteum-derived cells growing in culture are exposed to 10 to 20 mg of demineralized bone per T-25 tissue culture flask containing 5 to 15 mls of an appropriate medium. After about 4 to 5 days in culture, the cells are washed and harvested and assayed for levels of alkaline phosphatase using commercially available kits (see for example Sigma Chemical Company, St. Louis, Miss.). The Atomic Absorption Assay (AA) is well know to those of ordinary skill in the art with this assay. The Arzenazo III assay involves the binding of this dye to calcium ion with a shift in absorption characteristics where the absorbance can be monitored using a spectrophotometer and absorbance is a linear function of calcium ion concentration. The alkaline phosphatase (Apase) assay involves the conversion of para-nitrophenolphosphate (PNPP) to para-nitrophenol (PNP) by the enzyme alkaline phosphate at alkaline pH. The para-nitrophenol exhibits a unique absorbance and the amount of PNP formed per unit time interval can be quantitated by comparison of changes in absorbance to a standard curve of PNP. Both assays are well know in the art and both are commercially available assays from, for example, Sigma Chemical Company, St. Louis, Miss.
REFERENCES:
patent: 4485097 (1984-11-01), Bell
patent: 4516276 (1985-05-01), Mittelmeier et al.
patent: 5275954 (1994-01-01), Wolfinbarger et al.
patent: 5531791 (1996-07-01), Wolfinbarger, Jr.
Hopkins Susanne M.
Lifenet
O'Hara Kelly
Yu Mickey
LandOfFree
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