Method of making trabecular bone
Inventors
Blair, Harry Colbert • Larrouture, Quitterie • Tourkova, Irina L. • Nelson, Deborah J. • Schlesinger, Paul
Assignees
University of Chicago • University of Pittsburgh • US Department of Veterans Affairs • Washington University in St Louis WUSTL
Publication Number
US-11060060-B2
Publication Date
2021-07-13
Expiration Date
2036-09-16
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Abstract
Provided herein are methods of producing trabecular bone material, such as trabecular bone or trabecular bone matrix and cells useful in preparing the trabecular bone material.
Core Innovation
Provided herein is a method of producing trabecular bone material by culturing CLC3 knockdown osteoblasts ex vivo in culture medium for a length of time sufficient to produce bone material. The method includes obtaining CLC3 knockdown osteoblasts by culturing CLC3 knockdown mesenchymal stem cells or osteoblast precursor cells in osteoblast differentiation medium. The bone material produced, which may be live bone or devitalized bone matrix, has a trabecular three-dimensional structure, such as hydroxyapatite deposits, and is suitable for implantation.
The problem addressed is the lack of suitable methods for preparing bone tissue, particularly trabecular bone tissue, for treating osteoporosis, traumatic injury, or bone defects. Prior attempts using bioreactors with mesenchymal stem cells and scaffolds have failed to reliably produce three-dimensional bone tissue with meaningful trabecular architecture. Difficulties include expanding mesenchymal stem cells effectively and decoupling cell growth from bone matrix formation. Thus, reliable generation of trabecular bone in vitro remains a substantial challenge.
The invention is based on the discovery that loss or knockdown of CLC3, a chloride voltage-gated channel expressed in mineralizing osteoblasts, leads to novel trabecular pattern mineralization in vitro. While knockout of CLC3 alone results in mild bone phenotype due to compensatory CLC5 expression, combined CLC3 knockout and CLC5 knockdown abolishes mineralization. This indicates that regulated acid export mediated by chloride-proton exchangers is essential for normal mineralization and patterning. Using CLC3 knockdown mesenchymal stem cells cultured in osteoblast differentiation medium produces osteoblasts capable of forming trabecular bone material reliably in vitro.
Claims Coverage
The patent includes one independent claim directed to a method for producing trabecular bone material and dependent claims detailing specific aspects of the method. The following inventive features are identified:
Method of producing trabecular bone material by culturing CLC3 knockout osteoblasts
A method comprising culturing CLC3 knockout osteoblasts ex vivo in culture medium for a sufficient duration to produce trabecular bone material.
Obtaining CLC3 knockout osteoblasts from CLC3 knockout mesenchymal stem cells or precursors
The CLC3 knockout osteoblasts are obtained by culturing CLC3 knockout mesenchymal stem cells or osteoblast precursor cells in an osteoblast differentiation medium for a sufficient length of time such that bone material is produced.
Use of human or murine mesenchymal stem cells or osteoblast precursors
The mesenchymal stem cells or osteoblast precursor cells used are human or murine in origin.
Composition of osteoblast differentiation medium
The osteoblast differentiation medium comprises ascorbic acid and a phosphate source such as glycerol-2-phosphate, and may not contain glucocorticoids.
Preparation of CLC3 knockout osteoblasts via differentiation
A method comprising isolating CLC3 knockout mesenchymal stem cells or osteoblast precursors from a CLC3 knockout transgenic animal and differentiating them to an osteoblast phenotype capable of producing trabecular bone material.
The claims cover methods of producing trabecular bone material by culturing CLC3 knockout osteoblasts derived from CLC3 knockout mesenchymal stem cells or precursors under osteoblast differentiation conditions, highlighting specific medium compositions and species, and methods for preparing the knockout cells from transgenic animals.
Stated Advantages
The method enables reliable production of trabecular bone material, which is a substantial challenge with prior approaches.
Utilizing CLC3 knockdown osteoblasts reduces dependency on scaffolds and increases efficiency of bone matrix formation.
The produced bone material can be used directly or devitalized for implantation, supporting orthopedic reconstruction and healing of large defects.
Documented Applications
Implantation to support engraftment of joints and other orthopedic and reconstructive applications such as titanium rods for teeth.
Support healing of nonunion fractures.
Support healing of large bone defects, including skull defects.
Treatment of osteoporosis by introduction of the bone material at sites of trabecular bone loss such as spine, hip, or wrist.
Combination with metallic, ceramic, or polymeric medical implant devices including screws, pins, artificial joint implants, or prostheses.
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