The Journal of Bone and Joint Surgery (American) 83:1032-1039 (2001)
© 2001 The Journal of Bone and Joint Surgery, Inc.
Effect of Bone Morphogenetic Protein-2-Expressing Muscle-Derived Cells on Healing of Critical-Sized Bone Defects in Mice
Joon Yung Lee, MD,
Douglas Musgrave, MD,
Dalip Pelinkovic, MD,
Kazumasa Fukushima, MD, PhD,
James Cummins, BSc,
Arvydas Usas, MD,
Paul Robbins, PhD,
Freddie H. Fu, MD and
Johnny Huard, PhD
Investigation performed at the Children’s Hospital of
Pittsburgh, Pittsburgh, Pennsylvania
Joon Yung Lee, MD
Douglas Musgrave, MD
Dalip Pelinkovic, MD
Kazumasa Fukushima, MD, PhD
James Cummins, BSc
Arvydas Usas, MD
Paul Robbins, PhD
Freddie H. Fu, MD
Johnny Huard, PhD
Growth and Development Laboratory, Department of Orthopaedic
Surgery (J.Y.L., D.M., D.P., K.F., J.C., A.U., and J.H.) and Department
of Molecular Genetics and Biochemistry (P.R. and J.H.), and Division
of Sports Medicine, Department of Orthopaedic Surgery (F.H.F.),
Children’s Hospital of Pittsburgh and University of Pittsburgh,
Pittsburgh, PA 15261
No benefits in any form have been received or will be received
from a commercial party related directly or indirectly to the subject
of this article. No funds were received in support of this study.
Background: Cells that express bone morphogenetic
protein-2 (BMP-2) can now be prepared by transduction with adenovirus
containing BMP-2 cDNA. Skeletal muscle tissue contains cells that
differentiate into osteoblasts on stimulation with BMP-2. The objectives
of this study were to prepare BMP-2-expressing muscle-derived cells
by transduction of these cells with an adenovirus containing BMP-2
cDNA and to determine whether the BMP-2-expressing muscle-derived
cells would elicit the healing of critical-sized bone defects in
mice.
Methods: Primary cultures of muscle-derived cells
from a normal male mouse were transduced with adenovirus encoding
the recombinant human BMP-2 gene (adBMP-2). These cells (5 ¥ 105)
were implanted into a 5-mm-diameter critical-sized skull defect
in female SCID (severe combined immunodeficiency strain) mice with
use of a collagen sponge as a scaffold. Healing in the treatment and
control groups was examined grossly and histologically at two and
four weeks. Implanted cells were identified in vivo with
use of the Y-chromosome-specific fluorescent in situ hybridization
(FISH) technique, and their differentiation into osteogenic cells
was demonstrated by osteocalcin immunohistochemistry.
Results: Skull defects treated with muscle cells
that had been genetically engineered to express BMP-2 had >85% closure
within two weeks and 95% to 100% closure within
four weeks. Control groups in which the defect was not treated (group
1), treated with collagen only (group 2), or treated with collagen and
muscle cells without adBMP-2 (group 3) showed at most 30% to
40% closure of the defect by four weeks, and the majority
of the skull defects in those groups showed no healing. Analysis
of injected cells in group 4, with the Y-chromosome-specific FISH
technique showed that the majority of the transplanted cells were
located on the surfaces of the newly formed bone, but a small fraction (approximately
5%) was identified within the osteocyte lacunae of the
new bone. Implanted cells found in the new bone stained immunohistochemically
for osteocalcin, indicating that they had differentiated in
vivo into osteogenic cells.
Conclusions: This study demonstrates that cells
derived from muscle tissue that have been genetically engineered to
express BMP-2 elicit the healing of critical-sized skull defects
in mice. The cells derived from muscle tissue appear to enhance
bone-healing by differentiating into osteoblasts in vivo.
Clinical Relevance: Ex vivo gene
therapy with muscle-derived cells that have been genetically engineered
to express BMP-2 may be used to treat nonhealing bone defects. In
addition, muscle-derived cells appear to include stem cells, which
are easily obtained with muscle biopsy and could be used in gene
therapy to deliver BMP-2.
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