Patrick Bosch

Growth factors improve muscle healing in vivo

Injury to muscles is very common. We have previously observed that basic fibroblast growth factor (b-FGF), insulin growth factor type 1 (IGF-1) and nerve growth factor (NGF) are potent stimulators of the proliferation and fusion of myoblasts in vitro. We therefore injected these growth factors into mice with lacerations of the gastrocnemius muscle. The muscle regeneration was evaluated at one week by histological staining and quantitative histology. Muscle healing was assessed histologically and the contractile properties were measured one month after injury. Our findings showed that b-FGF, IGF and to a less extent NGF enhanced muscle regeneration in vivo compared with control muscle. At one month, muscles treated with IGF-1 and b-FGF showed improved healing and significantly increased fast-twitch and tetanus strengths. Our results suggest that b-FGF and IGF-1 stimulated muscle healing and may have a considerable effect on the treatment of muscle injuries.

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (beta-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.

Ex Vivo Gene Therapy to Produce Bone Using Different Cell Types

Gene therapy and tissue engineering promise to revolutionize orthopaedic surgery. This study comprehensively compares five different cell types in ex vivo gene therapy to produce bone. The cell types include a bone marrow stromal cell line, primary muscle derived cells, primary bone marrow stromal cells, primary articular chondrocytes, and primary fibroblasts. After transduction by an adenovirus encoding for bone morphogenetic protein-2, all of the cell types were capable of secreting bone morphogenetic protein-2. However, the bone marrow stromal cell line and muscle derived cells showed more responsiveness to recombinant human bone morphogenetic protein-2 than did the other cell types. In vivo injection of each of the cell populations transduced to secrete bone morphogenetic protein-2 resulted in bone formation. Radiographic and histologic analyses corroborated the in vitro data regarding bone morphogenetic protein-2 secretion and cellular osteocompetence. This study showed the feasibility of using primary bone marrow stromal cells, primary muscle derived cells, primary articular chondrocytes, primary fibroblasts, and an osteogenesis imperfecta stromal cell line in ex vivo gene therapy to produce bone. The study also showed the advantages and disadvantages inherent in using each cell type.

Human skeletal muscle cells in ex vivo gene therapy to deliver bone morphogenetic protein-2

We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised beta-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised beta-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches.

Biomechanical analysis of pinning techniques for pediatric supracondylar humerus fractures.

Background: Closed reduction and percutaneous pin fixation is the recommended treatment of displaced (Gartland types 2 and 3) supracondylar humerus fractures. The need for a medial pin for maximal stability remains controversial. The purpose of this study was to develop a model of supracondylar humerus fractures simulating medial column comminution and to evaluate the torsional stability of various pin configurations recommended in the current literature. Methods: Transverse cuts were made in synthetic humeri with a wedge taken from the medial aspect of the proximal fracture fragment in one half of the specimens to simulate medial column comminution. Each fracture was then reduced and fixed with 1 of 4 pin configurations using 0.062 in K-wires. The fixed specimens were then subjected to a torsional load producing internal rotation of the distal fragment. Rotation in degrees and the corresponding torque was recorded for statistical analysis. Results: Specimens with the medial wedge removed demonstrated less torsional stability than their identically fixed counterparts with the intact medial column. In specimens with the intact medial column, the greatest torsional stability was achieved with the 2 lateral divergent and medial cross pin configuration followed by 3 lateral pins, then standard crossed pins with 2 lateral divergent pins demonstrating the least torsional stability. For the medial comminution group the 2 lateral, 1 medial pin construct again had the greatest torsional stability and 2 lateral pins the least. The standard crossed pin and 3 lateral pin constructs were not significantly different in the presence of medial comminution. Conclusions: In a synthetic humerus model of supracondylar humerus fractures, medial comminution was shown to reduce torsional stability significantly in all pin configurations. There was no statistical difference in torsional stability between 3 lateral pins and standard crossed pins in specimens with medial comminution.

The efficiency of muscle-derived cell-mediated bone formation.

The development of new clinically applicable methods for the delivery of bone morphogenic protein (BMP) is an area of intensive research. Cell-mediated gene therapy approaches are being explored as a potential delivery vehicle. Primary muscle-derived cells isolated from an adult mouse were transduced with an adenoviral–BMP-2 construct. These cells were injected into the triceps surae of severe combined immune deficient (SCID) mice where they induced heterotopic bone formation. BMP-2 expression by these muscle-derived cell constructs was measured in vitro to estimate in vivo BMP-2 delivery. In vitro expression of BMP-2 by 3 × 105 muscle-derived cells was 87.89 ng/72 h. These results suggest that the efficiency of muscle cell-based gene delivery of BMP-2 exceeds the direct delivery of recombinant BMP-2 protein.

Effect of Cultural Factors on Outcome of Ponseti Treatment of Clubfeet in Rural America

BACKGROUND Nonoperative management of clubfoot with the Ponseti method has proven to be effective, and it is the accepted initial form of treatment. Although several studies have shown that problems with compliance with the brace protocol are principally responsible for recurrence, no distinction has been made with regard to whether the distance from the site of care affects the early recurrence rate. We compared early recurrence after Ponseti treatment between rural and urban ethnically diverse North American populations to analyze whether distance from the site of care affects compliance and whether certain patient demographic characteristics predict recurrence. METHODS One hundred consecutive infants with a total of 138 clubfeet treated with the Ponseti method were followed prospectively for at least two years from the beginning of treatment. Early recurrence, defined as the need for subsequent cast treatment or surgical treatment, and compliance, defined as strict adherence to the brace protocol described by Ponseti, were analyzed with respect to the distance from the site of care, age at presentation, number of casts needed for the initial correction, need for tenotomy, and family demographic variables. RESULTS Of eighteen infants from a rural area who had early recurrence, fourteen were Native American. The families of these children, like those of all of the children with early recurrence, discontinued orthotic use earlier than was recommended by the physician. Discontinuation of orthotic use was related to recurrence, with an odds ratio of 120 (p < 0.0001), in patients living in a rural area. Native American ethnicity, unmarried parents, public or no insurance, parental education at the high-school level or less, and a family income of less than

The effect of bone morphogenetic protein-2 expression on the early fate of skeletal muscle-derived cells

20,000 were also significant risk factors for recurrence in patients living in a rural area. Intrinsic factors of the clubfoot deformity were not correlated with recurrence or discontinuation of bracing. CONCLUSIONS Compliance with the orthotic regimen after cast treatment is imperative for the Ponseti method to succeed. The striking difference in outcome in rural Native American patients as compared with the outcomes in urban Native American patients and children of other ethnicities suggests particular problems in communicating to families in this subpopulation the importance of bracing to maintain correction. An examination of communication styles suggested that these communication failures may be culturally related.

Biologic intervention in muscle healing and regeneration

The identification of bone morphogenetic proteins (BMPs) has stimulated intense interest in BMP delivery approaches. Ex vivo BMP-2 gene delivery has recently been described using skeletal muscle-derived cells. Skeletal muscle-derived cells, because of proven efficient transgene delivery and osteocompetence, represent an attractive cell population on which to base ex vivo BMP-2 gene delivery. However, the early in vivo fate of BMP-2-expressing muscle-derived cells is unknown. This study investigates the in vivo effects of BMP-2 secretion on skeletal muscle-derived cells in terms of cell survival and cell differentiation. The first experiment compared survival of BMP-2-expressing cells with control cells during the first 48 h after in vivo implantation. The results demonstrate that BMP-2 secretion did not adversely affect cell survival 8, 24, or 48 h after intramuscular implantation. The second experiment histologically compared the fate of BMP-2-expressing muscle-derived cells to the same cells not expressing BMP-2. The results show that BMP-2 expression prevented in vivo myogenic differentiation and promoted osteogenic differentiation of the transduced cells. This study further supports the existence of osteoprogenitor cells residing within skeletal muscle. Moreover, it is demonstrated that BMP-2 secretion does not adversely affect early cell survival of muscle-derived cells. These data are important for future investigations into BMP-2 gene delivery approaches to the musculoskeletal system.

Prospective, surgeon-randomized evaluation of crossed pins versus lateral pins for unstable supracondylar humerus fractures in children.

Muscle injuries are a challenging problem in traumatology and the most frequently occurring injuries in sports medicine. Even though muscles retain their ability to regenerate after injury, the healing process of muscles after such injuries has been found to be slow and often leads to an incomplete muscle recovery. In an attempt to develop approaches to improve muscle healing after injury, the authors have developed reproducible injury models for muscle contusion, strain, and laceration. The authors show that muscle regeneration occurs after those injuries, but the development of scar tissue greatly limits the natural healing process. It is likely that an enhancement of muscle growth and regeneration can be used to improve muscle healing after injuries. The authors have then identified growth factors that enhance myoblast proliferation and differentiation in vitro and muscle regeneration in the injured muscles, which improves muscle healing after injuries. Furthermore, different gene transfer systems, including cell and gene therapy, have been found successful in delivering genes into injured muscles and may open new opportunities to deliver growth factors and improve muscle healing after lacerations, contusions, and strains.