Attenuated human bone morphogenetic protein-2-mediated bone regeneration in a rat model of composite bone and muscle injury.

Extremity injuries involving large bone defects with concomitant severe muscle damage are a significant clinical challenge often requiring multiple treatment procedures and possible amputation. Even if limb salvage is achieved, patients are typically left with severe short- and long-term disabilities. Current preclinical animal models do not adequately mimic the severity, complexity, and loss of limb function characteristic of these composite injuries. The objectives of this study were to establish a composite injury model that combines a critically sized segmental bone defect with an adjacent volumetric muscle loss injury, and then use this model to quantitatively assess human bone morphogenetic protein-2 (rhBMP-2)-mediated tissue regeneration and restoration of limb function. Surgeries were performed on rats in three experimental groups: muscle injury (8-mm-diameter full-thickness defect in the quadriceps), bone injury (8-mm nonhealing defect in the femur), or composite injury combining the bone and muscle defects. Bone defects were treated with 2 μg of rhBMP-2 delivered in the pregelled alginate injected into a cylindrical perforated nanofiber mesh. Bone regeneration was quantitatively assessed using microcomputed tomography, and limb function was assessed using gait analysis and muscle strength measurements. At 12 weeks postsurgery, treated bone defects without volumetric muscle loss were consistently bridged. In contrast, the volume and mechanical strength of regenerated bone were attenuated by 45% and 58%, respectively, in the identically treated composite injury group. At the same time point, normalized muscle strength was reduced by 51% in the composite injury group compared to either single injury group. At 2 weeks, the gait function was impaired in all injury groups compared to baseline with the composite injury group displaying the greatest functional deficit. We conclude that sustained delivery of rhBMP-2 at a dose sufficient to induce bridging of large segmental bone defects failed to promote regeneration when challenged with concomitant muscle injury. This model provides a platform with which to assess bone and muscle interactions during repair and to rigorously test the efficacy of tissue engineering approaches to promote healing in multiple tissues. Such interventions may minimize complications and the number of surgical procedures in limb salvage operations, ultimately improving the clinical outcome.

[1]  M. Allen,et al.  Functional recovery of the plantarflexor muscle group after hindlimb unloading in the rat , 2004, European Journal of Applied Physiology.

[2]  David J Mooney,et al.  Quantitative assessment of scaffold and growth factor‐mediated repair of critically sized bone defects , 2007, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[3]  John F Kragh,et al.  Combat wounds in operation Iraqi Freedom and operation Enduring Freedom. , 2008, The Journal of trauma.

[4]  Ann Sandison,et al.  Comparison of the Vascularity of Fasciocutaneous Tissue and Muscle for Coverage of Open Tibial Fractures , 2009, Plastic and reconstructive surgery.

[5]  T A Einhorn,et al.  Enhancement of fracture-healing. , 1995, The Journal of bone and joint surgery. American volume.

[6]  R. Burns,et al.  Limb salvage versus traumatic amputation. A decision based on a seven-part predictive index. , 1991, Annals of surgery.

[7]  A Daluiski,et al.  The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. , 1999, The Journal of bone and joint surgery. American volume.

[8]  Renjing Liu,et al.  Myogenic progenitors contribute to open but not closed fracture repair , 2011, BMC musculoskeletal disorders.

[9]  R. Carano,et al.  Angiogenesis and bone repair. , 2003, Drug discovery today.

[10]  T. Walters,et al.  Functional assessment of skeletal muscle regeneration utilizing homologous extracellular matrix as scaffolding. , 2010, Tissue engineering. Part A.

[11]  D. Hutmacher,et al.  Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects. , 2011, Bone.

[12]  M. Schaffler,et al.  Prevention of fracture healing in rats by an inhibitor of angiogenesis. , 2001, Bone.

[13]  F. Wei,et al.  One-stage reconstruction of composite bone and soft-tissue defects in traumatic lower extremities. , 2004, Plastic and reconstructive surgery.

[14]  P. Kostenuik,et al.  Transient muscle paralysis degrades bone via rapid osteoclastogenesis , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[15]  Jay R Lieberman,et al.  The role of growth factors in the repair of bone. Biology and clinical applications. , 2002, The Journal of bone and joint surgery. American volume.

[16]  John F Kragh,et al.  Characterization of Extremity Wounds in Operation Iraqi Freedom and Operation Enduring Freedom , 2007, Journal of orthopaedic trauma.

[17]  F. Shapiro,et al.  Bone development and its relation to fracture repair. The role of mesenchymal osteoblasts and surface osteoblasts. , 2008, European cells & materials.

[18]  O. Reikerås,et al.  Poor muscle coverage delays fracture healing in rats , 2002, Acta orthopaedica Scandinavica.

[19]  James F Kellam,et al.  An analysis of outcomes of reconstruction or amputation after leg-threatening injuries. , 2002, The New England journal of medicine.

[20]  Robert E Guldberg,et al.  In vitro and in vivo osteoblastic differentiation of BMP‐2‐ and Runx2‐engineered skeletal myoblasts , 2007, Journal of cellular biochemistry.

[21]  Tien-Min G. Chu,et al.  Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2. , 2007, Biomaterials.

[22]  Konstantinos N Malizos,et al.  The healing potential of the periosteum molecular aspects. , 2005, Injury.

[23]  R. Guldberg,et al.  Effects of in vivo mechanical loading on large bone defect regeneration , 2012, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[24]  W. Virkus,et al.  Healing of rat femoral segmental defect with bone morphogenetic protein-2: a dose response study. , 2012, Journal of musculoskeletal & neuronal interactions.

[25]  J. C. McDermott,et al.  Identification of secreted proteins during skeletal muscle development. , 2007, Journal of proteome research.

[26]  L. Claes,et al.  Moderate soft tissue trauma delays new bone formation only in the early phase of fracture healing , 2006, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[27]  O. Reikerås,et al.  Influence of Extensive Muscle Injury on Fracture Healing in Rat Tibia , 2003, Journal of orthopaedic trauma.

[28]  M. Febbraio,et al.  Muscles, exercise and obesity: skeletal muscle as a secretory organ , 2012, Nature Reviews Endocrinology.

[29]  M. McKee,et al.  Managing Bone Defects , 2011, Journal of orthopaedic trauma.

[30]  L. Liang,et al.  Recombinant myostatin (GDF-8) propeptide enhances the repair and regeneration of both muscle and bone in a model of deep penetrant musculoskeletal injury. , 2010, The Journal of trauma.

[31]  Ann Sandison,et al.  Comparison of the healing of open tibial fractures covered with either muscle or fasciocutaneous tissue in a murine model , 2008, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[32]  Andrés J. García,et al.  Effects of protein dose and delivery system on BMP-mediated bone regeneration. , 2011, Biomaterials.

[33]  C. Edwards,et al.  Severe open tibial fractures. Results treating 202 injuries with external fixation. , 1988, Clinical orthopaedics and related research.

[34]  C. Krettek,et al.  Nonreamed interlocking nailing of closed tibial fractures with severe soft tissue injury. , 1995, Clinical orthopaedics and related research.

[35]  A Ignatius,et al.  Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting. , 2011, Bone.

[36]  A. Masquelet,et al.  Muscle reconstruction in reconstructive surgery: soft tissue repair and long bone reconstruction , 2003, Langenbeck's Archives of Surgery.

[37]  P. Mcneil,et al.  Role of muscle-derived growth factors in bone formation. , 2010, Journal of musculoskeletal & neuronal interactions.

[38]  R. Hayda,et al.  Risk factors for and results of late or delayed amputation following combat-related extremity injuries. , 2010, Orthopedics.

[39]  A. Schindeler,et al.  The contribution of different cell lineages to bone repair: exploring a role for muscle stem cells. , 2009, Differentiation; research in biological diversity.

[40]  Robert E Guldberg,et al.  Mechanical regulation of vascular growth and tissue regeneration in vivo , 2011, Proceedings of the National Academy of Sciences.

[41]  T. Guda,et al.  Improving bone formation in a rat femur segmental defect by controlling bone morphogenetic protein-2 release. , 2011, Tissue engineering. Part A.

[42]  Tohru Kiyono,et al.  Osteogenic properties of human myogenic progenitor cells , 2008, Mechanisms of Development.

[43]  C. Mann,et al.  Aberrant repair and fibrosis development in skeletal muscle , 2011, Skeletal Muscle.

[44]  H. Tscherne,et al.  [Pathophysiology and classification of soft tissue damage in fractures]. , 1983, Der Orthopade.

[45]  David J Mooney,et al.  An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects. , 2011, Biomaterials.

[46]  P. Harrington Open tibial fractures with severe soft-tissue loss. Limb salvage compared with below-the-knee amputation. , 1994, The Journal of bone and joint surgery. American volume.

[47]  S. Bain,et al.  The effect of muscle dysfunction on bone mass and morphology. , 2010, Journal of musculoskeletal & neuronal interactions.

[48]  V. Rosen,et al.  THE HEALING OF SEGMENTAL BONE DEFECTS, INDUCED BY RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN (rhBMP‐2): A RADIOGRAPHIC, HISTOLOGICAL, AND BIOMECHANICAL STUDY IN RATS , 1993 .

[49]  V. Rosen,et al.  The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. , 1992, The Journal of bone and joint surgery. American volume.

[50]  W. Evans,et al.  Sarcopenia and age-related changes in body composition and functional capacity. , 1993, The Journal of nutrition.

[51]  J. Dwek The periosteum: what is it, where is it, and what mimics it in its absence? , 2010, Skeletal Radiology.

[52]  James F Kellam,et al.  Long-term persistence of disability following severe lower-limb trauma. Results of a seven-year follow-up. , 2005, The Journal of bone and joint surgery. American volume.

[53]  Brandon J. Ausk,et al.  Cortical bone resorption following muscle paralysis is spatially heterogeneous. , 2012, Bone.

[54]  M. Carreira,et al.  The GH/IGF1 axis and signaling pathways in the muscle and bone: mechanisms underlying age-related skeletal muscle wasting and osteoporosis. , 2010, The Journal of endocrinology.

[55]  R. Gustilo,et al.  The management of open fractures. , 1990, The Journal of bone and joint surgery. American volume.