Clinical Orthopaedics and Related Research ®

A Publication of The Association of Bone and Joint Surgeons ®

Published in
Clinical Orthopaedics and Related Research®
Volume 467 | Issue 12 | Dec, 2009

The Classic: Bone Morphogenetic Protein

Marshall R. Urist MD, Basil S. Strates [object Object]

Levels of Expression for BMP-7 and Several BMP Antagonists May Play an Integral Role in a Fracture Nonunion: A Pilot Study

Marc Fajardo MD, Chuan-Ju Liu PhD, Kenneth Egol MD Delays in bone healing or even the development of a nonunion could be related to the concentrations and/or functions of the bone morphogenetic proteins (BMPs). The RNA expression profile of the BMPs within fracture nonunion tissue is unknown. This preliminary descriptive study was performed to define the RNA profiles of the BMPs, their receptors, and their inhibitors within human fracture nonunion tissue and correlate them to matched healing bone. All patients had hypertrophic nonunions. Tissue samples taken from the nonunion site of 15 patients undergoing surgical treatment for an established nonunion were analyzed. The RNA expression patterns of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8; BMP receptor Types IA, IB, and II; and the BMP inhibitors chordin, Noggin, Drm (Gremlin), and follistatin were determined in the nonunion (fibrous tissue) and healing bone (callus tissue) using quantitative real-time PCR. Comparison between the nonunion and healing bone samples revealed substantially elevated concentrations of BMP-4, Drm/Gremlin, follistatin, and Noggin in nonunion tissue when compared to healing bone. In contrast, BMP-7 concentration was higher in the healing bone. Our data suggest inhibition of BMP-7, by Drm (Gremlin), follistatin, and Noggin and upregulation of BMP-4 may play an integral role in the development of nonunions.

Cancellous Bone Properties and Matrix Content of TGF-β2 and IGF-I in Human Tibia: A Pilot Study

Yener N. Yeni PhD, X. Neil Dong PhD, Bingbing Zhang MD, Gary J. Gibson PhD, David P. Fyhrie PhD [object Object]

Stimulation of Ectopic Bone Formation in Response to BMP-2 by Rho Kinase Inhibitor: A Pilot Study

Hideki Yoshikawa MD, PhD, Kiyoko Yoshioka, Takanobu Nakase MD, PhD, Kazuyuki Itoh MD, PhD The small GTPase Rho and Rho-associated protein kinase (Rho kinase, ROCK) signal participates in a variety of biological functions including vascular contraction, tumor invasion, and penile erection. Evidence also suggests Rho-ROCK is involved in signaling for mesenchymal cellular differentiation. However, whether it is involved in osteoblastic differentiation is unknown. We therefore asked whether Rho-ROCK signaling participates in recombinant human bone morphogenetic protein (rhBMP-2)-induced osteogenesis both in vitro and in vivo. Continuous delivery of a specific ROCK inhibitor (Y-27632) enhanced ectopic bone formation induced by rhBMP-2 impregnated into an atelocollagen carrier in mice without affecting systemic bone metabolism. Treatment with Y-27632 also enhanced the osteoblastic differentiation of cultured murine neonatal calvarial cells. These effects were associated with increased expression of BMP-4 gene. Expression of a dominant negative mutant of ROCK in ST2 cells promoted osteoblastic differentiation, while a constitutively active mutant of ROCK attenuated osteoblastic differentiation and the ROCK inhibitor reversed this phenotype. Thus, ROCK inhibits osteogenesis, and a ROCK inhibitor in combination with the local delivery of rhBMP/collagen composite may be clinically applicable for stimulating bone formation.

rhBMP-2 Modulation of Gene Expression in Infected Segmental Bone Defects

Katherine E. Brick BS, Xinqian Chen MD, Jamie Lohr MD, Andrew H. Schmidt MD, Louis S. Kidder PhD, William D. Lew MS The osteoinductive capability of BMPs appears diminished in the setting of acute infection. We applied rhBMP-2 to a segmental defect in a rat femur and measured the expression of key bone formation genes in the presence of acute infection. Types I and II collagen, osteocalcin, and BMP Type II receptor mRNA expression were characterized in 72 Sprague-Dawley rats, which received either bovine collagen carrier with 200 μg rhBMP-2 plus Staphylococcus aureus, carrier with bacteria only, carrier with rhBMP-2 only, or carrier alone. Six animals from each group were euthanized at 1, 2, and 4 weeks. Total RNA was isolated and extracted, and mRNA was determined by real-time comparative quantitative PCR. Infected defects had little expression of collagen I and II and osteocalcin mRNAs, while BMP receptor II expression with infection was greater than carrier-only controls at Weeks 2 and 4. Notably, all four genes were upregulated in infected defects in the presence of rhBMP-2. Thus, in a clinical setting with a high risk of infection and nonunion, such as a compound fracture with bone loss, rhBMP-2 may increase the rate and extent of bone formation. Even if infection does occur, rhBMP-2 may allow a quicker overall recovery time.

OP-1 Augments Glucocorticoid-inhibited Fracture Healing in a Rat Fracture Model

Robert S. Gilley DVM, PhD, DACVS, Larry J. Wallace DVM, MS, DACVS, Craig A. Bourgeault BS, Louis S. Kidder PhD, Joan E. Bechtold PhD Glucocorticoids inhibit bone remodeling and fracture healing. We sought to determine whether osteogenic protein 1 (OP-1) can overcome this inhibition in a closed fracture model in the rat. Time-released prednisolone or placebo pellets were implanted subcutaneously; closed femoral fractures were created 2 weeks later in rats. Fractures received sham, OP-1 and collagen, or collagen-only implants. Femurs were harvested at 3, 10, 21, 28, and 42 days postfracture. Fractures were examined radiographically for amount of hard callus; mechanically for torque and stiffness (also expressed as a percentage of the contralateral intact femur); and histomorphometrically for amount of cartilaginous and noncartilaginous soft callus, hard callus, and total callus. Glucocorticoid administration inhibited fracture healing. The application of a devitalized Type I collagen matrix mitigated the inhibitory effects of prednisolone on fracture healing However, further increases in indices of fracture healing were observed when OP-1 was added to the collagen matrix compared with collagen alone. OP-1 and collagen was more effective than collagen alone.

Can rhBMP-2 Containing Collagen Sponges Enhance Bone Repair in Ovariectomized Rats?: A Preliminary Study

Sezgin Sarban MD, Alparslan Senkoylu MD, U. Erdem Isikan MD, Petek Korkusuz MD, PhD, Feza Korkusuz MD With an aging population the frequency of postmenopausal fractures is increasing. Methods to enhance the repair of osteoporotic bone repair therefore become more important to reduce the society burden of care. We asked if absorbable collagen sponges containing recombinant human bone morphogenetic protein-2 (rhBMP-2) have the potential to enhance bone repair. We randomly assigned 40 rats into the ovariectomy and sham operation groups. A segmental defect was created in the right tibia 12 weeks after ovariectomy. rhBMP-2-containing absorbable collagen sponges were implanted into the defect in half of the animals in each group. We analyzed radiographs and histological sections and performed three-point bending tests to assess repair. Radiological scores in the rhBMP-2 applied rats were higher than those in controls at the end of 8 weeks after tibial osteotomy. The specimens failed under higher loads in the rhBMP-2-applied groups and histology revealed a higher fracture healing score, including callus formation, bone union, marrow changes, and cortex remodeling. We observed no adverse tissue responses such as fibrous connective tissue formation and inflammatory cellular infiltration. rhBMP-2 in absorbable collagen sponges enhanced bone repair in segmental tibial defects of ovariectomized rats. The sponges with rhBMP-2 appeared to enhance bone repair.

Alendronate Enhances Osteogenic Differentiation of Bone Marrow Stromal Cells: A Preliminary Study

Hyung Keun Kim PhD, Ji Hyun Kim MS, Azlina Amir Abbas MS (Ortho), Taek Rim Yoon MD Alendronate inhibits osteoclastic activity. However, some studies suggest alendronate also has effects on osteoblast activity. We hypothesized alendronate would enhance osteoblastic differentiation without causing cytotoxicity of the osteoblasts. We evaluated the effect of alendronate on the osteogenic differentiation of mouse mesenchymal stem cells. D1 cells (multipotent mouse mesenchymal stem cells) were cultured in osteogenic differentiation medium for 7 days and then treated with alendronate for 2 days before being subjected to various tests using MTT assays, Alizarin Red, enzyme-linked immunosorbent assay, energy-dispersive xray spectrophotometry, reverse transcriptase–polymerase chain reaction, confocal microscopy, and flow cytometric analysis. D1 cells differentiated into osteoblasts in the presence of osteogenic differentiation medium as confirmed by positive Alizarin Red S staining, increased alkaline phosphatase activity and osteocalcin mRNA expression, a calcium peak by energy-dispersive xray spectrophotometry, and by positive immunofluorescence staining against CD44. Osteogenic differentiation was enhanced after treatment with alendronate as confirmed by Alizarin Red S staining, elevated alkaline phosphatase activity and osteocalcin mRNA expression, a greater calcium peak by energy-dispersive xray spectrophotometry, and by immunofluorescence staining against CD44 by flow cytometric analysis. These data suggest alendronate enhances osteogenic differentiation when treated with mouse mesenchymal stem cells in osteogenic differentiation medium.

Nonviral Delivery of Basic Fibroblast Growth Factor Gene to Bone Marrow Stromal Cells

Başak Açan Clements PhD, Charlie Y. M. Hsu BSc, Cezary Kucharski DVM, Xiaoyue Lin MD, Laura Rose BSc, Hasan Uludağ PhD [object Object]

Starch-poly-є-caprolactone Microparticles Reduce the Needed Amount of BMP-2

E. R. Balmayor MSc, G. A. Feichtinger MSc, H. S. Azevedo PhD, M. Griensven MD, PhD, R. L. Reis PhD, DSc BMP-2 is currently administered clinically using collagen matrices often requiring large amounts of BMP-2 due to burst release over a short period of time. We developed and tested a novel injectable drug delivery system consisting of starch-poly-є-caprolactone microparticles for inducing osteogenesis and requiring smaller amounts of BMP-2. We evaluated BMP-2 encapsulation efficiency and the in vitro release profile by enzyme-linked immunosorbent assay. BMP-2 was rapidly released during the first 12 hours, followed by sustained release for up to 10 days. We then evaluated the osteogenic potential of dexamethasone (standard osteogenic induction agent) and BMP-2 after incorporation and during release using an osteo/myoblast cell line (C2C12). Alkaline phosphatase activity was increased by released BMP-2. Mineralization occurred after stimulation with BMP-2-loaded microparticles. A luciferase assay for osteocalcin promoter activity showed high levels of activity upon treatment with BMP-2-loaded microparticles. In contrast, no osteogenesis occurred in C2C12 cells using dexamethasone-loaded microparticles. However, human adipose stem cells exposed to the microparticles produced high amounts of alkaline phosphatase. The data suggest starch-poly-є-caprolactone microparticles are suitable carriers for the incorporation and controlled release of glucocorticoids and growth factors. Specifically, they reduce the amount of BMP-2 needed and allow more sustained osteogenic effects.

Synthetic Alginate is a Carrier of OP-1 for Bone Induction

Katsuhiko Nanno MD, Kenjiro Sugiyasu MD, Takashi Daimon PhD, Hideki Yoshikawa MD, PhD, Akira Myoui MD, PhD Bone morphogenetic proteins (BMPs) can induce bone formation in vivo when combined with appropriate carriers. Several materials, including animal collagens and synthetic polymers, have been evaluated as carriers for BMPs. We examined alginate, an approved biomaterial for human use, as a carrier for BMP-7. In a mouse model of ectopic bone formation, the following four carriers for recombinant human OP-1 (BMP-7) were tested: alginate crosslinked by divalent cations (DC alginate), alginate crosslinked by covalent bonds (CB alginate), Type I atelocollagen, and poly-D,L-lactic acid-polyethyleneglycol block copolymer (PLA-PEG). Discs of carrier materials (5-mm diameter) containing OP-1 (3–30 μg) were implanted beneath the fascia of the back muscles in six mice per group. These discs were recovered 3 weeks after implantation and subjected to radiographic and histologic studies. Ectopic bone formation occurred in a dose-dependent manner after the implantation of DC alginate, atelocollagen, and PLA-PEG, but occurred only at the highest dose implanted with CB alginate. Bone formation with DC alginate/OP-1 composites was equivalent to that with atelocollagen/OP-1 composites. Our data suggest DC alginate, a material free of animal products that is already approved by the FDA and other authorities, is a safe and potent carrier for OP-1. This carrier may also be applicable to various other situations in the orthopaedic field.

Vitamin-D Binding Protein Does Not Enhance Healing in Rat Bone Defects: A Pilot Study

Jui-Sheng Sun MD, PhD, Pei-Yu Chen MD, Yang-Hwei Tsuang MD, PhD, Ming-Hong Chen MD, PhD, Po-Quang Chen MD, PhD Vitamin D-binding protein (DBP) has an anabolic effect on the skeleton and reportedly enhances bone ingrowth. We used an in vivo critical bone defect model to determine whether local administration of DBP promotes bone defect healing. We created a 5-mm segmental bone defect in the radial shaft in a rat model. Forty-eight rats were assigned to eight groups: local application of 1 μg, 5 μg, 10 μg, or 50 μg DBP (DBP-1, DBP-5, DBP-10, DBP-50), autogenous bone marrow mononuclear cells with or without 10 μg DBP (BM-DBP-10, BM), 80 μg BMP-2 delivered in gelatin sponge (BMP-2), and the sham operated group. Radiographic evaluation, histological stains, and epifluorescence microscopy were performed. Grossly, all bone gaps of the BMP-2 group were solidly bridged by callus, while all those in the sham operated group remained unhealed by 9 weeks. Only one specimen of the BM-DBP-10 and DBP-50 groups and three specimens of the BM group were solidly healed; pseudarthroses occurred in all of the other specimens. Histological study and radiographs of the specimens showed similar results. We did not observe the enhanced bone healing reported in a previous study.

Enhanced Meniscal Repair by Overexpression of hIGF-1 in a Full-thickness Model

Haining Zhang PhD, MD, Ping Leng PhD, Jie Zhang MD The importance of the menisci to the well-being of the normal knee is well-documented. However, there is no ideal repair or reconstructive approach for damaged menisci. Gene therapy provides one promising alternative strategy, especially when combined with injectable tissue engineering to achieve minimally invasive clinical application. We asked whether the introduction of human insulin-like growth factor 1 (hIGF-1) gene could improve the repair of full-thickness meniscal defects. We created full-thickness meniscal defects in the “white area” of the anterior horn in 48 goats. Bone marrow stromal cells with the transfection of hIGF-1 gene and injectable calcium alginate gel were mixed together to repair the defects; three control groups included cells without transfection, gel without cells, and defects left empty. After 4, 8, and 16 weeks, the animals were euthanized and the excised defects were examined by macroscopic assessment, histological analysis, electron microscopy, proteoglycan determination, and MRI. Sixteen weeks after surgery the repaired meniscal defects were filled with white tissue similar to that in normal meniscal fibrocartilage. The repair tissue was composed of cells embedded within matrix that filled the spaces of the fibers. The proteoglycan content in the gene-enhanced tissue engineering group was higher than those in the control groups, and less than that in the normal meniscus. The results suggest full-thickness meniscal defects in regions without blood supply can be reconstructed with hIGF-1-enhanced injectable tissue engineering.

Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

Hai-ning Zhang PhD, MD, Ping Leng PhD, Ying-zhen Wang MD, Jie Zhang MD The menisci are intraarticular fibrocartilaginous structures essential to the normal function of the knee that lack the ability to self-repair. Human meniscal fibrochondrocytes may respond to beneficial genes like human insulin growth factor-1 (hIGF-1) and the meniscal cell may be a feasible donor for gene therapy. To explore this possibility, we amplified the hIGF-1 gene sequence in full length and cloned it into a bicistronic plasmid. This gene was then transfected into cultured human meniscal fibrochondrocytes by the liposome FuGene 6. Green fluorescence was expressed in part of the cells 6 hours after transfection and increased gradually, with a peak concentration of the hIGF-1 in the supernatants to 22.68 ng/mL 56 hours after transfection. Phenotypes of some cells changed and the proliferation accelerated after transfection. Flow cytometry analysis demonstrated upregulation of cell numbers in the G2 and S stages after hIGF-1 gene introduction. We conclude the hIGF-1 gene can be transfected into the human meniscal cell efficiently by liposome and it causes accelerated proliferation and differentiation. Within 10 days after transfection, the cytokine appears to be secreted into supernatants with the bioactivity and promotes the proliferation of the NIH 3T3 cell line.

Are BMPs Involved in Normal Nerve and Following Transection?: A Pilot Study

Masaya Tsujii MD, PhD, Koji Akeda MD, PhD, Takahiro Iino MS, Atsumasa Uchida MD, PhD Bone morphogenic proteins (BMPs) may have neurotrophic functions but there is limited evidence of these functions in the peripheral nervous system. We therefore investigated the expression of BMPs and BMP receptors (BMPRs) in normal and injured peripheral nerves. In 10 of 15 Sprague-Dawley rats, a 3-mm segment of sciatic nerve was resected at the trifurcation in the thigh. One day (n = 5) and 7 days (n = 5) after transection, proximal and distal stumps were removed and immunohistochemically analyzed for BMP-2, -7, BMPR-1A, -1B, and -2. The other five animals served as normal controls. In normal nerves, BMP-2 expression was localized at Ranvier’s node, and BMP-7 and BMPR-1B were expressed in several axon-Schwann cell units, whereas other receptors were not expressed. After nerve transection, BMP-7 expression was upregulated at both proximal and distal stumps along with Schwann cell columns during Wallerian degeneration. BMPRs were also upregulated compared with the normal nerve. The upregulation in BMP expression after nerve transection suggests that BMPs may play a role in the healing response of the peripheral nerve.

Are Endogenous BMPs Necessary for Bone Healing during Distraction Osteogenesis?

Norine Alam MS, René St-Arnaud PhD, Dominique Lauzier AS, Vicki Rosen PhD, Reggie C. Hamdy MD, FRCSC [object Object]

Distraction Osteogenesis Enhances Remodeling of Remote Bones of the Skeleton: A Pilot Study

Julia F. Funk MD, Gert Krummrey MD, Carsten Perka PhD, Michael J. Raschke PhD, Hermann J. Bail PhD [object Object]

Successful Spinal Fusion by E. coli-derived BMP-2-adsorbed Porous β-TCP Granules: A Pilot Study

Sho Dohzono MD, Yuuki Imai MD, PhD, Hiroaki Nakamura MD, PhD, Shigeyuki Wakitani MD, PhD, Kunio Takaoka MD, PhD Bone morphogenetic proteins (BMPs) were originally identified as osteoinductive proteins. With cloning of BMP genes, studies of BMPs and their clinical application have advanced. However, with increasing clinical applications, drug delivery systems and production costs have become more important issues. To address these issues, we asked whether E. coli-derived rhBMP-2 (E-BMP-2)-adsorbed porous β-TCP granules could achieve posterolateral lumbar fusion in a rabbit model similar to autogenous bone grafts. Lumbar spinal fusion masses were evaluated by 3-D computed tomography, mechanical testing, and histological analyses 8 weeks after surgery. By these measures E-BMP-2-adsorbed β-TCP granules achieved lumbar spinal fusion in dose-dependent fashion in a rabbit model as well as autogenous bone graft. Our preliminary findings suggest E-BMP-2-adsorbed porous β-TCP could be a novel, effective alternative to autogenous bone grafting for generating new bone and promoting regenerative repair of bone, and potentially utilizable in the clinical setting for treating spinal disorders.

Correlation of Computed Tomography with Histology in the Assessment of Periprosthetic Defect Healing

Stephen D. Cook PhD, Laura P. Patron BSE, Samantha L. Salkeld MSE, Kirk E. Smith BS, Bruce Whiting PhD, Robert L. Barrack MD Computed tomography (CT) may more accurately assess the healing of grafted osteolytic lesions around acetabular components compared with plain radiographs, although clinical validation is lacking. To determine whether clinical or micro-CT imaging could assess accurately the grafted lesion compared with histology, we therefore quantified bone healing and ingrowth to determine an effective rhBMP-2 dose and ratio to allograft bone when grafted adjacent to a cementless porous-coated component. We grafted surgically created acetabular defects in canines (n = 20) before uncemented total hip arthroplasty. At 6 weeks, embedded acetabula were imaged and the CT slice images matched to histology section images. The percentage of bone in the defect and growth into the porous surface was assessed quantitatively. Low-dose rhBMP-2 with allograft (1:5 ratio) resulted in a higher percentage of defect healing (43.8%) than rhBMP-2 alone (29.2%) and a higher percentage of bone ingrowth (15.7%) than allograft bone alone (1.1%) as measured by histology. Micro-CT measurements were similar to histologic measurements of defect healing, whereas clinical CT overestimated periprosthetic bone by 38%. Neither clinical CT nor micro-CT techniques are adequate for assessing ingrowth or the bone-implant interface with metal artifacts.

Use of Bone Morphogenic Protein-7 as a Treatment for Osteoarthritis

Neil Badlani MD, Yasushi Oshima MD, PhD, Rob Healey BS, Richard Coutts MD, David Amiel PhD Osteoarthritis is a degenerative disorder resulting from breakdown of articular cartilage. Previous work has shown bone morphogenic protein-7 has a potential protective effect on cartilage during the development of osteoarthritis. The purpose of this study was to determine whether bone morphogenic protein-7 could decrease the amount of cartilage degradation in preexisting osteoarthritis. The rabbit ACLT model was used as a model of osteoarthritis. Bone morphogenic protein-7 was delivered via Alzet osmotic pump to the joint 4 weeks after anterior cruciate ligament transection; thus cartilage injury was preexisting. The experimental group showed less cartilage degradation than the controls, with an average Outerbridge score of 1.9 versus 2.6 for the controls. Histomorphometry showed a trend toward less cartilage degradation in the bone morphogenic protein-7 group when compared with controls. Semiquantitative real-time polymerase chain reaction showed a considerably greater expression of aggrecan in the bone morphogenic protein-7-treated cartilage when compared with controls and less expression of matrix metalloproteinase-3 and matrix metalloproteinase-13, important catabolic mediators. The synovial tissue of the experimental group also showed considerably less expression of matrix metalloproteinase-3, matrix metalloproteinase-13, and aggrecanase. These results indicate bone morphogenic protein-7 may reduce degradation of articular cartilage in osteoarthritis.

Enhancement of Difficult Nonunion in Children with Osteogenic Protein-1 (OP-1): Early Experience

Bruno Dohin MD, Noémi Dahan-Oliel MSc, François Fassier MD, Reggie Hamdy MD Numerous studies have described the use of osteogenic protein-1 (OP-1) in adults, but there are few reports in children. The objectives of this short-term followup cohort study were (1) to examine clinical and radiographic healing of persistent nonunions after OP-1 application in children; and (2) to determine the safety of OP-1 use in this sample. Clinical healing was defined by absence of pain and tenderness at the nonunion site and the ability to fully weight bear on the affected limb. Radiographic healing was determined by bony bridging of the nonunion site in at least one view. Safety was defined as the absence of major adverse events, including allergic reactions, infections, local inflammatory reactions, and heterotopic ossification. OP-1 was used in 19 patients who had an operative procedure for the bridging of persistent nonunions between 1999 and 2007. The mean age was 11.6 years (range, 4.8–20.3 years). Thirteen patients had persistent nonunion after one or more previous surgeries, prior to the initial OP-1 application. A single dose of 3.5 mg of OP-1 mixed with 1 g of Type I bovine collagen was applied to 23 sites of 19 patients. Three patients received additional OP-1 applications. Healing occurred clinically and radiographically in 17 of the 23 sites. Complications included four superficial pin site infections, one deep infection, and two fractures. No major local adverse event related to OP-1 application was noted in our sample. Our findings suggest OP-1 stimulates healing of persistent nonunions without major adverse events in our patient population.,[object Object]

The Synergistic Effect of Autograft and BMP-7 in the Treatment of Atrophic Nonunions

Peter V. Giannoudis MD, EEC (Ortho), Nikolaos K. Kanakaris MD, Rozalia Dimitriou MSc, MD, Ian Gill MB, BS, Vinod Kolimarala MB, BS, Richard J. Montgomery FRCS Combining autologous bone graft and recombinant human bone morphogenetic protein-7 (BMP-7) to treat long-bone fracture aseptic atrophic nonunions theoretically could promote bone healing at higher rates than each of these grafting agents separately. We retrospectively reviewed prospectively collected data on patient general characteristics, clinical outcomes, and complications over 3 years to determine the healing rates and the incidence of complications and adverse events of this “graft expansion rationale.” There were 45 patients (32 male) with a median age of 43 years (range, 19–76 years). Minimum followup was 12 months (mean, 24.5 months; range, 12–65 months). There were seven humeral, 19 femoral, and 19 tibial nonunions. The median number of prior operations was two (range, 1–7). All fractures united. Clinical and radiographic union occurred within a median of 5 months (range, 3–14 months) and 6 months (range, 4–16 months), respectively. Thirty-nine (87%) patients returned to their preinjury occupation at a mean of 4.2 months (range, 3–6 months). The median visual analog scale pain score was 0.9 (range, 0–2.8; maximum 10), and the median functional score was 86 (range, 67–95; maximum 100) at the final followup. BMP-7 as a bone-stimulating agent combined with conventional autograft resulted in a nonunion healing rate of 100% in these 45 patients.,[object Object]

Osteogenic Protein-1 Overcomes Inhibition of Fracture Healing in the Diabetic Rat: A Pilot Study

Louis S. Kidder PhD, Xinqian Chen MD, Andrew H. Schmidt MD, William D. Lew MS Type I diabetes mellitus inhibits fracture healing and leads to an increase in complications. As a pilot study, we used a closed fracture model in the diabetic rat to address the question of whether osteogenic protein-1 (OP-1) in a collagen carrier can overcome this inhibition by increasing the area of the newly mineralized callus and femoral torque to failure compared with diabetic animals with fractures treated without OP-1. Diabetes was created in 54 rats by injection of streptozotocin. After 2 weeks, a closed femur fracture was created using a drop-weight impaction device. Each fracture site was immediately opened and treated with or without 25 μg OP-1 in a collagen carrier. Animals were euthanized after 2 or 4 weeks. Fracture healing was assessed by callus area from high-resolution radiographs, callus strength from torsional failure testing, and undecalcified histologic analysis. The area of newly mineralized callus was greater in diabetic animals treated with 25 μg OP-1/carrier compared with diabetic animals with untreated fractures and with fractures treated with carrier alone. This increase in callus area did not translate into an equivalent increase in torque to failure. Osteogenic protein-1 showed some evidence of overcoming the inhibition of fracture healing in the diabetic rat.

Complications of Recombinant Human BMP-2 for Treating Complex Tibial Plateau Fractures: A Preliminary Report

Sreevathsa Boraiah MD, Omesh Paul MD, David Hawkes MBChB, Matthew Wickham MBChB, Dean G. Lorich MD Bone morphogenic proteins (BMPs) are potent osteoinductive agents. Their use in fracture surgery is still being studied and the clinical indications are evolving. Heterotopic bone after BMP use in spine surgery is a known complication. While some literature describes the ability of BMP to enhance fracture healing, few articles describe complications of BMP. In tibial plateau fractures, after elevating the cartilage en mass, a subchondral void may be created in these fractures. Structural support provided by bone void-filling agents can be augmented with osteoinduction achieved by BMP. We asked whether heterotopic bone formation would occur more frequently with BMP-2 when used in tibial plateau fractures and whether BMP-2 enhanced the ability to maintain surgically restored subchondral bone integrity. Heterotopic bone developed more frequently in patients receiving BMP (10 of 17) than in patients not receiving BMP (one of 23). Four patients receiving BMP and no patients not receiving BMP underwent removal of heterotopic bone. Maintenance of subchondral bone integrity was similar without and with the use of BMP. BMP is a potent osteoinductive agent; however, when used for an off-label indication in periarticular situations, complications such as heterotopic bone are common and increase reoperation rates.,[object Object]

Mesenchymal Stem Cell and Nucleus Pulposus Cell Coculture Modulates Cell Profile

Chi-Chien Niu MD, Li-Jen Yuan MD, Song-Shu Lin MS, Lih-Huei Chen MD, Wen-Jer Chen MD Spontaneous cell fusion can occur in cocultured stem cells. We examined whether telomerase activity change and cell fusion occurred in mesenchymal stem cell (MSC) and nucleus pulposus cell (NPC) coculture. MSCs and NPCs were labeled with PKH26 and PKH67 dyes and cocultured at a 50:50 ratio. An equal number of MSCs or NPCs were used as the control. After 14 days, cells were evaluated by cell growth, telomerase activity, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and histologic observation. Cell fusion was confirmed by microscopic observation and fluorescence-activated cell sorter (FACS) analysis. The results suggested cell growth rate and telomerase activity were higher in cocultured cells than in NPCs cultured alone. The mRNA expression levels of the Type II collagen and aggrecan were elevated in cocultured cells. Immunohistochemical analysis revealed positive staining for Type II collagen and keratan sulfate in NPCs cultured alone and in a proportion of cocultured cells. Histologic observation revealed binucleated cocultured cells expressed both PKH dyes in the same location and slide focus. The FACS analysis revealed 42% of cocultured cells were double-stained. Cocultured cells partially maintained the NPC phenotype. The partially maintained phenotype of the NPCs may be attributable to spontaneous cell fusion in association with increased telomerase activity.

Differential Expression of Vascular Endothelial Growth Factor in Glucocorticoid-related Osteonecrosis of the Femoral Head

Deike Varoga MD, Wolf Drescher MD, PhD, Melanie Pufe MD, Godo Groth, Thomas Pufe PhD VEGF plays a role in bone remodeling. Ingrowth of reparative arterioles can be observed in late-stage osteonecrosis. To explore the reparative processes, we quantified the most important angiogenesis factor (VEGF) in different zones of late-stage glucocorticoid-induced osteonecrosis. We treated primary nonosteonecrosis osteoblasts with glucocorticoids in vitro as a model for the bone cells in early-stage steroid-related osteonecrosis. We obtained six late-stage (ARCO Stage IV) osteonecrosis femoral heads and six osteoarthritic femoral heads during THA. The expression of vascular endothelial growth factor was analyzed by reverse-transcription PCR, ELISA, or immunohistochemistry. Osteoblasts from the reactive interface (penumbra) of osteonecrosis femoral heads exhibited increased immunoreactivity to VEGF compared to those from the periphery. ELISA confirmed VEGF upregulation in the penumbra from osteonecrosis femoral heads. Primary osteoblasts derived from osteoarthritic femoral heads exhibited downregulation of VEGF after 24 hours of coincubation with glucocorticoids. The increase in VEGF in the reactive interface (penumbra) of osteonecrosis in late-stage femoral head may reflect a secondary phenomenon. The observed high amount of VEGF in later-stage osteonecrosis might stimulate the ingrowth of reparative arterioles into the femoral head.

Prophylaxis of Heterotopic Ossification of the Hip: Systematic Review and Meta-Analysis

Patrick Vavken MD, MSc, Lorenzo Castellani MD, Thomas P. Sculco MD Heterotopic ossification (HO) is a potentially severe, if infrequent, complication in hip surgery, and uncertainty exists regarding whether to use NSAIDs or radiation in its prevention. Thus, we systematically reviewed the literature in MedLine, EMBASE, CINAHL, and the Cochrane Controlled Trial Register and, after ruling out publication bias and data heterogeneity, performed a meta-analysis of randomized, controlled trials to assess effectiveness and complications of NSAIDs and radiation in the prevention of HO. We identified nine studies reporting on effectiveness and complications including a total of 1295 patients. The pooled risk ratio for the effectiveness in HO prevention was 0.96 (95% confidence interval, 0.88–1.06) and was independent of the type of surgery (THA or open reduction and internal fixation). There was no association with gender, age, length of followup, or year of publication. The risk ratio for associated complications was 0.79 (95% confidence interval, 0.45–1.41), and, again, was independent of the aforementioned factors. We found no evidence for a statistically significant or clinically important difference between NSAIDs or radiation in preventing HO.

Femoral Head Size and Wear of Highly Cross-linked Polyethylene at 5 to 8 Years

Paul F. Lachiewicz MD, Daniel S. Heckman MD, Elizabeth S. Soileau BSN, Jimmi Mangla MD, John M. Martell MD

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Clinical Anatomy of the Quadriceps Femoris and Extensor Apparatus of the Knee

Andrew C. Waligora BS, Norman A. Johanson MD, Bruce Elliot Hirsch PhD Most descriptions of the extensor mechanism of the knee do not take into account its complexity and variability. The quadriceps femoris insertion into the patella is said to be through a common tendon with a three-layered arrangement: rectus femoris (RF) most superficially, vastus medialis (VM) and lateralis (VL) in the intermediate layer, and vastus intermedius (VI) most deeply. We dissected 20 limbs from 17 cadavers to provide a more detailed description of the anterior components of the knee: the tendon, the patellar retinacula, and the patellofemoral ligaments. Only three of the 20 specimens exhibited the typically described quadriceps pattern. The remainder had bilaminar and even more complex trilaminar and tetralaminar fiber arrangements. We found an oblique head of the vastus lateralis (VLO), separated from the longitudinal head by a layer of fat or fascia, in 60% of the specimens. However, we found no distinct oblique head of the vastus medialis (VMO) in any specimen. The medial patellofemoral ligament (MPFL) was more common than the lateral (LPFL), supporting its suggested role as the principal passive medial stabilizer of the patella. Because the quadriceps muscle group plays a direct role in patellofemoral joint function, investigation into the clinical applications of its highly variable anatomy may be worthwhile with respect to joint dysfunction and failures of TKAs.

One-step Bone Marrow-derived Cell Transplantation in Talar Osteochondral Lesions

Sandro Giannini MD, Roberto Buda MD, Francesca Vannini MD, PhD, Marco Cavallo MD, Brunella Grigolo PhD The ideal treatment of osteochondral lesions is debatable. Although autologous chondrocyte implantation provides pain relief, the need for two operations and high costs has prompted a search for alternatives. Bone marrow-derived cells may represent the future in osteochondral repair. Using a device to concentrate bone marrow-derived cells and collagen powder or hyaluronic acid membrane as scaffolds for cell support and platelet gel, a one-step arthroscopic technique was developed for cartilage repair. We performed an in vitro preclinical study to verify the capability of bone marrow-derived cells to differentiate into chondrogenic and osteogenic lineages and to be supported onto scaffolds. In a prospective clinical study, we investigated the ability of this technique to repair talar osteochondral lesions in 48 patients. Minimum followup was 24 months (mean, 29 months; range, 24–35 months). Clinical results were evaluated using the American Orthopaedic Foot and Ankle Society (AOFAS) score and the influence of scaffold type, lesion area, previous surgeries, and lesion depth was considered. MRI and histologic evaluation were performed. The AOFAS score improved from 64.4 ± 14.5 to 91.4 ± 7.7. Histologic evaluation showed regenerated tissue in various degrees of remodeling although none showed entirely hyaline cartilage. These data suggest the one-step technique is an alternative for cartilage repair, permitting improved functional scores and overcoming the drawbacks of previous techniques.,[object Object]

Objective Guidelines for Removing an External Fixator after Tibial Lengthening Using Pixel Value Ratio: A Pilot Study

Li Zhao MD, PhD, Qing Fan MD, K. P. Venkatesh MS, Man S. Park, Hae Ryong Song MD, PhD During limb lengthening over an intramedullary nail, decisions regarding external fixator removal and weightbearing depend on the amount of callus seen at the lengthening area on radiographs. However, this method is subjective and objective evaluation of the amount of callus likely would minimize nail or interlocking screw breakage and refracture after fixator removal. We asked how many cortices with full corticalization of the newly formed bone at the lengthening area are needed to allow fixator removal and full weightbearing and how to radiographically determine the stage of corticalization. We retrospectively reviewed 17 patients (34 lengthenings) who underwent bilateral tibial lengthenings over an intramedullary nail. The average gain in length was 7.2 ± 3.4 cm. We determined the pixel value ratio (ratio of pixel value of regenerate versus the mean pixel value of adjacent bone) of the lengthened area on radiographs. There were no nail or screw breakage and refracture. Partial weightbearing with crutches was permitted when the pixel value ratio was 1 in two cortices and full weightbearing without crutches was permitted when the pixel value ratio was 1 in three cortices. The pixel value ratio on radiographs can be an objective parameter for callus measurement and may provide guidelines for the timing of external fixator removal. We cannot determine from our limited data the minimum pixel value in how many cortices would suggest safe removal, but we can say our criteria were not associated with subsequent refracture.

New Equations for Predicting Postoperative Risk in Patients with Hip Fracture

Jun Hirose MD, PhD, Junji Ide MD, PhD, Hiroki Irie MD, PhD, Kenshi Kikukawa MD, PhD, Hiroshi Mizuta MD, PhD Predicting the postoperative course of patients with hip fractures would be helpful for surgical planning and risk management. We therefore established equations to predict the morbidity and mortality rates in candidates for hip fracture surgery using the Estimation of Physiologic Ability and Surgical Stress (E-PASS) risk-scoring system. First we evaluated the correlation between the E-PASS scores and postoperative morbidity and mortality rates in all 722 patients surgically treated for hip fractures during the study period (Group A). Next we established equations to predict morbidity and mortality rates. We then applied these equations to all 633 patients with hip fractures treated at seven other hospitals (Group B) and compared the predicted and actual morbidity and mortality rates to assess the predictive ability of the E-PASS and Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity (POSSUM) systems. The ratio of actual to predicted morbidity and mortality rates was closer to 1.0 with the E-PASS than the POSSUM system. Our data suggest the E-PASS scoring system is useful for defining postoperative risk and its underlying algorithm accurately predicts morbidity and mortality rates in patients with hip fractures before surgery. This information then can be used to manage their condition and potentially improve treatment outcomes.,[object Object]

The Use of Confidence Intervals in Reporting Orthopaedic Research Findings

Patrick Vavken MD, MSc, Klemens M. Heinrich, Christian Koppelhuber, Stefan Rois, Ronald Dorotka MD Conflict between clinical importance and statistical significance is an important problem in medical research. Although clinical importance is best described by asking for the effect size or how much, statistical significance can only suggest whether there is any difference. One way to combine statistical significance and effect sizes is to report confidence intervals. We therefore assessed the reporting of confidence intervals in the orthopaedic literature and factors influencing this frequency. In parallel, we tested the predictive value of statistical significance for effect size. In a random sample of predetermined size, we found one in five orthopaedic articles reported confidence intervals. Participation of an individual trained in research methods increased the odds of doing so fivefold. The use of confidence intervals was independent of impact factor, year of publication, and significance of outcomes. The probability of statistically significant results to predict at least a 10% between-group difference was only 69% (95% confidence interval, 55%–83%), suggesting that a high proportion of statistically significant results do not reflect large treatment effects. Confidence intervals could help avoid such erroneous interpretation by showing the effect size explicitly.

Are Away Rotations Critical for a Successful Match in Orthopaedic Surgery?

Keith Baldwin MD, MPH, MSPT, Zachary Weidner BS, Jaimo Ahn MD, PhD, Samir Mehta MD [object Object]

Case Report: Primary Squamous Cell Carcinoma of a Tarsal Bone

Czar Louie L. Gaston MD, Ariel M. Vergel de Dios MD, Tammy L. Dela Rosa MD, Edward H. M. Wang MD, MSc We describe a rare case of primary squamous cell carcinoma of the cuneiform bone of the foot in a 57-year-old man. In the appendicular skeleton, epithelial carcinomas of bone are usually metastatic deposits, primary squamous cell carcinomas of bone being found more frequently in the skull. A review of the English literature revealed only two other reported cases of primary squamous cell carcinoma outside the skull—one in the ilium and one in the tibia. In our patient, extensive metastatic workup and monitoring during more than 2 years showed no primary focus, supporting the rare presentation of a primary squamous cell carcinoma of bone in the appendicular skeleton.

Case Report: Longitudinal Stress Fracture of the Humerus: Imaging Features and Pitfalls

Colm J. McMahon MB, Sanjay K. Shetty MD, Megan E. Anderson MD, Mary G. Hochman MD Longitudinal stress fractures are an uncommon injury in which a diaphyseal fracture line occurs parallel to the long axis of a bone in the absence of direct trauma. They have been described in the tibia and less commonly in the femur but apparently not in the upper limb. We report a longitudinal stress fracture occurring in the humerus of a 62-year-old woman who had a history of osteoporosis and had undergone recent surgery of the contralateral wrist. We present the radiographic, MRI, and CT features of the case and emphasize the difficulties in diagnosis caused by negative findings on early radiographs and by nonspecific bone marrow edema pattern on MRI. The risk of a contralateral upper extremity stress fracture from activities of daily living in a patient with osteoporosis whose other upper extremity is immobilized also is highlighted.

Erratum to: PATCHED-ONE or SMOOTHENED Gene Mutations Are Infrequent in Chondrosarcoma

Taiqiang Yan MD, Mark Angelini MD, FRCSC, Benjamin A. Alman MD, FRCSC, Irene L. Andrulis PhD, Jay S. Wunder MD, MSc, FRCS(C)

Journal Scan: Spine

Andrea S. Bauer MD, Jay M. Zampini MD, Kevin J. McGuire MD, MSCE

An 11-year-old Boy with a Patella Fracture

Jason A. Nydick DO, Martin J. Herman MD, Jean-Pierre Chadarévian MD
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