Clinical Orthopaedics and Related Research ®

A Publication of The Association of Bone and Joint Surgeons ®

Symposium: Tribute to Dr. Marshall Urist: Musculoskeletal Growth Factors 30 articles

Articles

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.

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.

The Classic: Bone Morphogenetic Protein

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

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]

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]

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.

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.

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.

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.

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]

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.

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.

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.

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]

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]

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.

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]

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.

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.

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]

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.

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.

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.

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.

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.

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.