Outcome satisfaction in long-term survivors of oncologic limb salvage procedures.

OBJECTIVE: We present data from our recently completed study that suggests that joint salvage is the key parameter in keeping the limb salvage ethos relevant. METHODS: We reviewed all patients operated on nationally over 30 years (1978 to 2008). We identified 265 survivors of which 162 were available for evaluation at the time of survey. RESULTS: There were 92 males and 70 females followed an average 9.1±SD3.1 years. Average age at survey was 38±SD17 years. ANOVA for mental health and TESS degree of importance for ADL identified that all categories of joint preservation were similar to an average group of people from the population. Physical health scores were significantly better amongst joint-preserved versus joint replacement patients (p = 0.003). Nevertheless, there was no significant difference between amputees with respect to physical health scores compared to arthrodesed and joint-replaced individuals. Biological reconstructions scored significantly better than metallic segmentary reconstructions (p < 0.001). Dissatisfaction appeared to correlate best with the presence of pain following reconstruction (p < 0.001). CONCLUSIONS: While our study supports the finding of amputations being as satisfactory as arthrodeses and joint replacement salvage surgery, joint salvage was superior to all other categories. Assessments of equivalence should incorporate joint salvage and materials used as evaluable parameters.

Structural Adaptations of the Primate Flexor Carpi Ulnaris After Tendon Transfer.

PURPOSE: Muscle remodeling occurs after tendon transfer. However, it is not known whether these adaptations are permanent and clinically significant. This study examined the early and late structural adaptations following a standard tendon transfer in a primate model. METHODS: A flexor carpi ulnaris (FCU) to extensor digitorum communis (EDC) transfer was performed in 8 adult monkeys. A sham operation was performed in the contralateral forearm. Four animals were sacrificed at 5 months (early cohort) and 4 at 16 months (late cohort). The transferred FCU, contralateral FCU, and EDC muscles were removed for analysis. Fiber length (FL), physiological cross-sectional area (PCSA), and gross morphology of the transferred FCU were compared with the contralateral EDC and FCU. RESULTS: In the early cohort, the FL of the transferred FCU was longer than the control FCU and similar to the contralateral EDC. The PCSA of the transferred FCU was lower than that of the control FCU but greater than the control EDC. In the late cohort, the difference in FL and PCSA between the transferred FCU and the control FCU persisted. The PCSA of the transferred FCU was similar to that of the control EDC. The bipennate transferred FCU had also undergone gross morphological changes to resemble the multipennate EDC. CONCLUSIONS: This study demonstrates, in a primate model, that the FCU undergoes structural adaptations to resemble the EDC following an FCU-to-EDC transfer. However, these adaptations are incomplete and not sustained over time. CLINICAL RELEVANCE: This study demonstrates that there is muscle plasticity in tendon transfers in a primate model. However, it is important to match potential donor muscles to the recipient during tendon transfer.

Revisiting the anatomy and biomechanics of the anconeus muscle and its role in elbow stability.

BACKGROUND: Recent studies have designated the anconeus muscle as an option for use as a pedicled flap for covering soft tissue defects about the elbow, with reported minimal risk of morbidity. This has raised the question as to the importance of the anconeus muscle and as to whether this is truly an accessory muscle that can be sacrificed, or whether the anconeus muscle significantly contributes to elbow and forearm stability? This study revisits the anatomy and biomechanics of the anconeus muscle and aims to investigate the neuromuscular compartments of the anconeus muscle and to determine the changes in the muscle length, fibre length and moment arm over a range of elbow flexion angles for each compartment. METHODS: An anatomical study on 8 human cadavers (51-77 years of age) was done and a 2-dimensional kinematic elbow model developed to determine changes in the muscle length and moment arm of the muscle related to changes in elbow flexion angles. FINDINGS: The muscle was modelled with two possible lines of action, one along the posterior and another on the anterior edge of the muscle as they had different muscle fibre lengths (posterior: average of 32 mm, anterior: average of 20 mm). The anterior edge also had an aponeurosis which was 70% of its length. From 0 to 120° elbow flexion, the length of the posterior and anterior edges increased with a maximum change recorded at 90° elbow flexion (31.7±1.0 mm and 65.3±1.4 mm, respectively). The moment arm is 14-mm at 0° flexion, but between the posterior and anterior edges it decreases at different rates with increasing elbow flexion angle. Beyond 80°, the anterior edge behaves as an elbow flexor, while the posterior edge remains an elbow extensor. The study demonstrates that the anconeus muscle has two neuromuscular compartments each with distinct intramuscular innervations and muscle fibre lengths. INTERPRETATION: The posterior and deep aspect of the muscle functions as an elbow extensor decreasing in influence with increasing elbow flexion angle. The anterior superficial aspect which is adjacent and parallel to the lateral collateral ligaments, would most likely work in unison to provide constraint to the posterolateral stability of the elbow.

Allografts with autogenous platelet-rich plasma for tibial defect reconstruction: a rabbit study.

PURPOSE: To evaluate the effect of autogenous platelet-rich plasma (PRP) for fresh-frozen allografts in tibial defect reconstruction in rabbits. METHODS: 40 adult New Zealand white rabbits underwent tibial defect reconstruction with autografts (n=12), allografts without PRP (n=12), or allografts with PRP (n=12) and were observed for 12, 16, and 24 weeks (4 for each period). Tibias of the remaining 4 rabbits were used as donor allografts, and the remaining allografts were procured from recipient rabbits. A 1.5- cm cortical segment of the tibia was osteotomised, and then fixed with a 9-hole mini-compression plate and 2 cerclage wires. Allografts were stripped off the periosteum and soft tissues and medullary contents, and then stored in a freezer at -80 ºC. All allografts were deep frozen for at least 4 weeks before transplantation. 7 ml of whole blood was drawn to prepare 1 ml of PRP. The PRP was then mixed with 1.0 ml of human thrombin to form a platelet gel. The PRP gel was then packed into the medullary canal of the allograft and applied on the cortical surface before tibial defect reconstruction. Rabbits were sacrificed at 12, 16, and 24 weeks. The specimens were assessed for bone union at host-graft junctions and for bone resorption, new bone formation, callus encasement, and viable osteocyte counts. RESULTS: There were 4 specimens in each group at each observation period. Osteoid bridging the gap at host-graft junctions was noted in all specimens in the autograft and allograft-with-PRP groups at week 12 and in the allograft-without-PRP group at week 24. Bone union in allografts without PRP was delayed. All indices for biological incorporation (resorption index, new bone formation index, callus encasement index, and viable osteocyte count) were significantly greater in the autograft than allograft-without-PRP groups, except for the resorption index at week 24, whereas the differences were not significant between the autograft and allograft-with-PRP groups. The differences between the 2 allograft groups were usually not significant, except for the resorption index. CONCLUSION: PRP-augmented allografts behaved similarly to autografts for tibial defect reconstruction in rabbits. PRP increased bone union and bone resorption.

Trans-iliosacral plating for vertically unstable fractures of sacral spine associated with spinopelvic dissociation: A cadaveric study.

BACKGROUND: The treatment algorithm for sacral fracture associated with vertical shear pelvic fracture has not emerged. Our aim was to study a new approach of fixation for comminuted and vertically unstable fracture pattern with spinopelvic dissociation to overcome inconsistent outcome and avoid complications associated with fixations. We propose fixation with well-contoured thick reconstruction plate spreading across sacrum from one iliac bone to another with fixation points in iliac wing, sacral ala and sacral pedicle on either side. Present biomechanical study tests the four fixation pattern to compare their stiffness to vertical compressive forces. MATERIALS AND METHODS: Dissection was performed on human cadavers through posterior midline paraspinal approach elevating erector spinae from insertion with two flaps. Feasibility of surgical exposure and placement of contoured plate for fixation was evaluated. Ten age and sex matched computed tomography scans of pelvis with both hips were obtained. Reconstructions were performed with advantage windows 4.2 (GE Light Speed QX/I, General Electric, Milwaukee, WI, USA). Using the annotation tools, direct digital CT measurement (0.6 mm increments) of three linear parameters was carried out. Readings were recorded at S2 sacral level. Pelvic CT scans were extensively studied for entry point, trajectory and estimated length for screw placement in S2 pedicle, sacral ala and iliac wing. Readings were recorded for desired angulation of screw in iliac wing ala of sacrum and sacral pedicle with respect to midline. The readings were analyzed by the values of mean and standard deviation. Biomechanical efficacy of fixation methods was studied separately on synthetic bone. Four fixation patterns given below were tested to compare their stiffness to vertical compressive forces: 1) Single S1 iliosacral screw (7.5 mm cancellous screw), 2) Two S1 and S2 iliosacral screws, 3) Isolated trans-iliosacral plate, 4) Trans-iliosacral plate + single S1 iliosacral screw. STATISTICAL ANALYSIS: Mean of desired angulation for inserting screws and percentage of displacement on biomechanical testing was evaluated. RESULTS: Mean angulations for inserting sacral pedicel were 12.3° (SD 2.7°) convergent to midline and divergent of 14° (SD 2.3°) for sacral ala screw and 23° (SD 4.9°) for iliac wing screw. All screws needed to be inserted at an angle of 90° to sacral dorsum to avoid violation of root canals. Cross headed displacement across fracture site was measured and plotted against the applied vertical shear load of 300 N in five cycles each for all the four configurations. Also, the force required for cross headed displacement of 2.5 mm and 5 mm was recorded for all configurations. Transmitted load across both ischial tuberosities was measured to resolve unequal distribution of forces. Taking one screw construct (configuration 1) as standard base reference, trans-iliosacral plate construct (configuration 3) showed equal rigidity to standard reference. Two screw construct (configuration 2) was 12% stronger and trans-iliosacral plate (configuration 4) with screw was 9% stronger at 2.5 mm displacing on 300 N force, while it showed 30% and 6%, respectively, at 5 mm cross-headed displacement. CONCLUSIONS: Trans-iliosacral plating is feasible anatomically, biomechanically and radiologically for sacral fractures associated with vertical shear pelvic fractures. Low profile of plate reduces the risk of hardware prominence and decreases the need for implant removal. Also, the fixation pattern of plate allows to spare mobile lumbosacral junction which is an important segment for spinal mobility. Biomechanical studies revealed that rigidity offered by plate for cross headed displacement across fracture site is equal to sacroiliac screws and further rigidity of construct can be increased with addition of one more screw. There is need for precountered thicker plate in future.

Interstitial fluid pressure as an alternate regulator of angiogenesis independent of hypoxia driven HIF-1α in solid tumors.

We previously showed that interstitial fluid pressure (IFP) may be an alternate regulator of angiogenesis in solid tumors. Given the accepted link between hypoxia-induced factor and angiogenesis this study investigated the effect of IFP on hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in human osteosarcoma xenografts in SCID mice and in different hypoxic environments. Tumors were grown either at heterotopic (flank) or orthotopic (medullary canal of the proximal tibia) sites in the host animal. Microfluidic probes determined pH, O(2)-saturation, IFP, and peripheral blood flow perfusion continuously. We assessed tumor growth in the orthotopic site (n = 15) by softex radiographs weekly, 3D microCT, histological evaluation, and for molecular responses. An increased cytoplasmic immunohistostaining of cells for HIF-1α (p = 0.03) and VEGF-A (p = 0.004) on the outer periphery was noted compared to the tumor center, with VEGFR2 uniformly stained throughout. This paralleled a raised state of interstitial hypertension (p = 0.007) in the tumor center relative to the peripheral surface but was inconsistent with a state of hypoxia (p = 0.03) in the tumor center. In vitro culture of human osteosarcoma cell lines (HOS, U2OS) and a human osteoblast control at 0- and 20-mmHg of hydrostatic pressure revealed suppression of HIF-1α (p = 0.02) and VEGF-A (p = 0.02) gene expression when IFP was raised, while the effect on VEGFR1 was equivocal. This study proposes an alternative regulatory angiogenic pathway via the influence of IFP on cancer cell function. The identification of a mechanistic cellular link to the physical parameter becomes an important tool to evaluate cancer cell growth within solid tumors.

Intramuscular nerve damage in lacerated skeletal muscles may direct the inflammatory cytokine response during recovery.

The expression of inflammatory cytokines and growth factors in surgically repaired lacerated muscles over a 12-week recovery phase was investigated. We hypothesized that these expression levels are influenced by both neural and muscular damage within lacerated muscles. Microarrays were confirmed with reverse transcription-polymerase chain reaction assays and histology of biopsies at the lesion of three simulated lacerated muscle models in 130 adult rats. The lacerated medial gastrocnemius with the main intramuscular nerve branch either cut (DN), crushed but leaving an intact nerve sheath (RN); or preserved intact (PN) were compared. At 4 weeks, DN had a higher number of interleukins up-regulated. DN and RN also had a set of Bmp genes significantly expressed between 2 and 8 weeks (P ≤ 0.05). By 12 weeks, DN had a poorer and slower myogenic recovery and greater fibrosis formation correlating with an up-regulation of the Tgf-ß gene family. DN also showed poorer re-innervation with higher mRNA expression levels of nerve growth factor (Ngf) and brain-derived neurotrophin growth factor (Bdnf) over RN and PN. This study demonstrates that the inflammatory response over 12 weeks in lacerated muscles may be directed by the type of intramuscular nerve damage, which can influence the recovery at the lesion site. Inflammatory-related genes associated to the type of intramuscular nerve damage include Gas-6, Artemin, Fgf10, Gdf8, Cntf, Lif, and Igf-2. qPCR also found up-regulation of Bdnf (1-week), neurotrophin-3 (2w), Lif (4w), and Ngf (4w, 8w) mRNA expressions in DN, making them possible candidates for therapeutic treatment to arrest the poor recovery in muscle lacerations (250).

Limited forearm motion compensated by thoracohumeral kinematics when performing tasks requiring pronation and supination.

This study investigates the altered thoracohumeral kinematics when forearm rotation is restricted while performing five activities requiring pronation and supination. Two splints simulated both a fixed-supinated or fixed-neutral forearm in six healthy subjects; the three-dimensional coupled relationship among motion about the forearm, elbow, and shoulder were analyzed. In using a screwdriver, the normal range of forearm rotation of 77.6° (SD = 30.8°) was reduced in the fixed-supinated to 11.3° (SD = 2.9°) and fixed-neutral to 18.2° (SD = 6.2°). This restriction from the fixed-supinated and fixed-neutral forearms was compensated at the shoulder by a significant increase in the total range of (1) ad/abduction by 57.3° and 62.8° respectively (p < .001), (2) forward-reverse flexion (24.3° and 18.2° respectively; p < .05) and (3) internal-external rotation (37.1° and 44.2° respectively; p < .001). A similar result was demonstrated for the doorknob activity. The elbow did not significantly contribute to forearm rotation (p = .14), and is believed to be due to the elbow axis being orthogonal and oblique to the forearm axis. For open kinetic-chain activities, with a fixed-supinated forearm performing there was a significant coupled increase in ad/abduction (p < .05) and int/external rotation (p < .05) for the phone and feeding tasks, with the phone task also having a significantly increased forward shoulder flexion (p < .05). For the fixed-neutral forearm, significant compensatory movement was only seen in the feeding task with increased ad/abduction and internal-external shoulder rotation (p < .05) and the card inserting task with increased ad/abduction and forward-reverse shoulder flexion. Limited forearm function requires compensatory motion from adjacent joints to perform activities that require pronation and supination.

Collagen membranes for host-graft integration: an animal study.

PURPOSE: To evaluate the biocompatibility of collagen membranes for host-graft integration. METHODS: 18 Achilles tendons in 9 rats were used. Five tendons were controls and repaired with prolene 3-0 sutures only. The remaining 13 tendons were cases, and a 10-mm section was excised. A 15x5 mm bovine pericardial collagen membrane (Tutomesh; Tutogen Medical, Germany) was laid circumferentially around the tendon and secured with prolene 3-0 sutures. Tendons were harvested after 4 and 6 weeks. Only the repair interfaces (i.e. cut ends and immediate surrounding tissue) were evaluated. Integration of the membranes to the tendons was evaluated using a semi-quantitative wound maturation scale (1-4) based on the presence of inflammatory cells, vascularisation, fibroblasts, and the amount and alignment of collagen fibrils. The presence of fibroblasts and vascularisation were positive parameters, whereas inflammatory cell ratios were regarded as negative parameters. Immunohistochemical study was also performed. RESULTS: There was no host-graft reaction or wound complication (infection, abscess or seroma). Histological and immunohistochemical assessment confirmed re-approximation of the cut tendon ends and incorporation of the membrane onto the tendon as early as week 4. At week 4, the mean maturation scale scores were 2.7 for controls and 3.3 for cases (p=0.11). At week 6, the corresponding values were 3.8 and 3.0 (p=0.004). A reparative process involving formation of blood vessels and invasion by fibroblasts was noted in both control and case tendons. In controls, T- and B-lymphocytes were present. In cases, inflammatory cells were noted at the junction of the host and membrane without invasion of the graft material. There was no lymphocytic infiltration on the graft. The foreign body reaction was localised and minimal. CONCLUSION: The collagen membrane achieved favourable host-graft integration as early as week 4, with a minimal foreign body reaction.

Collagen membranes for host-implant integration: a pilot clinical study.

PURPOSE: To evaluate host-implant integration with collagen membranes in 14 patients who underwent limb salvage surgery for musculoskeletal oncological disease. METHODS: 8 females and 6 males aged 10 to 69 (mean, 30) years underwent limb savage surgery with collagen membranes (Tutomesh; Tutogen Medical, Germany) for osteosarcoma (n=7), chondrosarcoma (n=3), giant cell tumour (n=1), malignant fibrous histiocytoma (n=1), arteriovenous malformation (n=1), and pigmented villonodular synovitis (n=1). The procedures performed were proximal humeral resection (n=3), partial scapulectomy (n=1), proximal femoral resection (n=2), total femoral resection (n=2), proximal tibial resection (n=3), and wide resection of soft tissues of the knee (n=3). In addition, 10 patients underwent endoprosthesis reconstruction. Reconstruction of musculoskeletal defects was classified into type I (intercalary, n=2), type II (joint, n=4), and type III (both, n=8). Graft incorporation and local recurrence were monitored. Clinical outcome measures entailed the Short Form-36, Toronto Extremity Salvage Score (TESS), and Musculoskeletal Tumor Society Score (MSTS). RESULTS: Two patients with proximal tibial resection and one with total femoral resection had wound healing problems. No patient had any infection or any foreign body reaction necessitating implant removal. Eight patients with type II or III reconstruction were followed up for a mean of 11 (range, 1-23) months. Their scores in the Short Form-36, TESS, and MSTS were similar to those who had undergone reconstructions without the membrane, with the exception of type II reconstructions for which the membrane conferred good results. CONCLUSION: The Tutomesh membrane facilitated host-implant integration and provided a feasible anatomic reconstruction for ligaments in the shoulder, knee, and hip.

Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility.

INTRODUCTION: Metastatic breast cancer cells frequently and ectopically express the transcription factor RUNX2, which normally attenuates proliferation and promotes maturation of osteoblasts. RUNX2 expression is inversely regulated with respect to cell growth in osteoblasts and deregulated in osteosarcoma cells. METHODS: Here, we addressed whether the functional relationship between cell growth and RUNX2 gene expression is maintained in breast cancer cells. We also investigated whether the aberrant expression of RUNX2 is linked to phenotypic parameters that could provide a selective advantage to cells during breast cancer progression. RESULTS: We find that, similar to its regulation in osteoblasts, RUNX2 expression in MDA-MB-231 breast adenocarcinoma cells is enhanced upon growth factor deprivation, as well as upon deactivation of the mitogen-dependent MEK-Erk pathway or EGFR signaling. Reduction of RUNX2 levels by RNAi has only marginal effects on cell growth and expression of proliferation markers in MDA-MB-231 breast cancer cells. Thus, RUNX2 is not a critical regulator of cell proliferation in this cell type. However, siRNA depletion of RUNX2 in MDA-MB-231 cells reduces cell motility, while forced exogenous expression of RUNX2 in MCF7 cells increases cell motility. CONCLUSIONS: Our results support the emerging concept that the osteogenic transcription factor RUNX2 functions as a metastasis-related oncoprotein in non-osseous cancer cells.

Effect of autologous mesenchymal stem cells on biological healing of allografts in critical-sized tibial defects simulated in adult rabbits.

INTRODUCTION: This study evaluated the effect of autologous bone marrow derived adult Mesenchymal Stem Cells (MSCs) on the biological healing of weight bearing diaphyseal bone allograft in the tibia of adult rabbits. MATERIALS AND METHODS: Forty Adult New Zealand White Rabbits divided into 3 groups (Autograft, Allograft or Allograft impregnated with MSCs) with 12 rabbits in each group were used for the study. A 1.5 cm of cortical bone segment was excised from the rabbit's right tibia. The segment was replaced by an Autograft, Allograft or Allograft loaded with MSCs, depending on which group the rabbit was assigned. Internal fixation was performed using a 9-hole Mini-compression Plate and Cerclage Wires. Rabbits were sacrificed at end of observation periods of 12, 16 and 24 weeks. Specimens procured were assessed clinically and radiologically and fixed in 10% buffered formalin. For each specimen, 5 µm undecalcified sections were cut and stained with Von Kossa and Toluidine Blue stains. Histomorphometery was then performed. RESULTS: Our study showed that addition of autologous MSCs to diaphyseal allograft segments enhances and accelerates not just the union at host graft junctions and also the biological incorporation of the allograft segment as shown by Resorption Index, New-Bone Formation Index and Osteocyte Index. CONCLUSIONS: The addition of autologous MSCs to deep frozen cortical allograft segments improved the host - allograft union rate and biological incorporation of diaphyseal allografts as shown by resorption activity, new bone formation and osteocyte cell counts.

Myosin heavy chain isoform profiles remain altered at 7 months if the lacerated medial gastrocnemius is poorly reinnervated: a study in rabbits.

Lacerated skeletal muscles often do not recover full function after repair. Denervated muscles with altered myosin heavy chain isoform (MHC) profiles are known to result in functional impairment. We studied the functional recovery of lacerated muscles, assessing MHC profile changes in association to the involvement of the intramuscular nerve (IM). We tested three lacerated models using the rabbit's medial gastrocnemius where the IM was either cut (NNR), repaired (NR), or preserved intact (NP). Muscles were assessed 7 months after repair for muscle atrophy, isometric contraction (by electrical stimulation), and fibrosis formation at the lesion site. Changes in myofibrillar actomyosin adenosine triphosphatase activity, MHC profile, regenerating myofibers and reinnervation were assessed by Western blot, histology, or immunohistology. Lacerated muscles with a repaired (NR) or an intact (NP) IM showed good recovery, with no significant changes in the MHC profile. Muscles where the IM was not repaired (NNR) resulted in significant scar area at the lesion site (p < 0.05), muscle atrophy (67%, p < 0.05) and loss in contractile properties (63% of the uninjured side, p < 0.05). At 7 months, all muscles were reinnervated. However, the NNR had an inappropriate (polyneural) and poorly distributed reinnervation, the presence of regenerating myofibers, and demonstrated a fast-to-slow MHC transition (71%:29% to 44%:56%, ANOVA, p = 0.018). This was associated to the cut IM when the NNR muscle was lacerated. Poor reinnervation in lacerated skeletal muscles alters the myosin heavy chain profile permanently. This study provides a rationale to also consider biological solutions to improve nerve regeneration and reinnervation in the surgical repair of lacerated muscles.

Runx2, p53, and pRB status as diagnostic parameters for deregulation of osteoblast growth and differentiation in a new pre-chemotherapeutic osteosarcoma cell line (OS1).

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS1 have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS1 had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio (P < 0.05). Absence of p53 correlates with increased Runx2 expression, while the slow proliferation of OS1 cells is perhaps attenuated by pRB retention. OS1 express mesenchymal stem cell markers (CD44, CD105) and differ in relative expression of CD29, CD63, and CD71 to SAOS-2. (P < 0.05). Cell cycle synchronization with nocodazole did not affect Runx2 and CDK1 levels but decreased cyclin-E and increased cyclin-A (P < 0.05). Xenotransplantion of OS1 in SCID mice yields spontaneous tumors that were larger and grew faster than SAOS-2 transplants. Hence, OS1 is a new osteosarcoma cell culture model derived from a pre-chemotherapeutic ethnic Chinese patient, for mechanistic studies and development of therapeutic strategies to counteract metastasis and deregulation of mesenchymal development.

Impaired cell cycle regulation of the osteoblast-related heterodimeric transcription factor Runx2-Cbfbeta in osteosarcoma cells.

Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfbeta heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G(1). Runx2 levels are modulated during the cell cycle, which are maximal in G(1) and minimal beyond the G(1)/S phase transition (S, G(2), and M phases). It is not known whether Cbfbeta gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfbeta are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfbeta gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G(1) (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfbeta protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfbeta levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G(1) and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma.

Novel approach of predicting fracture load in the human proximal femur using non-invasive QCT imaging technique.

This paper presents an analysis of predicting the load-bearing capacities of human femurs using quantitative computer tomography (QCT)-based beam theory. Cross-sectional images of 12 human cadaver femurs (intact bones, age: 39-77 years; male = 8, female = 4) were scanned in conjunction with a calcium hydroxyapatite phantom which has five chambers of known densities. The apparent densities obtained from the scans were used to evaluate the Young's modulus (E) by applying the established empirical relationships. The fracture load of a configuration that simulated single-legged stance was measured experimentally and compared with the predicted failure load using a composite beam theory, plane stress model of the femur. In this model, the failure was assumed to occur at the weakest cross-section through the bone determined from QCT-based structural analysis. In contrast to the other experimental investigations, the setup used in this study considers the entire length of a human femur and also incorporates a novel mechanical jig to mimic the realistic physiological scenario. In one of our earlier studies, simulated lytic defects of varying size were created at the inter-trochanteric region of femurs and their load-bearing capacities were calculated based on their structural properties. Both the results obtained from the current study as well as the ones from our previous study were used to assess the viability of the methodology. A high degree of correlation was observed when the predicted failure loads obtained from the intact femurs and previously studied defective femurs were compared with the ex vivo fracture loads. The coefficients of determination (R(2)) of QCT-derived predicted loads with respect to the measured failure loads were 0.80 for the intact femurs and 0.87 for the defective femurs. The results suggest that the QCT-derived beam analysis provides a viable approach for the assessment of load-bearing capacity in various clinical scenarios.

Unconfined compression properties of a porous poly(vinyl alcohol)-chitosan-based hydrogel after hydration.

A poly(vinyl alcohol) (PVA) hydrogel composite scaffold containing N,O-carboxymethylated chitosan (NOCC) was tested to assess its potential as a scaffold for cartilage tissue engineering in a weight-bearing environment. The mechanical properties under unconfined compression for different hydration periods were investigated. The effect of supplementing PVA with NOCC (20wt.% PVA:5vol.% NOCC) produced a porosity of 43.3% and this was compared against a non-porous PVA hydrogel (20g PVA: 100ml of water, control). Under non-hydrated conditions, the porous PVA-NOCC hydrogel behaved in a similar way to the control non-porous PVA hydrogel, with similar non-linear stress-strain response under unconfined compression (0-30% strain). After 7days' hydration, the porous hydrogel demonstrated a reduced stiffness (0.002kPa, at 25% strain), resulting in a more linear stiffness relationship over a range of 0-30% strain. Poisson's ratio for the hydrated non-porous and porous hydrogels ranged between 0.73 and 1.18, and 0.76 and 1.33, respectively, suggesting a greater fluid flow when loaded. The stress relaxation function for the porous hydrogel was affected by the hydration period (from 0 to 600s); however the percentage stress relaxation regained by about 95%, after 1200s for all hydration periods assessed. No significant differences were found between the different hydration periods between the porous hydrogels and control. The calculated aggregate modulus, H(A), for the porous hydrogel reduced drastically from 10.99kPa in its non-hydrated state to about 0.001kPa after 7days' hydration, with the calculated shear modulus reducing from 30.92 to 0.14kPa, respectively. The porous PVA-NOCC hydrogel conformed to a biphasic, viscoelastic model, which has the desired properties required for any scaffold in cartilage tissue engineering.

Elevated expression of Runx2 as a key parameter in the etiology of osteosarcoma.

To understand the molecular etiology of osteosarcoma, we isolated and characterized a human osteosarcoma cell line (OS1). OS1 cells have high osteogenic potential in differentiation induction media. Molecular analysis reveals OS1 cells express the pocket protein pRB and the runt-related transcription factor Runx2. Strikingly, Runx2 is expressed at higher levels in OS1 cells than in human fetal osteoblasts. Both pRB and Runx2 have growth suppressive potential in osteoblasts and are key factors controlling competency for osteoblast differentiation. The high levels of Runx2 clearly suggest osteosarcomas may form from committed osteoblasts that have bypassed growth restrictions normally imposed by Runx2. Interestingly, OS1 cells do not exhibit p53 expression and thus lack a functional p53/p21 DNA damage response pathway as has been observed for other osteosarcoma cell types. Absence of this pathway predicts genomic instability and/or vulnerability to secondary mutations that may counteract the anti-proliferative activity of Runx2 that is normally observed in osteoblasts. We conclude OS1 cells provide a valuable cell culture model to examine molecular events that are responsible for the pathologic conversion of phenotypically normal osteoblast precursors into osteosarcoma cells.

Role of multi-drug resistance-associated protein-1 transporter in statin-induced myopathy.

This study investigated the effects of probenecid to inhibit the multi-drug resistance-associated protein-1 (MRP-1) in mediating the efflux and myotoxicity in rat skeletal muscles, with administration of rosuvastatin. Male Sprague-Dawley rats were administered daily, for 15 days, with either rosuvastatin (50, 100 or 200 mg/kg) or probenecid (100 mg/kg) alone, or with a combination of rosuvastatin (50, 100 or 200 mg/kg) and probenecid (100 mg/kg). Skeletal muscle toxicity was elevated with probenecid administered with 200 mg/kg/day of rosuvastatin, with the elevation of creatine kinase by 12-fold, alanine aminotrasferase by 10-fold and creatinine by 9-fold at day 15, with no adverse effects observed when probenecid was given alone. Mitochondria ultrastructural damage with enlargement, disruption, cristolysis and vaculation was seen in the soleus and plantaris of animals administered with probenecid and high dosages of statin. These muscles were also expressing more succinic dehydrogenase (SDH)-positive and cytochrome oxidase (CyOX)-positive fibers. Although generally well-tolerated, statins produce a variety of adverse skeletal muscle events. Hydrophilic statins, with reduced levels of non-specific passive diffusion rates into extra-hepatic tissues, are still seen to produce myopathy. This highlights the important roles of transport mechanisms in statin transport at the skeletal muscles. Excessive influx, reduced efflux or the combination of the two could result in elevated cellular levels of statins at the skeletal muscles, resulting in toxicity. This study provides preliminary evidence that the MRP-1 transporter and efflux at skeletal muscles possibly play significant roles in statin-induced myopathy.

An autologous cell lysate extract from human embryonic stem cell (hESC) derived osteoblasts can enhance osteogenesis of hESC.

It was recently demonstrated that osteogenesis of hESC was more efficient without the initial embryoid body formation step. This study sought to further improve this direct differentiation culture system, by developing an autologous osteogenic-inducing culture supplement extracted from hESC-derived osteogenic cells themselves. A whole cell lysate was prepared from hESC-derived osteogenic cells, simply by exposure to deionized water followed by free-thawing and subsequent filtration. The product was used to coat the surface of cell culture dishes together with gelatin, prior to culture of hESC under osteogenic-inducing conditions. The results showed that the autologous cell lysate extract promoted the aggregation and clustering of cells to form nodule-like structures. Immunohistochemical staining on day 9 demonstrated that these cellular aggregates strongly expressed STRO-1, while on day 14 the nodule-like structures stained positively for both osteocalcin and osteonectin (SPARC). By contrast, the negative control (gelatin coating alone) showed much less prominent cellular aggregation and clustering, and was stained much less intensely for these markers. Additionally, Von Kossa staining on day 14 was also more intense in the presence of the autologous cell lysate extract. Hence, this product can be used to further enhance the osteogenesis of hESC. This would save costs from the use of highly-expensive cytokines, growth factors and matrix components, as well as avoid pathogenic transmission from animal and human products.