Differential effects of bone morphogenetic protein-2 and transforming growth factor-beta1 on gene expression of collagen-modifying enzymes in human adipose tissue-derived mesenchymal stem cells.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) in combination with bone morphogenetic protein-2 (BMP-2) or transforming growth factor-beta1 (TGF-beta1) are under evaluation for bone tissue engineering. Posttranslational modification of type I collagen is essential for functional bone tissue with adequate physical and mechanical properties. We investigated whether BMP-2 (10-100 ng/mL) and/or TGF-beta1 (1-10 ng/mL) affect gene expression of alpha2(I) procollagen and collagen-modifying enzymes, that is, lysyl oxidase and lysyl hydroxylases 1, 2, and 3 (encoded by PLOD1, 2, and 3), by human AT-MSCs. BMP-2, but not TGF-beta1, increased alkaline phosphatase activity after 28 days, indicating osteogenic differentiation of AT-MSCs. At day 4, both BMP-2 and TGF-beta1 upregulated alpha2(I) procollagen and PLOD1, which was downregulated at day 28. TGF-beta1, but not BMP-2, downregulated PLOD3 at day 28. Lysyl oxidase was upregulated by TGF-beta1 at day 4 and by BMP-2 at day 7. Neither BMP-2 nor TGF-beta1 affected PLOD2. In conclusion, these results suggest that AT-MSCs differentially respond to BMP-2 and TGF-beta1 with changes in gene expression of collagen-modifying enzymes. AT-MSCs may thus be able to appropriately modify type I collagen to form a functional bone extracellular matrix for tissue engineering, dependent on the growth factor added.

Intravenous administration of the conditionally replicative adenovirus Ad5-Delta24RGD induces regression of osteosarcoma lung metastases.

Metastatic osteosarcoma (OS) has a very poor prognosis. New treatments are therefore wanted. The conditionally replicative adenovirus Ad5-Delta24RGD has shown promising anti-tumor effects on local cancers, including OS. The purpose of this study was to determine whether intravenous administration of Ad5-Delta24RGD could suppress growth of human OS lung metastases. Mice bearing SaOs-lm7 OS lung metastases were treated with Ad5-Delta24RGD at weeks 1, 2 and 3 or weeks 5, 6 and 7 after tumor cell injection. Virus treatment at weeks 1-3 did not cause a statistically significant effect on lung weight and total body weight. However, the number of macroscopic lung tumor nodules was reduced from a median of >158 in PBS-treated control mice to 58 in Ad5-Delta24RGD-treated mice (p = 0.15). Moreover, mice treated at weeks 5-7 showed a significantly reduced lung weight (decrease of tumor mass, p < 0.05), a significantly increased body weight gain (decrease of disease symptoms, p < 0.005) and a reduced number of macroscopic lung tumor nodules (median 60 versus > 149, p = 0.12) compared to PBS treated control animals. Adenovirus hexon expression was detected in lung tumor nodules at sacrifice three weeks after the last intravenous adenovirus administration, suggesting ongoing viral infection. These findings suggest that systemic administration of Ad5-Delta24RGD might be a promising new treatment strategy for metastatic osteosarcoma.

Long term follow-up of a patient with disseminated spinal hydatidosis.

A rare case of a 27-year-old male patient with disseminated sacral hydatidosis is presented. Because the diagnosis was missed initially, the patient underwent only partial resection of the tumour to obtain tissue for histology. The resection was followed by deep wound infection, and re-exploration had to be performed, thereby resecting the remaining cyst tissue and the S1-S3 vertebral bodies. Adjuvant anti-helminthic therapy was started postoperatively. Unfortunately, the hydatid cyst further progressed and could only be controlled with multiple decompression procedures and continuance of anti-helminthic therapy. We review the diagnosis, treatment and prognosis of this uncommon condition, which is a serious challenge for the spinal surgeon.

Enhanced tumor cell kill by combined treatment with a small-molecule antagonist of mouse double minute 2 and adenoviruses encoding p53.

Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer.

Sterilization and strength of 70/30 polylactide cages: e-beam versus ethylene oxide.

STUDY DESIGN: In vitro and in vivo studies on the degradation of 70/30 poly(L,DL-lactide) (PLDLLA) cages. OBJECTIVE: To evaluate the effect of e-beam and ethylene oxide sterilization on degradation and strength. SUMMARY OF BACKGROUND DATA: e-beam-sterilized PLDLLA cages were shown to maintain mechanical strength for at least 6 months during degradation studies in vitro. Yet failure of the cages was observed after only 3 months in vivo. We hypothesized that degradation characteristics and mechanical strength could be improved by sterilizing the cages through ethylene oxide (EtO) instead of e-beam. METHODS: PLDLLA cages were sterilized either by e-beam or EtO, and degraded in phosphate-buffered saline. Each month, cages were compressed until failure. Inherent viscosity was determined as a measure of degradation. For the in vivo evaluation, e-beam- or EtO-sterilized cages were implanted at L3-L4 in a standardized goat model. After 3 or 6 months, retrieved segments were scanned by high-resolution magnetic resonance imaging. Also, inherent viscosity of the polymer was measured. RESULTS: e-beam sterilization strongly decreased inherent viscosity of PLDLLA compared with EtO sterilization, but initial strength was only affected marginally. After 6 months, the strength of the e-beam-sterilized cages dropped, while that of EtO-sterilized cages was maintained. Degradation in vivo was slightly faster than in vitro. In both groups, however, mechanical failure occurred at 3 months after implantation. CONCLUSIONS: Inherent viscosity decreases with degradation time, but strength only decreases when inherent viscosity is below a certain threshold. Above this threshold, mechanical strength is a property of the polymer and independent of inherent viscosity. e-beam sterilization strongly decreases inherent viscosity and thus advances mechanical degradation. EtO sterilization delays degradation but does not increase initial strength. Early failure of PLDLLA cages in the goat model thus is unrelated to sterilization method and requires further study.

Internal thoracic vessels used as pedicle graft for anastomosis with vascularized bone graft to reconstruct C7-T3 spinal defects: a new technique.

STUDY DESIGN: A report of 4 cases of primary bone tumors (3 cases) or infection (1 case) at the cervicothoracic junction treated with resection-reconstruction. OBJECTIVES: To document a new technique using the internal thoracic vessels as recipient vessels for reconstruction of the cervicothoracic spine with free vascularized fibula grafts. SUMMARY OF BACKGROUND DATA: The cervicothoracic junction is a difficult region in reconstructive spinal surgery. Although nonvascularized fibula grafts can be used to reconstruct the osseous defect, compared with free vascularized fibula grafts they are biomechanical weaker, incorporate less well, are less resistant to infection, and remodel incomplete in time. However, when using free vascularized bone grafts, the selection of suitable recipient vessels remains one of the most critical decisions. MATERIALS AND METHODS: Four patients who had a primary tumor (3 cases) or a severe progressive kyphotic deformity and progressive neurologic symptoms due to tuberculosis (1 case) were treated by resection and vascularized reconstruction. In 3 patients, a staged anteroposterior en bloc resection of T1-T3 (2 cases) or T1-T2 (1 case) was performed; the ventral reconstruction of the osseous defect consisted of a vascularized fibula graft interposition between C7-T4 (2 cases) or C7-T3 (1 case). In another case, an axial slot was milled through the T1-T2 vertebral bodies to accept an osteotomized vascularized fibular graft. In all cases, a free vascularized fibula graft was used: the vascular anastomosis was performed between the peroneal and the dissected and rerouted internal thoracic vessels. The anterior construction was strengthened by a ventral plate-screw system. RESULTS: The resection-reconstruction procedures, including the dissection, rerouting, and anastomosis between the internal thoracic vessels and the peroneal vessels, were successfully performed. At present, all patients are alive, and there is no evidence of recurrent disease, unchanged, or improved neurologic with a mean follow-up of 28 months. All grafts are well incorporated. CONCLUSIONS.: A combined low anterolateral cervical and midsternal approach or a midline sternotomy allows not only a safe and excellent exposure to the cervicothoracic junction but also to the internal thoracic vessels. The internal thoracic vessels are appropriate donor vessels: its longevity, diameter, length, and rerouting capacity allow vascularized graft reconstruction of vertebral column defects of the low cervical (C6-C7) and/or upper thoracic (T1-T3) region.

Spinal tuberculosis in a 14-year-old immigrant in the Netherlands.

We present a case of Pott's disease, where the patient presented with neurological impairment due to vertebral granulomatous necrosis, needing immediate decompression and later stabilizing and reconstructive orthopaedic surgery, in order to create awareness for TB in general, especially this forgotten form of spinal tuberculosis.

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor.

Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer.

The clinical significance of lumbosacral transitional anomalies.

Lumbosacral transitional vertebrae (LSTV) are common congenital anomalies of the human spine. In LSTV, either the fifth lumbar vertebra may show assimilation to the sacrum (sacralisation), or the first sacral vertebra may show transition to a lumbar configuration (lumbarisation). Although the condition has an incidence of over 12% in the general population, knowledge about the exact clinical implications is still lacking. The association between LSTV and low back pain has been debated since it was first described by Bertolotti almost a century ago. Furthermore, several conflicting studies have been published regarding the association of LSTV with other spinal pathology. There seems to be a relation with early disc degeneration above the LSTV in young patients. However, these differences fade with age as they are masked by other degenerative changes of the spine. From a practical view-point, failure to recognise and to number LSTV during spinal surgery may have serious consequences.

Does bioresorbable cage material influence segment stability in spinal interbody fusion?

To reduce long term complications associated with nonresorbable interbody fusion cages, bioresorbable cages are being developed. We investigated the influence of bioresorbable cage material on segment stability, intervertebral disc height and fusion in vivo using radiostereometric analysis comparing 70/30 poly(L-lactide-co-D,L-lactide) (PLDLLA) cages with titanium cages. Twenty-eight goats were randomized to receive PLDLLA (n = 21) or a titanium control (n = 7) cage at L3-L4. Range of motion for flexion and extension and change in intervertebral disc height were measured before and after surgery and at followup (3, 6, and 12 months). Fusion was graded with a validated radiographic score. Although the PLDLLA cage could not provide the optimal environment for a successful high fusion rate, the range of motion of the PLDLLA segments gradually decreased in time and was similar to the titanium control group at 12 months. In addition the decrease of intervertebral disc height was similar for both PLDLLA (1.4 +/- 0.8 mm) and titanium (1.3 +/- 1.0 mm) specimens. Both results showed a bioresorbable cage does not lead to less decrease of motion or more loss of intervertebral disc height in time compared to titanium. This study therefore supports further development of a bioresorbable cage concept.

Polyamines modulate nitric oxide production and COX-2 gene expression in response to mechanical loading in human adipose tissue-derived mesenchymal stem cells.

For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) display a bone cell-like response to mechanical loading. We have shown earlier that this response includes increased nitric oxide (NO) production and cyclooxygenase-2 (COX-2) gene expression, both of which are intimately involved in mechanical adaptation of bone. COX-2 gene expression is likely regulated by polyamines, which are organic cations implicated in cell proliferation and differentiation. This has led to the hypothesis that polyamines may play a role in the response of adipose tissue-derived MSCs (AT-MSCs) to mechanical loading. The aim of this study was to investigate whether genes involved in polyamine metabolism are regulated by mechanical loading and to study whether polyamines modulate mechanical loading-induced NO production and COX-2 gene expression in human AT-MSCs. Human AT-MSCs displayed a bone cell-like response to mechanical loading applied by pulsating fluid flow (PFF), as demonstrated by increased NO production and increased gene expression of COX-2. Furthermore, PFF increased gene expression of spermidine/spermine N (1)-acetyltransferase, which is involved in polyamine catabolism, suggesting that mechanical loading modulates polyamine levels. Finally, the polyamine spermine was shown to inhibit both PFF-induced NO production and COX-2 gene expression, suggesting that polyamines modulate the response of human AT-MSCs to mechanical loading. In conclusion, this is the first study implicating polyamines in the response of human AT-MSCs to mechanical loading, creating opportunities for the use of polyamines in tissue engineering approaches targeting skeletal defects.

Mycoplasma hominis deep wound infection after neuromuscular scoliosis surgery: the use of real-time polymerase chain reaction (PCR).

Mycoplasma hominis is a commensal of the genitourinary tract. It mostly causes infections to associated structures of this system; however, occasionally it is a pathogen in nongenitourinary tract infections. Since, M. hominis strains require special growth conditions and cannot be Gram stained, they may be missed or delay diagnosis. This report describes a deep wound infection caused by M. hominis after neuromuscular scoliosis surgery; M. hominis was recovered by real-time polymerase chain reaction (PCR). An awareness of the role of M. hominis as an extragenital pathogen in musculoskeletal infections, especially in neuromuscular scoliosis, being a high-risk group for postoperative wound infection, it is necessary to identify this pathogen. Real-time PCR for postoperative deep wound infection, in patients with a history of genitourinary infections, decreases the delay in diagnosis and treatment. In these cases rapid real-time PCR on deep cultures should be considered.

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells.

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 microg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 microg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 microg/mL) and bone cell death (70% at 400 microg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.

Four-year follow-up of poly-L-lactic Acid cages for lumbar interbody fusion in goats.

BACKGROUND: New applications of bioabsorbable polymer implants demand for histologic evaluation because a host tissue response is elicited and late complications after polymer implantation have been reported. Furthermore, in load-bearing regions an accelerated polymer degradation and foreign body reaction may be observed. METHODS: Lumbar interbody fusion procedures were performed using poly-L-lactic acid (PLLA) and titanium cages in 43 goats. At 3, 6, 12, 24, 36, and 48 months after surgery, sequential histologic analysis of instrumented motion segments, lymph nodes, and nervous structures was performed. Blood samples were retrieved for laboratory analysis. RESULTS: No adverse local or distant histologic or systemic effects were observed during the absorption of the poly-L-lactic acid cages. Interbody fusion was maintained, and only a very mild inflammatory response was observed. In half the specimens complete absorption was observed, and in the remaining specimens an estimated 1-10% of the original PLLA was present at the 3-year follow-up. At the 4-year follow-up, five out of seven PLLA specimens showed no PLLA particles under polarized light microscopy. In the remaining two specimens an estimated 1% of the original PLLA could be observed. CONCLUSIONS: Poly-L-lactic acid cages are feasible for lumbar interbody fusion, and the biocompatibility under high load bearing conditions is excellent during the complete absorption of the PLLA interbody fusion cages.

Biodegradable X-ray markers of controlled radio-opacity. Temporary position measurements in bone.

In order to analyze X-ray markers for potential use in biodegradable implants or radiostereogrammatic analysis (RSA), we combined iopromide contrast fluid with biodegradable calcium phosphate cement. The radio-opacity of 10 x 10 mm markers containing different iodine concentrations (0, 120, 240, 360 and 720 mg per gram cement) was compared to an aluminium wedge of increasing (1-10 mm) thickness. The addition of iopromide increased the radio-opacity in a dose-dependent manner, which was comparable to 9-mm aluminium at concentrations of 240-720 mg/g. Radiographs of markers placed in explanted rabbit and in human femora were made to investigate the clinical accuracy for position determination. Markers of 1 x 1 mm (120 mg/g) were clearly discernable in all femora, and could be used to adequately measure distances of 5-45 mm (accuracy 0.10-2.19 mm). These markers might be embedded in biodegradable implants or used as temporary markers in the bone to analyze postoperative position on radiographs.

Osteoid osteoma of the spine: a novel technique using combined computer-assisted and gamma probe-guided high-speed intralesional drill excision.

STUDY DESIGN: A report of five cases of thoracolumbar osteoid osteoma treated with combined computer-assisted and gamma probe-guided high-speed drill excision. OBJECTIVES: To document the surgical technique consisting of a combination of both computer-assisted and gamma probe-guided high-speed drill excision for osteoid osteoma of the spine. SUMMARY OF BACKGROUND DATA: Curative treatment of spinal osteoid osteoma is performed by surgical intralesional excision of the nidus, but intraoperative localization of the nidus is often difficult. Although intraoperative gamma-probe guidance facilitates accurate localization of the nidus, wide surgical resection of the bony structure is still mandatory to ensure removal of the nidus. Computer-assisted surgery has been proven to facilitate surgical intervention in spinal surgery. However, there is no clinical report regarding the application and usefulness of computer-assisted intralesional excision of the osteoid nidus. Excision of the nidus with a computer-assisted high-speed drill and intraoperative gamma probe control may result in complete intralesional excision without sacrificing more bone than necessary. METHODS: One day before surgery, patients were injected with radioactive mTc-oxidronate. With a computed tomography-based electro-optical navigation system, real-time virtual images of the osteoid osteoma were generated by matching the intraoperative surface with preoperative computed tomography images. The osteoid osteoma was excised with the use of an image-guided high-speed drill, and complete excision was controlled with a gamma detection probe. RESULTS: Excision of the nidus was confirmed by relief of symptoms, postexcision computed tomography scans, and histologic evaluation on clinical and radiographic follow-up observation. All five patients reported immediate complete relief of characteristic pain and no evidence of recurrence after 6 to 33 months of follow-up observation. There were no complications. CONCLUSIONS: The combination of both computer-assisted surgery and gamma probe-guided high-speed drill excision for osteoid osteoma of the spine helps to localize and excise the nidus of the osteoid osteoma with minimal bone resection of the posterior spinal structures.

Long-term follow-up of 15 patients with non-metastatic Ewing's sarcoma and a skip lesion.

UNLABELLED: BACKGROUND Skip lesions in Ewing's sarcoma of the bone seem to be rare; to our knowledge only 7 cases have been published in the English medical literature. METHODS: We retrospectively reviewed imaging and histological data relating to 235 patients with non-metastatic Ewing's sarcoma of the bone who participated in the cooperative Ewing's sarcoma study (CESS 86 and CESS 91), and we identified 15 patients with a skip lesion at diagnosis. RESULTS: The skip lesion was located in the same bone as the primary tumor in 13 patients, and in an adjacent juxtaarticular bone in 2 cases. The average follow-up was 11 years. Despite aggressive treatment including surgery in all cases, tumor relapse occurred in 9 patients, and 7 of these patients died due to metastatic disease. INTERPRETATION: Skip lesions in patients with otherwise non-metastatic skeletal Ewing's sarcoma may be of the same consequence as the molecular detection of marrow metastases and possibly confer a worse prognosis. Newer imaging modalities (for example PET) and careful staging work-up may indicate that skip metastases in Ewing's sarcoma are more common than previously suspected.

Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation.

To engineer bone tissue, mechanosensitive cells are needed that are able to perform bone cell-specific functions, such as (re)modeling of bone tissue. In vivo, local bone mass and architecture are affected by mechanical loading, which is thought to provoke a cellular response via loading-induced flow of interstitial fluid. Adipose tissue is an easily accessible source of mesenchymal stem cells for bone tissue engineering, and is available in abundant amounts compared with bone marrow. We studied whether adipose tissue-derived mesenchymal stem cells (AT-MSCs) are responsive to mechanical loading by pulsating fluid flow (PFF) on osteogenic stimulation in vitro. We found that ATMSCs show a bone cell-like response to fluid shear stress as a result of PFF after the stimulation of osteogenic differentiation by 1,25-dihydroxyvitamin D3. PFF increased nitric oxide production, as well as upregulated cyclooxygenase-2, but not cyclooxygenase-1, gene expression in osteogenically stimulated AT-MSCs. These data suggest that AT-MSCs acquire bone cell-like responsiveness to pulsating fluid shear stress on 1,25-dihydroxyvitamin D3-induced osteogenic differentiation. ATMSCs might be able to perform bone cell-specific functions during bone (re)modeling in vivo and, therefore, provide a promising new tool for bone tissue engineering.

In vivo comparison of Dhvar-5 and gentamicin in an MRSA osteomyelitis prevention model.

OBJECTIVES: The continued rise in drug-resistant pathogens has led to global research efforts into new antimicrobial agents. A promising class of new agents are the antimicrobial peptides. The aim of the study was to investigate the efficacy of the antimicrobial peptide Dhvar-5 in a prophylactic, methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis model. METHODS: Dhvar-5 (12 mg or 24 mg/rabbit) was incorporated into polymethyl methacrylate (PMMA) beads as a local drug delivery system. For comparison, plain beads (control) and beads containing gentamicin as a sulphate (10 mg or 24 mg per rabbit) were also prepared. The beads were inserted into the inoculated femoral cavity of 36 rabbits, and 1 week later they were killed. The presence and severity of MRSA osteomyelitis was assessed by culture and histology. RESULTS: Both the 24 mg Dhvar-5 beads and the 24 mg gentamicin sulphate beads significantly reduced the bacterial load of the inoculated femora compared with the control chain. Although a 24 mg Dhvar-5 dose inhibited MRSA growth, it did not completely sterilize the femora. Sterilization occurred only in some of the gentamicin-treated specimens. CONCLUSION: We conclude that both the gentamicin beads and the Dhvar-5 beads were only partially effective at preventing MRSA infection in this model.