Effects of mesenchymal stromal cells versus serum on tendon healing in a controlled experimental trial in an equine model.

BACKGROUND: Mesenchymal stromal cells (MSC) have shown promising results in the treatment of tendinopathy in equine medicine, making this therapeutic approach seem favorable for translation to human medicine. Having demonstrated that MSC engraft within the tendon lesions after local injection in an equine model, we hypothesized that they would improve tendon healing superior to serum injection alone. METHODS: Quadrilateral tendon lesions were induced in six horses by mechanical tissue disruption combined with collagenase application 3 weeks before treatment. Adipose-derived MSC suspended in serum or serum alone were then injected intralesionally. Clinical examinations, ultrasound and magnetic resonance imaging were performed over 24 weeks. Tendon biopsies for histological assessment were taken from the hindlimbs 3 weeks after treatment. Horses were sacrificed after 24 weeks and forelimb tendons were subjected to macroscopic and histological examination as well as analysis of musculoskeletal marker expression. RESULTS: Tendons injected with MSC showed a transient increase in inflammation and lesion size, as indicated by clinical and imaging parameters between week 3 and 6 (p < 0.05). Thereafter, symptoms decreased in both groups and, except that in MSC-treated tendons, mean lesion signal intensity as seen in T2w magnetic resonance imaging and cellularity as seen in the histology (p < 0.05) were lower, no major differences could be found at week 24. CONCLUSIONS: These data suggest that MSC have influenced the inflammatory reaction in a way not described in tendinopathy studies before. However, at the endpoint of the current study, 24 weeks after treatment, no distinct improvement was observed in MSC-treated tendons compared to the serum-injected controls. Future studies are necessary to elucidate whether and under which conditions MSC are beneficial for tendon healing before translation into human medicine.

[Tumor prostheses : Important in modern revision arthroplasty]. / Tumorendoprothetik : Stellenwert in der modernen Revisionsendoprothetik.

BACKGROUND: Due to demographic aging, an increasing number of revision arthroplasty procedures is expected during upcoming years. While the use of a megaprosthesis for bone reconstruction after tumor resection is gold standard, this type of prosthesis still meets with reservation in the field of revision arthroplasty because of numerous risk factors. OBJECTIVES: The purpose of this article is to present the importance of modular megaprostheses in revision arthroplasty, taking into consideration the risks of periprosthetic infection, aseptic loosening, material failure, and hip dislocation. RESULTS: Because of improvements in the field of megaprostheses during the last 30 years, the risks after implantation of this type of prosthesis have significantly decreased. The risk of periprosthetic infection has been reduced about 1/3 by the use of silver surface coating. Improvements in stem design, cement technique, and the additional use of locking screws have minimized the risk of aseptic loosening. Improvements in material composition have reduced the risk of material failure. The risk of hip dislocation could also be minimized by careful tissue preparation and appropriate suture technique. CONCLUSION: There is no need for the conservative use of megaprostheses in revision arthroplasty. There are many benefits in the use of megaprostheses in multimorbid patients (i.e., reduced operating time, the possibility of early full weight bearing, and a reduced risk of periprosthetic infection by the use of silver surface coating) instead of complex bone reconstruction during revision arthroplasty.

[Benign bone tumors. General principles]. / Gutartige Knochentumoren. Allgemeine Prinzipien.

BACKGROUND: Benign bone tumors and tumor-like lesions are much more frequent than malignant bone tumors among the total number of tumors of the skeleton. OBJECTIVE: This article gives a presentation of the characteristics and treatment modalities of benign bone tumors. MATERIAL AND METHODS: In this article in-house treatment principles are compared with those in the currently available literature. RESULTS: Benign bone tumors are frequently found incidentally; however, the term benign does not always signify that a purely observational role is needed. Benign bone tumors differ in their biological behavior and can be latent, active or aggressive which determines the treatment approach. Some benign bone tumors are just as aggressive locally as malignant tumors. The most important diagnostic feature is still conventional radiography and a thorough systematic analysis is necessary. Therapy options range from ignore, wait and see up to wide resection. In contrast to malignant tumors the radicalism of resection can be weighed against the accompanying local control and loss of function. CONCLUSION: The treatment of benign bone tumors depends on the histological type and the biological activity. Most benign bone tumors are diagnosed incidentally and do not necessitate any treatment.

The influence of tumor- and treatment-related factors on the development of local recurrence in osteosarcoma after adequate surgery. An analysis of 1355 patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols.

BACKGROUND: Local recurrence (LR) in osteosarcoma is associated with very poor prognosis. We sought to evaluate which factors correlate with LR in patients who achieved complete surgical remission with adequate margins. PATIENTS AND METHODS: We analyzed 1355 patients with previously untreated high-grade central osteosarcoma of the extremities, the shoulder and the pelvis registered in neoadjuvant Cooperative Osteosarcoma Study Group trials between 1986 and 2005. Seventy-six patients developed LR. RESULTS: Median follow-up was 5.56 years. No participation in a study, pelvic tumor site, limb-sparing surgery, soft tissue infiltration beyond the periosteum, poor response to neoadjuvant chemotherapy, failure to complete the planned chemotherapy protocol and biopsy at a center other than the one performing the tumor resection were significantly associated with a higher LR rate. No differences were found for varying surgical margin widths. Surgical treatment at centers with small patient volume and additional surgery in the primary tumor area, other than biopsy and tumor resection, were significantly associated with a higher rate of ablative surgery. CONCLUSIONS: Patient enrollment in clinical trials and performing the biopsy at experienced institutions capable of undertaking the tumor resection without compromising the oncological and functional outcome should be pursued in the future.

Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors.

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.

Characterisation and intracellular labelling of mesenchymal stromal cells derived from synovial fluid of horses and sheep.

Multipotent mesenchymal stromal cells (MSCs) derived from synovial fluid (SF) are considered to be a promising cell type for therapeutic applications in joint disease. However, despite their potential relevance for clinical and experimental studies, there is insufficient knowledge about SF-derived MSCs isolated from horses and sheep. In this study, cells were recovered from healthy SF and bone marrow (BM) of sheep, and from healthy and osteoarthritic SF of horses. Ovine SF-MSCs were used to assess the efficiency of intracellular labelling with quantum dots (QDs). Colony forming units, generation times, trilineage differentiation potential and expression of CD73, CD90 and CD105 at mRNA level were assessed. QD labelling was efficient, with >98% positive cells directly after labelling at 10 nmol/L and >95% positive cells directly after labelling at 2 nmol/L. The label decreased over 7 days of culture, with more persistence at the higher labelling concentration. No significant differences in proliferation were observed. All MSCs had trilineage differentiation potential, but adipogenesis was more distinct in equine samples and chondrogenesis was most pronounced in ovine SF-MSCs. CD73, CD90 and CD105 were expressed in equine and ovine MSCs.

Comparative RNA expression analyses from small-scale, single-donor platelet samples.

BACKGROUND: Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders. OBJECTIVES: We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT-PCR assay. We tested three methods to identify the differences in RNA between platelet sources. METHODS: Differential hybridization to cDNA macro-arrays and suppressive-subtractive hybridization PCR (SSH-PCR) were used to compare RNAs from normal platelets to those from a Bernard-Soulier syndrome (BSS) patient. Affymetrix GeneChip U133 plus 2.0 arrays were used to compare male and female platelet RNAs. RESULTS: Macroarrays identified approximately 7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH-PCR produced libraries almost exclusively of mitochondrial-derived transcripts, but included nuclear-encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small-scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs. CONCLUSIONS: This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small-scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.

The effect of rotationplasty on the ankle joint: long-term results.

In patients with rotationplasty the biomechanical conditions in the ankle joint are altered dramatically. By displacement and reduction of the weight-bearing area of the joint, the stress affecting its cartilage is increased. The use of an exoprothesis results in skin and soft tissue irritation. Due to these biomechanical changes, a prearthrotic deformity or skin problems could be expected. The current study examines changes in 21 patients treated with rotationplasty (mean follow-up 13.5 years) because of a malignant bone tumour or a femoral segmental defect. Local tenderness, skin and soft tissue changes, problems with exoprostheses, and pain was assessed by clinical examination and documented. Osseous changes were evaluated by plain X-ray. A MRI-scan was also obtained in five patients. Hardened skin and blisters were located at the main loading areas of the rotated foot. These changes could be reduced by optimizing the exoprosthetic fit. Radiographically, a slight asymptomatic attenuation of the articular space was observed in four patients and a slight coexistent subchondral sclerosis with small osteophytes in one patient. No degenerative changes were observed on X-ray and no cartilaginous changes were observed on MRI. The results suggest that the foot is able to adapt to the load changes after this procedure and that rotationplasty does not cause an inevitable arthrosis in the ankle joint.

Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells.

The molecular basis for the receptorless (r-) and activation-labile (act1) phenotypes of glucocorticoid-resistant mutants isolated from glucocorticoid-sensitive human leukemic CEM-C7 cells was determined. Clones isolated from a complementary DNA library prepared from r- ICR27TK.3 cells, in which one glucocorticoid receptor (GR) gene has been deleted, contained a single adenosine to thymidine transversion in the third position of codon 753, resulting in the substitution of phenylalanine for leucine. This mutant gene (GR753F) had only 13% of the trans-activating activity of the normal gene and produced a M(r) 92,000 receptor protein with the same r- phenotype seen in ICR27TK.3 cells. Analysis of complementary DNA clones isolated from a library prepared from parental glucocorticoid-sensitive 6TG1.1 cells showed that these cells express both a normal GR gene (GR+) and the GR753F gene. Thus, their genotype is GR+/GR753F. Analysis of clones isolated from a complementary DNA library prepared from glucocorticoid-resistant activation-labile 3R7. 6TG.4 cells revealed the presence of the GR753F gene and a second mutant gene (GR421Y) containing a guanosine to adenosine transition in the second position of codon 421, resulting in the replacement of the first cysteine of the proximal zinc finger of the DNA-binding domain by tyrosine. This mutant had no trans-activating activity but normal ligand-binding characteristics. Thus, the genotype of act1 3R7.6TG.4 cells is GR421Y/GR753F. Consequently, the sequence-specific DNA-binding activity of receptors in act1 cells is attributable to the GR753F gene, while the ligand-binding activity seen in intact cells is attributable to the GR421Y gene. These results provide a direct explanation for the r- and act1 phenotypes of glucocorticoid-resistant cells and demonstrate that glucocorticoid-sensitive cells derived from CEM-C7 cells contain a heterogeneous population of normal and mutant receptors.

Glucocorticoid receptor gene mutations in leukemic cells acquired in vitro and in vivo.

Glucocorticoid resistance was investigated in human leukemic CCRF-CEM cells. A mutation (L753F), which renders the human glucocorticoid receptor (hGR) gene functionally hemizygous, was identified in all CEM-derived cell lines analyzed. Allele-specific PCR identified the same mutation in lymph node biopsy material from patient CEM cells. Given the correlation between hGR concentration and glucocorticoid sensitivity, this suggests that loss of functional heterozygosity may result in resistance to glucocorticoid-based chemotherapy. The L753F mutation was probably not responsible for the ontogeny of the disease because it did not appear to be present in all leukemic cells. Thus, it is unlikely that hGR mutations would be detected in leukemic patients at presentation, but they may occur, and be selected for, during treatment. Deletions and point mutations in the hGR gene of cells selected for steroid resistance in vitro were investigated by PCR-single strand conformation polymorphism analysis. Loss of hGR mRNA expression resulted from 5'-deletion of the hGR gene and nonsense mutations in exon 6. These results provide the first evidence for somatic mutation in the hGR gene of a patient with acute lymphoblastic leukemia, offer a potential in vivo mechanism for acquisition of steroid resistance in leukemia, and suggest that screening for additional in vivo mutations will require analysis of genomic DNA.

Ewing's sarcoma of the ribs. A report from the cooperative Ewing's sarcoma study.

31 patients with primary Ewing's sarcoma of the ribs were treated according to the protocols of CESS 81, CESS 86P and CESS 86. The results of treatment were reviewed and analysed. 24 patients presented with localised disease and 7 with regional disease. 20 of 24 localised cases and 6 of 7 regional cases underwent tumour resection. All but 2 localised cases received irradiation. The cumulative relapse-free survival (RFS) rate of 31 patients was 61% at 12.8 years. Patients with poor prognosis had tumour of the upper ribs (P = 0.0338), the posterior component of the ribs (P = 0.0597), or regional disease (P = 0.0001). Tumour size, existence of pleural effusion, type of the surgical margin and response to chemotherapy were not significant prognostic factors. Most of the localised cases could be controlled by combined treatment, but in regional cases prognosis remained poor.

Radiotherapy in Ewing's sarcoma and PNET of the chest wall: results of the trials CESS 81, CESS 86 and EICESS 92.

PURPOSE: Treatment results and the pattern of relapse were evaluated in the multimodal treatment of Ewing's sarcomas of the chest wall. METHODS AND MATERIALS: In a retrospective analysis, 114 patients with non-metastatic Ewing's sarcoma of the chest wall were evaluated. They were treated in the CESS 81, CESS 86, or EICESS 92 studies between January 1981 and December 1993. The treatment consisted of polychemotherapy (VACA, VAIA, or EVAIA) and local therapy, either surgery alone (14 patients), radiotherapy alone (28 patients) or a combination of both (71 patients). The median follow-up was 46.6 months (range 5-170). A relapse analysis for all patients with local or combined relapses was performed. RESULTS: Overall survival was 60% after 5 years, event-free survival was 50%. Thirty-seven patients had a systemic relapse (32.4%), 11 patients had a local relapse alone (9.6%), and 3 patients had a combined local and systemic relapse (2.6%). The risk to relapse locally after 5 years was 0% after surgery alone, 19% after radiation alone, and 19% after postoperative irradiation. None of the 8 patients with preoperative irradiation have failed locally so far. With the introduction of central radiotherapy planning in CESS 86, local control of irradiated patients improved. Ten of 14 patients with local failure could be evaluated in the relapse analysis: 3 patients had an in-field relapse, 4 patients had a marginal relapse, 2 patients had a relapse outside the radiation fields, and 1 patient failed with pleural dissemination. Six treatment deviations were observed. CONCLUSION: Local control was best after surgery alone in a positively selected group of patients. Local control after radiation or combined radiation and surgery was good. With diligent performance of radiotherapy, it will be possible to further improve the results in the radiotherapy group.

Familial occurrence of osteosarcoma. A case report and review of the literature.

We treated two cases of osteosarcoma in siblings. After intensive chemotherapy and surgery, both patients are alive after 9 and 4 years without any signs of disease. We report the course of disease in these patients in detail and review the literature on 59 patients with familial osteosarcomas.

Aneurysmal bone cysts in children.

We analysed local recurrences in young children with aneurysmal bone cysts. Nine were boys and five were girls, aged 10 years or less. The sites of the lesions were distributed as follows: five femurs, four humerus, two tibia, one sacrum, one thoracic spine and one pubis. After intensive curettage, five patients underwent cementation and five patients bone grafting. Four patients underwent resection of the lesion. No patients received irradiation. One of the five patients who underwent cementation had a relapse and the lesion was controlled after the second curettage and cementation. One of the five patients who underwent bone grafting had a relapse, and he relapsed again after the second curettage and bone graft; finally his disease was controlled by resection of the lesion and bone grafting. After 55 months (median) of follow-up, all patients were disease-free. The local recurrence rate of aneurysmal bone cysts in children was not high in this hospital.

Computed direct magnification radiography of bone tumors.

The purpose of this study was to assess the diagnostic performance of computed direct magnification radiography in diagnosing bone tumors as compared with conventional radiography. Ninety-one patients with primary bone tumors and tumor-like lesions were radiographed with conventional and magnification techniques. All radiographs were analyzed by one orthopedic surgeon and two radiologists and the findings were correlated with histopathology. Two microfocal X-ray units were used for computed direct magnification radiography with a focal spot size of 20-130 microm. Using magnification versus conventional radiography, the diagnosis of benign and malignant lesions as well as the individual tumor diagnosis was obtained with higher accuracy (85% versus 71% and 69% versus 51%, respectively, P<0.01). Margins of destruction, periosteal reactions, and matrix patterns were evaluated with higher accuracy by all observers (P<0.01). We conclude that computed direct magnification radiography may improve evaluation and diagnosis of bone tumors.

Metastasis of chondrosarcoma.

This study was undertaken to analyse metastases of patients with intermediate- or high-grade chondrosarcomas. Out of 24 intermediate-grade tumours, 5 (21%) developed metastases, as did 6 of 10 high-grade tumours (60%) (P = 0.04). Four patients developed pulmonary metastasis only, 5 developed both pulmonary metastases and metastases of the other sites. Two patients showed a rare metastatic pattern: bone metastases only. The metastasis rate in the primary chondrosarcoma (42%) was higher than that in the secondary chondrosarcomas (0%) (P = 0.03). The metastasis rate was higher in patients with local recurrence (86%) than in those without local recurrence (19%) (P = 0.01). In 5 of 6 patients who had a local relapse and metastasis, the interval between the two relapses was a few months.

Metastasis of chondrosarcoma.

This study was undertaken to analyse metastases of patients with intermediate- or high-grade chondrosarcomas. Out of 24 intermediate-grade tumours, 5 (21%) developed metastases, as did 6 of 10 high-grade tumors (60%) (P = 0.04). Four patients developed pulmonary metastasis only, 5 developed both pulmonary metastasis and metastases of the other sites. Two patients showed a rare metastatic pattern: bone metastases only. The metastasis rate in the primary chondrosarcoma (42%) was higher than that in the secondary chondrosarcomas (0%) (P = 0.03). The metastasis rate was higher in patients with local recurrence (86%) than in those without local recurrence (19%) (P = 0.01). In 5 of 6 patients who had a local relapse and metastasis, the interval between the two relapses was a few months.

The impact of intraoperative brachytherapy on surgery of Ewing's sarcoma.

Surgery of Ewing's sarcoma sometimes results in an inadequate surgical margin. The influence of intraoperative brachytherapy on local control of the tumor, operation time, blood loss, and surgical complications was evaluated, comparing the results of 20 patients who received brachytherapy to a series of 42 patients receiving surgery without brachytherapy. The dose of intraoperative brachytherapy ranged between 9 Gy and 21 Gy. The average operation time was longer in 20 cases with brachytherapy (7.9 h) than in 42 cases without brachytherapy (4.3 h) (P < 0.0001). The average blood loss in the groups with (3531 ml) and without brachytherapy (3515 ml) was comparable (P = 0.3840). The surgical complication rate in patients receiving brachytherapy was also similar to that of untreated patients (30% versus 31%, P = 0.7690). Local relapse developed in 1 of 20 patients who received brachytherapy and 1 of 42 patients without brachytherapy. On the basis of this analysis, it can be concluded that this procedure is safe and does not increase of the acute complication rate. The latest results of local controls are awaited.

Surgical complications after preoperative irradiation of Ewing's sarcoma.

The influence of preoperative irradiation on surgical complications in 42 patients with Ewing's sarcoma was analysed. After preoperative irradiation and chemotherapy, 35 of 40 patients showed a good histological response and 25 of 40 patients had no viable tumour cells in the resected specimen. Local relapse alone did not develop, local relapse and metastasis developed in 2 patients and metastasis alone in 15 patients. Surgical complications appeared in 12 of 42 patients: 9 of 19 central tumours (19 pelvic lesions), 1 of 13 proximal and 2 of 10 distal tumours. Surgical complications after preoperative irradiation are distributed as follows: delayed wound healing 8, hematoma 2, thrombosis 2, skin infection 1, and abscess 1. On the other hand, complications appeared in 2 of 28 patients without preoperative irradiation: none in 9 patients having central tumours including 2 pelvic lesions, 1 in 12 patients with proximal tumours, and 1 in 7 patients with distal tumours. The multivariate regression test showed that the tumour site (central) is an influencing factor in the appearance of surgical complications. In central tumours, the surgical complication rate increases after preoperative irradiation; however, it is affected by the increase of the ratio of patients with pelvic tumours.