Ramses Forsyth

Morphological and immunophenotypic features of primary and metastatic giant cell tumour of bone

Giant cell tumour of bone (GCTB) is a primary tumour of bone that may rarely, in the absence of malignant cytological features, produce metastatic lesions, most commonly in the lungs. Whether these lung nodules represent true neoplastic secondaries or implants derived from the primary tumour is not certain. In this study, we have analysed the morphological and immunophenotypic features of 19 conventional GCTBs and corresponding lung nodules for expression of macrophage, osteoclast, proliferation and tumour-associated markers. A striking morphological feature of all GCTBs that produced lung secondaries was the presence of large areas of haemorrhage and thrombus formation; mononuclear and multinucleated cells of GCTB were frequently found within these areas of haemorrhage and thrombus. A similar pattern of CD14, CD33, HLA-DR and CD51 expression was seen in macrophages and giant cells in primary and secondary tumours. Smooth muscle actin expression was frequently noted in primary GCTBs that recurred and metastasised. No difference was seen in the expression of p53, p63, Ki-67, cyclin D1 or Bcl-2 in primary and secondary tumours. Our findings suggest that most lung nodules associated with primary conventional GCTBs are implants derived from tumour emboli formed in areas of haemorrhage and thrombus formation within the primary tumour.

Histologic Assessment of Acetabular Labrum Healing

PURPOSE The purpose of this study was to histologically examine the human healing response of arthroscopically repaired acetabular labrum tears. METHODS Biopsy specimens were retrieved from 6 patients during total hip arthroplasty after clinical failure of the index arthroscopic procedure. All patients were diagnosed as having femoroacetabular impingement with a concomitant labral tear. In all cases severe chondral damage was observed during arthroscopy (Beck grades 3 to 4). Despite successful technical repair of the labral tear, chondral damage in these patients was so advanced that the clinical progress after the procedure was unsatisfactory and arthroplasty of the joint was required. Biopsy specimens of the repaired acetabular labra were harvested during the arthroplasty surgery and processed for standard histologic evaluation. RESULTS Macroscopically and histologically, all repaired labra kept their triangular shape more or less and appeared to have healed. All harvested biopsy specimens displayed a typical fibrocartilaginous appearance with limited vascular supply. Calcifications were present in only 1 biopsy specimen. In 3 cases neovascularization of the labral tissue was noticed in the proximity of the sutures. In the superficial and deep parts of the labral body, small clefts were observed in all cases. CONCLUSIONS In this study the histologic aspects of arthroscopically repaired human labral tears were addressed. It was shown that human labral tears show healing potential after surgical repair. The surfaces of the labral tissues were intact, and neither remnants of the tear nor the presence of fibrovascular scar tissue was observed. However, some small clefts in the superior and deep parts of the repaired structures were noticed in all cases. LEVEL OF EVIDENCE Level IV, therapeutic case series.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions. This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

Tumor grafts derived from sarcoma patients retain tumor morphology, viability, and invasion potential and indicate disease outcomes in the chick chorioallantoic membrane model

The chick chorioallantoic membrane (CAM) assay was used to evaluate whether xenotransplanted sarcomas retain the histological characteristics and functional behavior of the original tumors. Metabolically active tumor tissue, identified by dynamic-contrast MRI, from 28 patients with a bone or soft-tissue tumors was applied to the CAM. Angiogenesis and graft and host behaviors were evaluated. The essential features and immunohistochemical characteristics of the original tumors were maintained, illustrating the diversity of sarcomas. Graft viability was inversely related to patient survival, but longer follow-up and more patients are needed to relate tumor graft behavior to natural history. We conclude that the CAM assay is a potential prognostic and predictive preclinical xenograft model for tumors that are difficult to culture in vitro, such as sarcomas; therefore, the use of the CAM assay may facilitate personalized medicine.

Chondrogenic Priming at Reduced Cell Density Enhances Cartilage Adhesion of Equine Allogeneic MSCs - a Loading Sensitive Phenomenon in an Organ Culture Study with 180 Explants

Background: Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain unanswered concerning mesenchymal stem cell (MSC) adhesion and incorporation into cartilage. Methods: To this end, peripheral blood (PB) MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs) for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x106 vs 1.0 x106). In total, 180 explants of six horses (30 per horse) were divided into five groups: no lesion (i), lesion alone (ii), lesion with naïve MSCs (iii), lesion with chondrogenically-induced MSCs (iv) and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v). Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14). Results: COMP expression was selectively increased by chondrogenic induction (p = 0.0488). PEMF stimulation (1mT for 10 minutes) further augmented COL II expression over induced values (p = 0.0405). On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9%) and lesion filling (3.7%) in all the different conditions (p < 0.0001). Remarkably, homogenous cell distribution was characteristic for chondrogenic induced MSCs (+/- PEMFs), whereas clump formation occurred in 39% of uninduced MSC treated cartilage explants. Finally, unloaded explants seeded with a moderately low density of MSCs exhibited greater lesion filling (p = 0.0022) and surface adherence (p = 0.0161) than explants seeded with higher densities of MSCs. In all cases, the overall amount of lesion filling decreased from day 5 to 14 (p = 0.0156). Conclusion: The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage.

Gene expression profiling of giant cell tumor of bone reveals downregulation of extracellular matrix components decorin and lumican associated with lung metastasis.

Giant cell tumor of bone (GCTB) displays worrisome clinical features such as local recurrence and occasionally metastatic disease which are unpredictable by morphology. Additional routinely usable biomarkers do not exist. Gene expression profiles of six clinically defined groups of GCTB and one group of aneurysmal bone cyst (ABC) were determined by microarray (n = 33). The most promising differentially expressed genes were validated by Q-PCR as potential biomarkers in a larger patient group (n = 41). Corresponding protein expression was confirmed by immunohistochemistry. Unsupervised hierarchical clustering reveals a metastatic GCTB cluster, a heterogeneous, non-metastatic GCTB cluster, and a primary ABC cluster. Balanced score testing indicates that lumican (LUM) and decorin (DCN) are the most promising biomarkers as they have lower level of expression in the metastatic group. Expression of dermatopontin (DPT) was significantly lower in recurrent tumors. Validation of the results was performed by paired and unpaired t test in primary GCTB and corresponding metastases, which proved that the differential expression of LUM and DCN is tumor specific rather than location specific. Our findings show that several genes related to extracellular matrix integrity (LUM, DCN, and DPT) are differentially expressed and may serve as biomarkers for metastatic and recurrent GCTB.

The histological appearance of the proximal aspect of the dorsal condylar sagittal ridge of the third metacarpal and metatarsal bone in young Warmblood horses: normal appearance and correlation with detected radiographic variations

The objective of this study is to describe the normal histological appearance of the dorsoproximal aspect of the sagittal ridge of the third metacarpal/metatarsal bone in young Warmblood horses, and to compare it to the different radiographic variations (irregular, indentation, lucency, notch) described at this level. A total of 25 metacarpo/metatarsophalangeal joints of 12 Warmblood horses were used. Five samples of each radiographically described group were selected for histological processing. Each category was compared with the normal control group. Each group showed a bone cortex, covered by hyaline cartilage and longitudinally aligned collagen fibres covered by loosely organized connective tissue proximally. The normal and irregular group showed a smooth bone cortex. In the indentation and lucency group, a depression in the cortex was detected. The notch group presented an expansion of the cortex. The collagen fibres and connective tissue were located in the depression in the indentation group whereas the location varied in the lucency and notch group. The radiologic detected differences are translated into detectable histological differences. Further research is warranted to determine whether these variations are developmental or congenital and to evaluate their potential influence on the joint function during hyperextension.