Modular Augmentation in Varus-Valgus-Constrained Knee Arthroplasty-Do We Need Sleeves to Avoid Femoral Loosening After Excessive Distal Augmentation?

BACKGROUND: In revision total knee arthroplasty, zonal fixation methods with a combination of augments, press-fit stems, and sleeves are popular. We hypothesized that high distal femoral augmentation with diaphyseal press-fit stems leads to an increased rate of early aseptic loosening and that femoral metaphyseal sleeves improve implant survival. Therefore, we retrospectively investigated implant survival in relation to augment heights and sleeves. METHODS: A total of 136 patients with mean clinical follow-up of 50 months (range, 28-85) who underwent modular total knee arthroplasty and revision total knee arthroplasty with semiconstrained implants between January 2012 and July 2018 were retrospectively evaluated. Implant survival with 4, 8, and 12 mm distal femoral augments was compared to no distal augmentation. Subsequently, a subgroup analysis was performed for femoral sleeve implantation. RESULTS: We observed an implant survival rate of 97.0%, 87.5%, and 69.2% for 4, 8, and 12 mm distal femoral augmentation, respectively (P = .73; P = .19; P = .008). The implant survival rate with femoral sleeves was 95.8% for the 8 mm augments and 85.7% for the 12 mm augments (P = .42; P = .96). Without femoral sleeves, the implant survival rate was 78.3% with the 8 mm augments and 50.0% with the 12 mm augments (P = .02; P < .001). CONCLUSION: Higher rates of aseptic femoral loosening were identified for distal femoral augmentation of 8 mm or more without metaphyseal sleeve fixation in semiconstrained implants. Thus, in cases with femoral metaphyseal bone damage requiring high distal femoral augmentation, metaphyseal sleeves should be used to avoid early aseptic femoral loosening.

Surgeons' expectations of osteotomies around the knee.

INTRODUCTION: High tibial osteotomy (HTO) is a valid and joint preserving surgical technique to treat medial degenerative osteoarthritis (OA) in young and active patients. A recent study shows that patients' expectations of osteotomy around the knee are high, but OA progression and potential conversion to a total knee arthroplasty (TKA) were underestimated. The aim of this study was to investigate surgeons' expectations of HTO and to compare the results to the patients' expectations and actual outcomes reported in the literature. METHODS: 461 surgeons were questioned online using the 'Hospital for Special Surgery Knee Surgery Expectations Survey (HFSS-KSES)' and a ten-item non-validated questionnaire to investigate the expectations of HTO. Two subgroups were formed to investigate differences regarding the surgeons' experience. Statistical analysis was performed using IBM SPSS Statistics. RESULTS: Surgeons' expectations of HTO were rated between very and little important with pain reduction being the most important item on the HFSS-KSES. Furthermore, 'improving the ability to walk', 'to perform daily activities', 'having confidence in the knee', and 'avoiding future degeneration' were rated of high importance. An important difference regarding the experience was the lower expectations on delay/prevention of TKA of less-experienced surgeons. CONCLUSION: Surgeons' expectations of HTO are high but nevertheless different to the patients' expectations reported in the literature. Also, expectations for the delay/prevention of TKA differed regarding the experience of surgeons. While pain reduction represents one of the most important items for surgeons and patients, the expected outcome regarding the delay/prevention of a TKA and returning to sports differs to the patients' expectations and to the actual outcome reported in the literature. This should be considered when performing the preoperative informed consent.

Inhibition of PLK1 by capped-dose volasertib exerts substantial efficacy in MDS and sAML while sparing healthy haematopoiesis.

INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.

Improved pre-operative diagnostic accuracy for low-grade prosthetic joint infections using second-generation multiplex Polymerase chain reaction on joint fluid aspirate.

BACKGROUND: A major obstacle for the treatment of prosthetic joint infection (PJI) is the identification of the underlying causative organism. While the diagnostic criteria ruling PJI in or out have become ever more accurate, the detection of the causative pathogen(s) still relies mostly on conventional and time-consuming microbial culture. The aim of this study was to evaluate the diagnostic potential of a second-generation multiplex PCR assay (Unyvero ITI G2, Curetis AG, Holzgerlingen, Germany) used on synovial fluid specimens. Our hypothesis was that the method would yield a higher diagnostic accuracy in the pre-operative workup than synovial fluid culture. Thus, a more precise classification of septic and aseptic prosthesis failure could be achieved before revision surgery. METHODS: Prospectively collected frozen joint fluid specimens from 26 patients undergoing arthroplasty revision surgery of the hip or knee were tested as per the manufacturer's protocol. Sensitivities, specificities, positive and negative predictive values as well as positive and negative likelihood ratios with corresponding confidence intervals were estimated using the statistical software R. A combination of the serum C-reactive protein (CRP) level, leukocyte count, erythrocyte sedimentation rate, joint fluid culture, tissue biopsy culture, and tissue biopsy histology served as the gold standard. RESULTS: Of the 26 patients included in the study, 15 were infected and 11 were aseptic. Conventional joint fluid culture showed a sensitivity of 0.67 and a specificity of 0.91. Joint fluid multiplex PCR yielded a sensitivity of 0.8 and a specificity of 1.0. CONCLUSIONS: Using the second-generation Unyvero ITI cartridge on joint fluid aspirate for the detection of prosthetic joint infection, we were able to achieve a higher diagnostic accuracy than with conventional culture. We conclude that to improve pathogen detection before revision surgery, this method represents a valuable and practicable tool.

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line.

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options.

Effects of thromboprophylaxis on mesenchymal stromal cells during osteogenic differentiation: an in-vitro study comparing enoxaparin with rivaroxaban.

BACKGROUND: Low-molecular-weight heparins (e.g. Enoxaparin) are widely used to prevent venous thromboembolism after orthopaedic surgery, but there are reports about serious side effects including reduction in bone density and strength. In recent years new oral antithrombotic drugs (e.g. direct Factor Xa-inhibitor, Rivaroxaban) have been used to prevent venous thromboembolism. However, there is lack of information on the effects of these new drugs on human mesenchymal stromal cells during osteogenic differentiation and, therefore, effects during postoperative bone healing. METHODS: We evaluated the effects of Rivaroxaban and Enoxaparin on the proliferation, mRNA and surface receptor expression as well as differentiation capacity of primary human mesenchymal stromal cells during their osteogenic differentiation. RESULTS: Enoxaparin, but not Rivaroxaban treatment significantly increased human mesenchymal stromal cell (hMSC) proliferation during the first week of osteogenic differentiation while suppressing osteogenic marker genes, surface receptor expression and calcification. CONCLUSIONS: This is the first paper to demonstrate that Rivaroxaban had no significant influence on hMSC differentiation towards the osteogenic lineage, indicating a less affected bone healing process compared with Enoxaparin in vitro. Based on these findings Rivaroxaban seems to be superior to Enoxaparin in early stages of bone healing in vitro.

Clinical challenge: fatal mucormycotic osteomyelitis caused by Rhizopus microsporus despite aggressive multimodal treatment.

BACKGROUND: Mucormycosis is an invasive mycotic disease caused by fungi in the zygomycetes class. Although ubiquitous in the environment, zygomycetes are rarely known to cause invasive disease in immunocompromised hosts with a high mortality even under aggressive antifungal and surgical therapy. Clinically, mucormycosis frequently affects the sinus occasionally showing pulmonary or cerebral involvement. However skeletal manifestation with Rhizopus microsporus (RM) osteomyelitis leading to emergency surgical proximal femoral resection with fatal outcome has not been described yet. CASE PRESENTATION: We report the case of a 73-year-old male suffering from myelodysplastic syndrome with precedent bone marrow transplantation. Six months after transplantation he consulted our internal medicine department in a septic condition with a four week history of painful swelling of the right hip. Radiography, computed tomography and magnetic resonance imaging revealed multiple bone infarcts in both femurs. In the right femoral head, neck and trochanteric region a recent infarct showed massive secondary osteomyelitis, breaking through the medial cortex. Emergency surgical proximal femoral resection was performed due to extensive bone and soft tissue destruction. Microbiological and basic local alignment search tool (BLAST) analysis revealed RM. Amphotericin B and posaconazole treatment with septic revision surgery was performed. However the disease ran a rapid course and was fatal two months after hospital admission. CONCLUSION: This alarming result with extensive RM osteomyelitis in the proximal femur of an immunocompromised patient may hopefully warn medical staff to perform early imaging and aggressive surgical supported multimodal treatment in similar cases.

Acute osteomyelitis of the humerus mimicking malignancy: Streptococcus pneumoniae as exceptional pathogen in an immunocompetent adult.

BACKGROUND: Chronic osteomyelitis due to direct bone trauma or vascular insufficiency is a frequent problem in orthopaedic surgery. In contrast, acute haematogenous osteomyelitis represents a rare entity that almost exclusively affects prepubescent children or immunodeficient adults. CASE PRESENTATION: In this article, we report the case of acute pneumococcal osteomyelitis of the humerus in an immunocompetent and otherwise healthy 44-year-old male patient presenting with minor inflammation signs and misleading clinical features. CONCLUSIONS: The diagnosis had to be confirmed by open biopsy which allowed the initiation of a targeted therapy. A case of pneumococcal osteomyelitis of a long bone, lacking predisposing factors or trauma, is unique in adults and has not been reported previously.

Total hip replacement in developmental dysplasia using an oval-shaped cementless press-fit cup.

PURPOSE: Acetabular roof deficiency due to subluxation of the femoral head (Hartofilakidis type II) increases the complexity of total hip arthroplasty. In these cases some form of support is usually required, to reach stable fixation of the acetabular component. Pursuing this aim, the oval-shaped cementless cranial socket could be an alternative to conventional treatment options. METHODS: Between 1998 and 2008, 37 patients (40 hips) underwent primary total hip arthroplasty using the cranial socket (mean follow-up 5.6 years, range 26 to 133 months). In a retrospective study we compared these clinical and radiological results with the results of a matched control group consisting of 35 patients (40 hips) treated with a standard cementless hemispherical cup in combination with bulk femoral autografting (mean follow-up 6.9 years, range 30 to 151 months). RESULTS: There were no statistically significant differences in the HHS (p=0.205) or the SF-36 (p=0.26) between both groups. There was no prosthesis failure due to septic or aseptic loosening. Time of surgery was significantly shorter in the cranial socket group (p<0.001). The acetabular component could be placed in the ideal rotational hip centre in 24 (60%) hips in the cranial socket group and 32 (80%) hips in the control group, respectively. CONCLUSIONS: Our study indicates, that the cranial socket can be an alternative treatment option for the reconstruction of acetabular deficiency in osteoarthritis secondary to developmental dysplasia.

Diagnostic accuracy of multiplex polymerase chain reaction on tissue biopsies in periprosthetic joint infections.

The diagnosis and treatment of periprosthetic joint infection (PJI) currently relies on cultures, which are time-consuming and often fail. Multiplex PCR assays promise reliable and prompt results, but have been heterogeneously evaluated. In this study, we analyse multiplex PCR in pathogen identification using only tissue biopsies. 42 patients after revision arthroplasty of the hip or knee were evaluated using multiplex PCR to identify microorganisms. The patients were classified according to the diagnostic criteria published by Zimmerli et al. and the results were compared to the respective microbiological cultures. PJI was detected in 15 patients and 27 revisions were aseptic. The multiplex PCR of tissue biopsies had a sensitivity of 0.3 (95% CI 0.12-0.62), a specificity of 1.0 (0.87-1.0), a positive predictive value of 1.0 (0.48-1.0) and a negative predictive value of 0.73 (0.56-0.86). The diagnostic accuracy of multiplex PCR on tissue biopsy samples is low in comparison to routine microbiological cultures. The evaluation of tissue biopsies using multiplex PCR was prone to false negative results. However, multiplex PCR assays have the advantage of rapid pathogen identification. We therefore recommend further investigation of multiplex PCR in the setting of suspected PJI with a careful choice of specimens.

Gorlin syndrome associated with small bowel carcinoma and mesenchymal proliferation of the gastrointestinal tract: case report and review of literature.

UNLABELLED: BACKGROUND AND CASE PRESENTATION: A patient with nevoid basal cell carcinoma syndrome (Gorlin syndrome) presented with two unusual clinical features, i.e. adenocarcinoma of the small bowel and extensive mesenchymal proliferation of the lower gastrointestinal tract. CONCLUSIONS: We discuss the possibility that these two features are pathogenetically linked to the formerly undescribed patient's PTCH germ line mutation.

Morphological adaptation of the tarso-metatarsal joints onto load transmission in the foot.

PURPOSE: Undefined middle foot pain among older individuals is an increasingly problem. Thereby the tarso-metatarsal joints (Lisfranc's joint, TMT joints) are particularly important for foot structure. The aim of this study was to obtain into force transmission in TMT joints by analysing degenerative morphological changes (DMC) of the articular cartilage and subchondral density patterns. MATERIALS AND METHODS: We used seven formalin-fixed foot preparations (average age 80.8) from the undergraduate dissection course. Applying the Collins schema, localisation of cartilaginous surface DMC was examined. For showing subchondral density patterns, the well-established method of CT-osteoabsorptiometry (CT-OAM) was used. RESULTS: The cartilaginous surface DMCs were frequently located at the medial edges. Maximum density areas were found at the medial and dorsal edges. There was a clear correlation of topical cartilage degeneration and subchondral density. CONCLUSION: In healthy feet, force transmission in the TMT joints appears to occur via the dorsal and medial edges. Considering the correlation of topical cartilaginous surface DMC and subchondral density patterns, clinical application of CT-OAM seems to be recommendable in the case of undefined middle foot pain.

Sclerosing epithelioid fibrosarcoma of the bone: a case report of high resistance to chemotherapy and a survey of the literature.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare soft tissue sarcoma mostly occurring in extraosseous sites. SEF represents a clinically challenging entity especially because no standardized treatment regimens are available. Intraosseous localization is an additional challenge with respect to the therapeutical approach. We report on a 16-year-old patient with SEF of the right proximal tibia. The patient underwent standardized neoadjuvant chemotherapy analogous to the EURAMOS-1 protocol for the treatment of osteosarcoma followed by tumor resection and endoprosthetic reconstruction. Histopathological analysis of the resected tumor showed >90% vital tumor cells suggesting no response to chemotherapy. Therefore, therapy was reassigned to the CWS 2002 High-Risk protocol for the treatment of soft tissue sarcoma. To date (22 months after diagnosis), there is no evidence of relapse or metastasis. Our data suggest that SEF may be resistant to a chemotherapy regimen containing Cisplatin, Doxorubicin, and Methotrexate, which should be considered in planning treatment for patients with SEF.

Diagnostic accuracy of MRI with metal artifact reduction for the detection of periprosthetic joint infection and aseptic loosening of total hip arthroplasty.

PURPOSE: To evaluate imaging characteristics obtained from magnetic resonance imaging (MRI) with metal artifact reduction (MAR) to differentiate between periprosthetic joint infection (PJI), aseptic loosening and cases without these pathologies after total hip arthroplasty (THA). METHODS: Patients with THA (n = 41; mean age 66.4 ±â€¯9.6 years; 25 women) undergoing 1.5 T MRI with high-bandwidth sequences and view angle tilting followed by revision surgery within 3 months were identified retrospectively. Imaging findings at the metal-bone interface, in the surrounding bone, the soft tissues and lymphadenopathy were assessed by three radiologists in a standardized fashion. Based on clinical and intraoperative findings, patients were categorized in groups with PJI (n = 15), aseptic loosening (n = 15) or without these pathologies (n = 11). Imaging findings were assessed in crosstabs, receiver-operating characteristics and classification and regression trees. RESULTS: Findings at the acetabular cup were specific for the presence of either PJI or aseptic loosening (specificity>0.765 for all), while findings at the stem were sensitive (sensitivity>0.824 for all except periostitis). To differentiate PJI versus aseptic loosening, soft tissue edema (sensitivity, 0.867/specificity>0.733), abnormalities at both, acetabular and femoral components (0.667/0.933-1.000) and enlarged lymph nodes (0.800/0.867) were accurate. CONCLUSION: Standardized assessment of MR imaging findings in THA patients facilitated the differentiation of PJI and aseptic loosening. This information can be helpful for therapy planning.

Antimicrobial peptides in human synovial membrane as (low-grade) periprosthetic joint infection biomarkers.

BACKGROUND: Safe diagnosis of periprosthetic joint infection (PJI) is of utmost importance for successful exchange arthroplasty. However, current diagnostic tools show insufficient accuracy in the clinically common and challenging chronic low-grade infections. To close this diagnostic gap, reliable (bio)markers display the most promising candidates. Antimicrobial peptides (AMPs) are part of the innate immune response towards microbial growth. Recently we could show significant intraarticular levels of human cathelicidin LL-37 and ß-defensin-3 (HBD-3) with high diagnostic accuracy in PJI synovial fluid. Consequently, these promising biomarkers were evaluated in PJI synovial membrane and synoviocytes, which may significantly facilitate histological diagnosis of PJI to improve outcome of septic joint replacement. METHODS: In this prospective single-center controlled clinical study (diagnostic level II), consecutive patients with total hip (THR) and knee (TKR) replacements were included undergoing primary arthroplasty (n = 8), surgical revision due to aseptic loosening (n = 9) and septic arthroplasty with coagulase-negative staphylococci (n = 8) according to the criteria of the Musculoskeletal Infection Society (MSIS). Semiquantitative immunohistochemical (IHC) analysis of LL-37, HBD-3 and HBD-2 in synovial membrane and isolated synoviocytes based on Total Allred Score (TS) and Immunoreactive Remmele and Stegner score (IRS) was performed. For statistical analysis, SPSS 26.0/R3.6.3 (p < 0.05) was used. RESULTS: The AMPs LL-37 and HBD-3 were significantly elevated (up to 20×) in synovial membranes from PJI compared to aseptic loosening or primary arthroplasty. The area under the curve (AUC) in a receiver operating characteristic curve analysis was equal to 1.0 for both scores revealing excellent diagnostic accuracy. Isolated synoviocytes as cellular AMP source showed comparable results with a significant LL-37/HBD-3-increase up to 3 × in PJI. In contrast, local HBD-2 levels were negligible (p > 0.23) upon PJI with a lower diagnostic accuracy (AUC = 0.65) in analogy to our previous findings with synovial fluid. CONCLUSIONS: Our results implicate AMPs as promising and specific biomarkers for the histological diagnosis of PJI.

A humanized bone microenvironment uncovers HIF2 alpha as a latent marker for osteosarcoma.

The quest for predictive tumor markers for osteosarcoma (OS) has not well progressed over the last two decades due to a lack of preclinical models. The aim of this study was to investigate if microenvironmental modifications in an original humanized in vivo model alter the expression of OS tumor markers. Human bone micro-chips and bone marrow, harvested during hip arthroplasty, were implanted at the flanks of NOD/scid mice. We administered recombinant human bone morphogenetic protein 7 (rhBMP-7) in human bone micro-chips/bone marrow group I in order to modulate bone matrix and bone marrow humanization. Ten weeks post-implantation, human Luc-SAOS-2 OS cells were injected into the humanized tissue-engineered bone organs (hTEBOs). Tumors were harvested 5 weeks post-implantation to determine the expression of the previously described OS markers ezrin, periostin, VEGF, HIF1α and HIF2α. Representation of these proteins was analyzed in two different OS patient cohorts. Ezrin was downregulated in OS in hTEBOs with rhBMP-7, whereas HIF2α was significantly upregulated in comparison to hTEBOs without rhBMP-7. The expression of periostin, VEGF and HIF1α did not differ significantly between both groups. HIF2α was consistently present in OS patients and dependent on tumor site and clinical stage. OS patients post-chemotherapy had suppressed levels of HIF2α. In conclusion, we demonstrated the overall expression of OS-related factors in a preclinical model, which is based on a humanized bone organ. Our preclinical research results and analysis of two comprehensive patient cohorts imply that HIF2α is a potential prognostic marker and/or therapeutic target. STATEMENT OF SIGNIFICANCE: This study demonstrates the clinical relevance of the humanized organ bone microenvironment in osteosarcoma research and validates the expression of tumor markers, especially HIF2α. The convergence of clinically proven bone engineering concepts for the development of humanized mice models is a new starting point for investigations of OS-related marker expression. The validation and first data set in such a model let one conclude that further clinical studies on the role of HIF2α as a prognostic marker and its potential as therapeutic target is a condition sine qua non.

Venetoclax with azacitidine targets refractory MDS but spares healthy hematopoiesis at tailored dose.

Patients with Myelodysplastic Syndromes (MDS) and secondary Acute Myeloid Leukemia (sAML) have a very poor prognosis after failure of hypomethylating agents (HMA). Stem cell transplantation is the only effective salvage therapy, for which only a limited number of patients are eligible due to age and comorbidity. Combination therapy of venetoclax and azacitidine (5-AZA) seems to be a promising approach in myeloid malignancies, but data from patients with HMA failure are lacking. Furthermore, a considerable concern of combination regimens in elderly AML and MDS patients is the toxicity on the remaining healthy hematopoiesis. Here, we report in vitro data showing the impact of venetoclax and 5-AZA, alone or in combination, in a larger cohort of MDS/sAML patients (n = 21), even after HMA failure (n = 13). We especially focused on the effects on healthy hematopoiesis and the impact on colony forming capacity as a parameter for long-term effects. To the best of our knowledge, we show for the first time that venetoclax in combination with capped dose of 5-AZA targets cell malignancies, while sparing healthy hematopoiesis.

Whole bone testing in small animals: systematic characterization of the mechanical properties of different rodent bones available for rat fracture models.

OBJECTIVES: Rat fracture models are extensively used to characterize normal and pathological bone healing. Despite, systematic research on inter- and intra-individual differences of common rat bones examined is surprisingly not available. Thus, we studied the biomechanical behaviour and radiological characteristics of the humerus, the tibia and the femur of the male Wistar rat-all of which are potentially available in the experimental situation-to identify useful or detrimental biomechanical properties of each bone and to facilitate sample size calculations. METHODS: 40 paired femura, tibiae and humeri of male Wistar rats (10-38 weeks, weight between 240 and 720 g) were analysed by DXA, pQCT scan and three-point-bending. Bearing and loading bars of the biomechanical setup were adapted percentually to the bone's length. Subgroups of light (skeletal immature) rats under 400 g (N = 11, 22 specimens of each bone) and heavy (mature) rats over 400 g (N = 9, 18 specimens of each bone) were formed and evaluated separately. RESULTS: Radiologically, neither significant differences between left and right bones, nor a specific side preference was evident. Mean side differences of the BMC were relatively small (1-3% measured by DXA and 2.5-5% by pQCT). Over all, bone mineral content (BMC) assessed by DXA and pQCT (TOT CNT, CORT CNT) showed high correlations between each other (BMC vs. TOT and CORT CNT: R2 = 0.94-0.99). The load-displacement diagram showed a typical, reproducible curve for each type of bone. Tibiae were the longest bones (mean 41.8 ± 4.12 mm) followed by femurs (mean 38.9 ± 4.12 mm) and humeri (mean 29.88 ± 3.33 mm). Failure loads and stiffness ranged from 175.4 ± 45.23 N / 315.6 ± 63.00 N/mm for the femurs, 124.6 ± 41.13 N / 260.5 ± 59.97 N/mm for the humeri to 117.1 ± 33.94 N / 143.8 ± 36.99 N/mm for the tibiae. Smallest interindividual differences were observed in failure loads of the femurs (CV% 8.6) and tibiae (CV% 10.7) of heavy animals, light animals showed good consistency in failure loads of the humeri (CV% 7.7). Most consistent results of both sides (left vs. right) in failure loads were provided by the femurs of light animals (mean difference 4.0 ± 2.8%); concerning stiffness, humeri of heavy animals were most consistent (mean difference of 6.2 ± 5%). In general, the failure loads showed strong correlations to the BMC (R2 = 0.85-0.88) whereas stiffness correlated only moderate, except for the humerus (BMC vs. stiffness: R2 = 0.79). DISCUSSION: Altogether, the rat's femur of mature specimens showed the most accurate and consistent radiological and biomechanical results. In synopsis with the common experimental use enabling comparison among different studies, this bone offers ideal biomechanical conditions for three point bending experiments. This can be explained by the combination of a superior aspect ratio and a round and long, straight morphology, which satisfies the beam criteria more than other bones tested.

Humanization of bone and bone marrow in an orthotopic site reveals new potential therapeutic targets in osteosarcoma.

BACKGROUND: Existing preclinical murine models often fail to predict effects of anti-cancer drugs. In order to minimize interspecies-differences between murine hosts and human bone tumors of in vivo xenograft platforms, we tissue-engineered a novel orthotopic humanized bone model. METHODS: Orthotopic humanized tissue engineered bone constructs (ohTEBC) were fabricated by 3D printing of medical-grade polycaprolactone scaffolds, which were seeded with human osteoblasts and embedded within polyethylene glycol-based hydrogels containing human umbilical vein endothelial cells (HUVECs). Constructs were then implanted at the femur of NOD-scid and NSG mice. NSG mice were then bone marrow transplanted with human CD34 + cells. Human osteosarcoma (OS) growth was induced within the ohTEBCs by direct injection of Luc-SAOS-2 cells. Tissues were harvested for bone matrix and marrow morphology analysis as well as tumor biology investigations. Tumor marker expression was analyzed in the humanized OS and correlated with the expression in 68 OS patients utilizing tissue micro arrays (TMA). RESULTS: After harvesting the femurs micro computed tomography and immunohistochemical staining showed an organ, which had all features of human bone. Around the original mouse femur new bone trabeculae have formed surrounded by a bone cortex. Staining for human specific (hs) collagen type-I (hs Col-I) showed human extracellular bone matrix production. The presence of nuclei staining positive for human nuclear mitotic apparatus protein 1 (hs NuMa) proved the osteocytes residing within the bone matrix were of human origin. Flow cytometry verified the presence of human hematopoietic cells. After injection of Luc-SAOS-2 cells a primary tumor and lung metastasis developed. After euthanization histological analysis showed pathognomic features of osteoblastic OS. Furthermore, the tumor utilized the previously implanted HUVECS for angiogenesis. Tumor marker expression was similar to human patients. Moreover, the recently discovered musculoskeletal gene C12orf29 was expressed in the most common subtypes of OS patient samples. CONCLUSION: OhTEBCs represent a suitable orthotopic microenvironment for humanized OS growth and offers a new translational direction, as the femur is the most common location of OS. The newly developed and validated preclinical model allows controlled and predictive marker studies of primary bone tumors and other bone malignancies.