Efficacy and safety of human mesenchymal stromal cells in healing of critical-size bone defects in immunodeficient rats.

To evaluate the preclinical efficacy and safety of human mesenchymal stem cells (hMSC) rapidly expanded in growth medium for clinical use with human serum and recombinant growth factors, we conducted a controlled, randomized trial of plasma clots with hMSC vs. plasma clots only in critical segmental femoral defects in rnu/rnu immunodeficient rats. X-ray, microCT and histomorphometrical evaluation were performed at 8 and 16 weeks. MSC were obtained from healthy volunteers and patients with lymphoid malignancy. Human MSC survived in the defect for the entire duration of the trial. MSC from healthy volunteers, in contrast to hMSC from cancer patients, significantly improved bone healing at 8, but not 16 weeks. However, at 16 weeks, hMSC significantly improved vasculogenesis in residual defect. We conclude that hMSC from healthy donors significantly contributed to the healing of bone defects at 8 weeks and to the vascularisation of residual connective tissue for up to 16 weeks. We found the administration of hMSC to be safe, as no adverse reaction to human cells at the site of implantation and no evidence of migration of hMSC to distant organs was detected.

In vitro characterization of self-assembled anterior cruciate ligament cell spheroids for ligament tissue engineering.

Tissue engineering of an anterior cruciate ligament (ACL) implant with functional enthesis requires site-directed seeding of different cell types on the same scaffold. Therefore, we studied the suitability of self-assembled three-dimensional spheroids generated by lapine ACL ligament fibroblasts for directed scaffold colonization. The spheroids were characterized in vitro during 14 days in static and 7 days in dynamic culture. Size maintenance of self-assembled spheroids, the vitality, the morphology and the expression pattern of the cells were monitored. Additionally, we analyzed the total sulfated glycosaminoglycan, collagen contents and the expression of the ligament components type I collagen, decorin and tenascin C on protein and for COL1A1, DCN and TNMD on gene level in the spheroids. Subsequently, the cell colonization of polylactide-co-caprolactone [P(LA-CL)] and polydioxanone (PDS) polymer scaffolds was assessed in response to a directed, spheroid-based seeding technique. ACL cells were able to self-assemble spheroids and survive over 14 days. The spheroids decreased in size but not in cellularity depending on the culture time and maintained or even increased their differentiation state. The area of P[LA-CL] scaffolds, colonized after 14 days by the cells of one spheroid, was in average 4.57 ± 2.3 mm(2). Scaffolds consisting of the polymer P[LA-CL] were more suitable for colonization by spheroids than PDS embroideries. We conclude that ACL cell spheroids are suitable as site-directed seeding strategy for scaffolds in ACL tissue engineering approaches and recommend the use of freshly assembled spheroids for scaffold colonization, due to their balanced proliferation and differentiation.

Incidental prostate cancer prevalence at radical cystoprostatectomy--importance of the histopathological work-up.

The reported incidental prostate cancer prevalence rates at radical cystoprostatectomy cover a range from 4 to 60 %. We investigated the influence of the histopathological work-up on prostate cancer prevalence rates. We identified 114 patients who had undergone cystoprostatectomy for bladder cancer between 2000 and 2012. Complete histopathological assessment was defined as follows: (i) complete embedding of the prostate gland, (ii) sectioning of 15 or more prostate sections, and (iii) processing as whole mount slides. Prostate cancer prevalence rates derived from complete and incomplete histopathological assessments were compared. The overall prostate cancer prevalence rate was 59.6 %. A mean of 14.4 macroscopic tissue sections (thickness 3-5 mm) were sectioned. Sectioning ≥15 sections resulted in a prostate cancer detection rate of 75 %, compared to 42.6 % when sectioning <15 sections (p < 0.001). Complete embedding yielded a prostate cancer detection rate of 72.3 and of 23.1 % for partly embedded prostates (p < 0.0001). Prostate cancer was detected in 68.8 % of the whole mounted samples and in 38.2 % of the samples sectioned as standard slides (p < 0.01); according to the criteria described by Epstein and Ohori, 44.1 % of the detected prostate cancers were clinically significant. The quality of the histopathological work-up significantly influences prostate cancer detection rates and might at least partially explain the highly variable reported incidental prostate cancer prevalence rates at cystoprostatectomy (CP). The high proportion of significant prostate cancer found in our series calls for a careful surgical approach to the prostate during CP.

Embroidered polymer-collagen hybrid scaffold variants for ligament tissue engineering.

Embroidery techniques and patterns used for scaffold production allow the adaption of biomechanical scaffold properties. The integration of collagen into embroidered polylactide-co-caprolactone [P(LA-CL)] and polydioxanone (PDS) scaffolds could stimulate neo-tissue formation by anterior cruciate ligament (ACL) cells. Therefore, the aim of this study was to test embroidered P(LA-CL) and PDS scaffolds as hybrid scaffolds in combination with collagen hydrogel, sponge or foam for ligament tissue engineering. ACL cells were cultured on embroidered P(LA-CL) and PDS scaffolds without or with collagen supplementation. Cell adherence, vitality, morphology and ECM synthesis were analyzed. Irrespective of thread size, ACL cells seeded on P(LA-CL) scaffolds without collagen adhered and spread over the threads, whereas the cells formed clusters on PDS and larger areas remained cell-free. Using the collagen hydrogel, the scaffold colonization was limited by the gel instability. The collagen sponge layers integrated into the scaffolds were hardly penetrated by the cells. Collagen foams increased scaffold colonization in P(LA-CL) but did not facilitate direct cell-thread contacts in the PDS scaffolds. The results suggest embroidered P(LA-CL) scaffolds as a more promising basis for tissue engineering an ACL substitute than PDS due to superior cell attachment. Supplementation with a collagen foam presents a promising functionalization strategy.

Comparison of colorectal cancer screening and diagnoses in HIV-positive and HIV-negative veterans.

Non-AIDS defining malignancies, including colorectal cancer (CRC), are emerging as significant problems in HIV-infected people. Some data suggest that HIV-positive patients have higher incidence of CRC at younger ages than those who are HIV-negative. This investigation examined CRC rates and screening types and trends between 943 HIV-infected cases and their age, race, and gender matched HIV-negative controls (n = 943) from 1 January 2005 to 31 December 2008 at the Atlanta VA Medical Center. The most common screening type among these patients was fecal occult blood testing (FOBT), but colonoscopies were more common in the controls (16.4% for cases, 27.5% for controls; p < 0.0001). Almost half of all patients included in this analysis did not have any screening for CRC during the four years of follow-up even though average age was 55 years. Fifty-one percent of cases had at least one screening test during follow-up compared to 48% of the controls; 7.6% of the cases had a screening each of the four years compared to only 2.4% of the controls (p < 0.0001). Ten HIV-positive patients were diagnosed with CRC during the study period compared to no CRC diagnoses among controls (p = 0.0015), though there was no difference in the diagnosis of colon polyps (4.6% vs. 5.1%, p = 0.5911). These data also suggest a discrepancy in CRC incidence between race and age groups: 80% of HIV-positive cases diagnosed with CRC during the study were black and two were less than 50 years of age. Future studies will need to address whether different recommendations are needed for screening based on HIV status, younger age, or race.

ECM inspired coating of embroidered 3D scaffolds enhances calvaria bone regeneration.

Resorbable polymeric implants and surface coatings are an emerging technology to treat bone defects and increase bone formation. This approach is of special interest in anatomical regions like the calvaria since adults lose the capacity to heal large calvarial defects. The present study assesses the potential of extracellular matrix inspired, embroidered polycaprolactone-co-lactide (PCL) scaffolds for the treatment of 13 mm full thickness calvarial bone defects in rabbits. Moreover the influence of a collagen/chondroitin sulfate (coll I/cs) coating of PCL scaffolds was evaluated. Defect areas filled with autologous bone and empty defects served as reference. The healing process was monitored over 6 months by combining a novel ultrasonographic method, radiographic imaging, biomechanical testing, and histology. The PCL coll I/cs treated group reached 68% new bone volume compared to the autologous group (100%) and the biomechanical stability of the defect area was similar to that of the gold standard. Histological investigations revealed a significantly more homogenous bone distribution over the whole defect area in the PCL coll I/cs group compared to the noncoated group. The bioactive, coll I/cs coated, highly porous, 3-dimensional PCL scaffold acted as a guide rail for new skull bone formation along and into the implant.

[Bone substitute. Transplants and replacement materials--an update]. / Knochenersatz. Transplantate und Ersatzmaterialien--ein Update.

Due to the special characteristics, autologous bone for bone grafting remains the gold standard for defect filling. Besides allogenic bone transplants, as an alternative a set of bone substitutes has been established. An overview of the bone substitutes presently on the market is almost lost due to the abundance of products. The present paper gives a review of the materials available on the market. Different classification systems regarding origin, vitality, biological priority and chemical composition are described as well as the individual materials including the advantages and disadvantages. Finally, a description of tissue engineering and gene therapy gives a view of future prospective.

Ovine bone marrow mesenchymal stem cells: isolation and characterization of the cells and their osteogenic differentiation potential on embroidered and surface-modified polycaprolactone-co-lactide scaffolds.

The current study was undertaken with the goal being isolation, cultivation, and characterization of ovine mesenchymal stem cells (oMSC). Furthermore, the objective was to determine whether biological active polycaprolactone-co-lactide (trade name PCL) scaffolds support the growth and differentiation of oMSC in vitro. The oMSC were isolated from the iliac crest of six merino sheep. Three factors were used to demonstrate the MSC properties of the isolated cells in detail. (1) Their ability to proliferate in culture with a spindle-shaped morphology, (2) presence of specific surface marker proteins, and (3) their capacity to differentiate into the three classical mesenchymal pathways, osteoblastic, adipogenic, and chondrogenic lineages. Furthermore, embroidered PCL scaffolds were coated with collagen I (coll I) and chondroitin sulfate (CS). The porous structure of the scaffolds and the coating with coll I/CS allowed the oMSC to adhere, proliferate, and to migrate into the scaffolds. The coll I/CS coating on the PCL scaffolds induced osteogenic differentiation of hMSC, without differentiation supplements, indicating that the scaffold also has an osteoinductive character. In conclusion, the isolated cells from the ovine bone marrow have similar morphologic, immunophenotypic, and functional characteristics as their human counterparts. These cells were also found to differentiate into multiple mesenchymal cell types. This study demonstrates that embroidered PCL scaffolds can act as a temporary matrix for cell migration, proliferation, and differentiation of oMSC. The data presented will provide a reliable model system to assess the translation of MSC-based therapy into a variety of valuable ovine experimental models under autologous settings.

Loss of inhibition over master pathways of bone mass regulation results in osteosclerotic bone metastases in prostate cancer.

Prostate cancer is the most common cancer among men in industrialised countries. Most patients with prostate cancer, however, will not die of it. As a result, many of them will experience symptomatic metastasis during the course of the disease. Prostate cancer has a high propensity to metastasize to bone. Unlike many other cancers prostate cancer cells induce a rather osteosclerotic than osteolytic reaction in the bone marrow by interfering with physiological bone remodelling. A proper understanding of the mechanisms of tumour cell-induced bone alterations and exaggerated bone deposition in prostate cancer may open new and urgently needed therapeutic approaches in the field of palliative care for affected patients. In this review we focus on the central role of two major regulators of bone mass, the wingless type integration site family members (WNTs) and the bone morphogenetic proteins (BMPs), in the development of osteosclerotic bone metastases.

[Internal fixation of meta- and diaphyseal intercalary bone defects after tumour resection with intramedullary nailing and porous polymethylmetacrylate (PMMA) spacer]. / Stabilisierung von meta- und diaphysären Segmentdefekten nach Tumorresektion durch Marknagelung und Polymethylmetacrylat- (PMMA-)Formkörper.

BACKGROUND: Radical resection of bone tumours localized in the meta- or diaphysis of long bones frequently results in segmental defects. Several stabilization techniques with preservation of the adjacent joint have been published, but the failure rate appears to be considerable. The objective of this study is the experimental and clinical testing of a new technique which combines unreamed nailing with support of the defect by a porous polymethylmetacrylate (PMMA) spacer. METHODS: For spacer preparation, PMMA spheres were adhered to a cylindrical spacer (Ø 32 mm) with interconnective porosity. Axial strength was determined, as was the stiffness/strengths of the combination osteosynthesis in human cadaver tibias (defect lengths 6 cm; empty defect served as a control). An experiment was also conducted with sheep. A 3 cm diaphyseal tibia defect was prepared and stabilized by nailing. For the control there was an empty defect. Group A had support of the defect with a PMMA spacer and group B a PMMA spacer coated with osteoconductive RGD-peptide. Evaluation after was made after 6 months including histology and a determination of relative torsional strength. In addition, a clinical study has been under way since October 1998, with 13 patients (defect lengths between 3-15 cm) being operated. RESULTS: Axial stability was 12,750+/-300 N (17.56+/-0.59 MPa). There was an enhancement of 4-point bending stiffness by 35% (P=0.028), of axial stiffness by 36% (ns) and of axial strength by 553% (P=0.028). Histology showed the formation of a new bone at the spacer/muscle interface. For the sheep, relative torsional strength was enhanced by 95% (P=0.08) in group A and by 91% (P=0.047) in group B. For the patients studied, the mean follow-up period was 16.1 months, max. 48 months. One mechanical failure occurred after 24 months, which was solved by callus distraction. CONCLUSIONS: Combination osteosynthesis is suitable for stabilizing segmental bone defects. The risk of mechanical failure appears to be low. Nevertheless, this technique should only be applied as a definitive solution if callus distraction is unfavourable due to advanced age or a poor lifetime prognosis. The method can also be used for temporary internal stabilization during prolonged postoperative chemotherapy.

Are autopsies of help to the parents of SIDS victims? A follow-up on SIDS families.

Little is known about what bereaved parents feel about the autopsy performed on their child. A multi-centre case control study of sudden infant death syndrome (SIDS) victims was carried out in Germany between 1998 and 2001, in which all infants had been autopsied. We performed a follow-up study 4-7 years after the parents had lost their child. A total of 141 parents filled in the questionnaire, which were sent to them by the study centre. Of these, 71% had had another child after the SIDS/sudden unexpected death in infancy. The majority (83%) of the participating parents found the autopsy helped them to cope better with the death. A large proportion (46%) did not want any professional help after the death, and 55% did not wish to have any contact with a self-help group. We conclude that the autopsy is helpful to the majority of bereaved parents. Professional help and self-help groups should be offered to the parents even if the majority in our study did not want to use either.

Urinary Interleukin-8 and 18 predict the response of superficial bladder cancer to intravesical therapy with bacillus Calmette-Guerin.

PURPOSE: We evaluate the predictive value of urinary cytokine levels of interleukin (IL) 8 and 18 for response in patients receiving intravesical bacillus Calmette-Guerin (BCG) for prevention of recurrences of superficial bladder cancer and treatment of carcinoma in situ. MATERIALS AND METHODS: In 28 patients with superficial bladder cancer treated with BCG IL-8 expression in the urine during the first 6 hours after the first BCG instillation was determined. In 17 patients IL-18 levels were also evaluated during the first 12 hours after BCG instillation. IL-8 and 18 levels were determined by solid phase double ligand enzyme-linked immunosorbent assay. RESULTS: In 12 of the 28 patients assessed for IL-8 expression disease recurred after a median followup of 66 months. Median IL-8 expression during the first 6 hours for these patients was 851 ng. (range 232 to 8,497). Median IL-8 expression during the first 6 hours in patients without recurrence was 4,200 ng. (range 432 to 12, 232). Of 8 patients with a followup of greater than 36 months 7 (88%) had no recurrent disease and IL-8 levels greater than 4,000 ng. Patients secreting more than 4,000 ng. IL-8 into the urine after BCG have a significantly higher chance of remaining disease-free (p <0.05), and those with elevated IL-18 expression have a significantly longer disease-free survival (p <0.05). After a median followup of 23 months (range 7 to 93) 6 of the 17 patients assessed for IL-18 expression had treatment failure. Median IL-18 expression in those patients during the first 12 hours was 2,632 pg. (range 860 to 8,298). Median IL-18 expression during the first 12 hours in patients without recurrence was 12,258 pg. (range 1,727 to 151,495). CONCLUSIONS: In this study we confirmed the value of quantitative IL-8 expression in the urine during the first 6 hours after BCG instillation for superficial bladder cancer to predict freedom of disease. Furthermore, to our knowledge we report for the first time the potential value of IL-18 expression in the urine during the first 12 hours after BCG to predict freedom from disease. These findings may help improve the treatment of patients with superficial bladder cancer, especially by identifying those with a high risk of disease recurrence and progression after BCG therapy.

[Biotransformation of the lipoxygenase inhibitor 2-hydroxy-5-methyl-laurophenone-oxime (FLM 5011)]. / Untersuchungen zur Biotransformation des Lipoxygenasehemmers 2-Hydroxy-5-methyl-laurophenon-oxim (FLM 5011).

2-Hydroxy-5-methyl-laurophenone-oxime (FLM 5011, 1) is an inhibitor of the lipoxygenase with antiinflammatory and antiallergic actions. The studies on the biotransformation using in vivo investigations and in vitro test systems resulted in finding of at least eight metabolites. Four of these compounds have been detected and identified in urine and faeces after p.o. administration in male Wistar rats. By means of cultures of hepatocytes, lymphocytes and myeloma cells additional metabolites were found and the main pathways of metabolism could be suggested. Furthermore it was possible to confirm the sequence of the metabolic reactions. First of all, 1 is hydroxylated in the omega-position of the lauryl side chain by the cytochrome P-450 system. The further oxidation to the carboxylated compound is followed by the stepwise degradation of the side chain by beta-oxidation similarly to the pathways of fatty acid metabolism. Simultaneously the oxime group is converted to the keto group. The metabolites and 1 partly occur as sulfate or glucuronide conjugates. Additionally all compounds produced by beta-oxidation are conjugated with other partners, probably amino acids. By omega-oxidation, compounds with higher inhibitory potency on the lipoxygenase than the parent compound are formed. These results suggest that the activity of 1 is partly caused by the initial metabolites.