Incorporation of osteoblasts (MG63) into 3D nanofibre matrices by simultaneous electrospinning and spraying in bone tissue engineering.

Nanofibre scaffolds are suitable tools for bone tissue engineering. Mimicking the extracellular matrix, they allow for cell growth and differentiation. However, in large 3D scaffolds, uniform cell colonisation presents an unsolved problem. Our aim was to design and analyse a method of colonising nanofibre scaffolds, combining electrospinning of fibres and electrospraying of cells, to determine its impact on cell survival, growth, and gene expression. The osteoblast-like cell line MG63 was suspended in medium and electrosprayed into growing scaffolds of poly-(l-lactic acid) (PLLA) or PLLA/Col-I blend nanofibres. Fluorescein diacetate (FDA) staining was used to determine survival and growth over a 22 d culture period. Expression of osteocalcin (OC) and type I collagen (Col-I) genes was determined by real time PCR. Fluorescence microscopy was used to analyse Col-I and OC deposition, as well as cell densities. While spraying distance and cell density in the spraying solution influenced survival and cell density, the combination of electrospinning and electrospraying did not negatively influence the maintenance of the osteoblast phenotype. Furthermore, VEGF induction in response to hypoxia was not suppressed, but modulated by polymer composition. Therefore, simultaneous electrospinning and electrospraying is a suitable tool in producing nanofibre based 3D cell seeded scaffolds.

Increased glenohumeral translation and biceps load after SLAP lesions with potential influence on glenohumeral chondral lesions: a biomechanical study on human cadavers.

PURPOSE: The aim of the study was to evaluate the stabilizing function of the long head of biceps tendon (LHB) and its tension, both without and with the presence of SLAP lesion to analyze a potentially occurring humeral chondral print of LHB with consecutive glenohumeral chondral lesions in SLAP lesions. METHODS: Testings were performed on 21 fresh frozen human cadaver shoulders with intact shoulder girdle by a 5 axis industrial robot with a force/moment sensor and 20 N joint compression, 50 N force in anterior, posterior, anterosuperior, and anteroinferior direction, and 0°, 30°, 60° of abduction. LHB was connected over a force measuring sensor with 5 N and 25 N preload. A type IIC SLAP lesion was created arthroscopically. RESULTS: A significant increase in anterior and anteroinferior translation was evaluated, whereas the LHB tension increased significantly in at most anterior and anterosuperior direction. The highest increase in translation and LHB tension after SLAP lesion was measured in anterior translation in at most 60° of abduction. The glenohumeral translation was significantly higher in SLAP lesions without LHB tenotomy than after isolated LHB tenotomy. CONCLUSIONS: SLAP lesions lead to increased glenohumeral translation and concurrently LHB tension and load in at most anterior direction. The increased anterior glenohumeral instability and the increased LHB load pressing on the humeral head might cause glenohumeral chondral lesions with a typical chondral print-like lesion on the humeral head underneath the LHB.

CD44: survival and metastasis in chondrosarcoma.

OBJECTIVE: Recent studies have shown abnormal expression of CD44s and some of its isoforms in many human malignancies, but little is known about the presence of CD44 in chondrosarcoma. In this study the expression of CD44s and two variant isoforms was evaluated. It was assumed that abnormalities in these receptor proteins may be associated with clinical outcome of the patients. METHOD: Thirty paraffin-embedded chondrosarcoma samples were immunostained with monoclonal antibodies for CD44s, CD44v5 and CD44v6. Two independent examiners who were unaware of the clinical status of the patients evaluated the immunohistochemical results. The percentage of CD44-positive cells was scored semiquantitatively. A rate of higher than 10% was considered as overexpression. RESULTS: Among the 30 patients (median age 50 years) there were 22 conventional chondrosarcomas, two dedifferentiated chondrosarcomas, two extraskeletal chondrosarcomas, and one periostal, mesenchymal, clear cell and myxoid chondrosarcoma each. In the immunochemistry staining overexpression (>10% of cells) of CD44s was shown in 56.7% (17 of 30), of CD44v5 in 43.3% (13 of 30) and of CD44v6 in 6.7% (two of 30) of the tumors. Four grade III chondrosarcomas (80%) and 10 (71.4%) grade II chondrosarcomas showed overexpression for CD44s, whereas CD44s was overexpressed in only three (27.3%) grade I chondrosarcomas. Cox regression suggests overexpression of CD44s to be an additional prognostic marker for chondroid bone tumors independent of grading and other covariates. CONCLUSIONS: Overexpression of CD44s correlated significantly with metastatic potential and with poorer survival in patients with chondrosarcoma. CD44s might be an independent additional marker, but small sample size remains to be considered.

Primary stability of tibial components in TKA: in vitro comparison of two cementing techniques.

In spite of improvements in cementing technique, migration of tibial component remains a problem in total knee arthroplasty. This study compares the primary stability of tibial components using two different cementing techniques with roentgen stereophotogrammetric analysis (RSA) in vitro. A total of 20 tibia specimens were matched into two groups, 10 specimens per group. Cementing technique was randomized to each group. In the first group only the base and in the second group the base and stem were cemented. The implants and the tibial metaphysis were marked with markers for the RSA analysis. All specimens were tested with an axial load of 2,000 N for 1,000 and 10,000 cycles and RSA analysis was performed. Endpoints for radiosterometric analysis were maximum total point motion, maximum subsidence, lift off, rotation and translation along the x-, y-, and z-axes. After 1,000 and 10,000 cycles, no significant differences could be found, but two tibial components of the surface cementing group showed a migration of more than 2 mm defined as failure compared to six failed tibial components in the full cementing group (P = 0.068). This higher number of failed arthroplasties in the fully cemented prosthesis group demonstrates a disadvantageous load distribution in the tibia apophysis which can cause an early component loosening.

Lack of obvious influence of PLLA nanofibers on the gene expression of BMP-2 and VEGF during growth and differentiation of human mesenchymal stem cells.

Growth factors like bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid) (PLLA) nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC) differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2-enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers.

Cloning, characterization and comparison of the Pseudomonas mendocina polyhydroxyalkanoate synthases Phac1 and PhaC2.

This study describes a comparison of the polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 of Pseudomonas mendocina. The P mendocina pha gene locus, encoding two PHA synthase genes [phaC1Pm and phaC2pm flanking a PHA depolymerase gene (phaZ)], was cloned, and the nucleotide sequences of phaC1Pm (1,677 bp), phaZ (1,034 bp), and phaC2pm (1,680 bp) were determined. The amino acid sequences deduced from phaC1Pm and phaC2pm showed highest similarities to the corresponding PHA synthases from other pseudomonads sensu stricto. The two PHA synthase genes conferred PHA synthesis to the PHA-negative mutants P. putida GPp104 and Ralstonia eutropha PHB-4. In P. putida GPp 104, phaC1Pm and phaC2Pm mediated PHA synthesis of medium-chain-length hydroxyalkanoates (C6-C12) as often reported for other pseudomonads. In contrast, in R. eutropha PHB-4, either PHA synthase gene also led to the incorporation of 3-hydroxybutyrate (3HB) into PHA. Recombinant strains of R. eutropha PHB-4 harboring either P. mendocina phaC gene even accumulated a homopolyester of 3HB during cultivation with gluconate, with poly(3HB) amounting to more than 80% of the cell dry matter if phaC2 was expressed. Interestingly, recombinant cells harboring the phaC1 synthase gene accumulated higher amounts of PHA when cultivated with fatty acids as sole carbon source, whereas recombinant cells harboring PhaC2 synthase accumulated higher amounts when gluconate was used as carbon source in storage experiments in either host. Furthermore, isogenic phaC1 and phaC2 knock-out mutants of P. mendocina provided evidence that PhaC1 is the major enzyme for PHA synthesis in P. mendocina, whereas PhaC2 contributes to the accumulation of PHA in this bacterium to only a minor extent, and then only when cultivated on gluconate.