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Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 5 - 5
1 Dec 2021
Alagboso F Mannala G Steinmann S Docheva D Rupp M Brochhausen C Alt V
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Aim

Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction.

Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment.

Method

Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 76 - 76
1 Dec 2021
Mannala G Rupp M Alagboso F Docheva D Alt V
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Aim

In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules involve in biofilm. Due to ethical restrictions, the use mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections. This model organism is easy to handle, cheap and ethical restriction free and could be used for the high through put screening of antimicrobial compounds to treat biofilm. To promote the use of this model in basic research we aimed to validate this based on the typical biofilm features such as less susceptible to the antibiotics, complexity of the biofilm structure and gene expression profile of biofilms.

Method

G. mellonella larvae are maintained at 30oC on artificial diet in an incubator. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. After sterilization with 100% alcohol, these K-wires were pre-incubated in S. aureus bacterial suspension (5×106 CFU/ml) for 30 min, washed in PBS and implanted inside the larva after with help of scalpel. The larvae were incubated at 37oC for two day for the survival analysis. To analyze the less susceptibility of the biofilms towards antibiotics, the larvae were treated with gentamicin and compared survival with planktonic infection in G. mellonella. To reveal the complex structure of biofilm, the implants were removed and processed for the MALDI analysis. Whole genome-based transcriptome of biofilm was performed to explore the changes in transcriptional landscapes.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_15 | Pages 87 - 87
1 Dec 2021
Mannala G Rupp M Walter N Brunotte M Alagboso F Docheva D Brochhausen C Alt V
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Aim

Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella.

Method

The bacteriophages (191219) were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages, and S. aureus EDCC 5055(1×107 CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37oC and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analysing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well plate. The eradication of biofilm was analysed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts at 2h, 4h and 24h time points of post treatment. In addition, we have analysed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Finally, experiments are performed to prove the effect of bacteriophages to clear in vivo biofilm using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis.


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 16 - 16
1 Jul 2020
Docheva D Lin D
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Tenomodulin (Tnmd) is the best known mature tendon factor for tendon and ligament tissues with reported important regulatory roles1. In addition, Tnmd C-terminal cysteine-rich domain has been descibed to exert anti-angiogenic functions in in vitro angiogenic assays as well as in vivo models of tendon injury and age-associated cardiac valve diseases1, 2. Interestingly, Tnmd expresson in the intervertebral disc (IVD), which is normally avascular tissue, has been also suggested3. Hence, the purpose of this study was first, to map the exact expression pattern of Tnmd during IVD development and aging and second, by implementing Tnmd-knockout mouse model, to examine if Tnmd plays a role in IVD homeostasis.

Histological analyses (hematoxylin/eosin, Safranin O, CD31 for endothelium, TUNEL for apoptosis and type X collagen and Runx2 for hypetrophy) were performed on Tnmd −/−, Tnmd −/− and chondromodulin I Chmd 1 −/− (Tnmd only homolog) double knockout and wild type mice WT (n = three to five) to examine IVD degeneration. Real time PCR was implemented to explore gene expression chnages in annulus fibrous (AF) between Tnmd −/− and WT mice. In addition, outer AF (OAF) cells were isolated from both genotypes to further determine cellular phenotype and assess effects on co-culture with human umbical vein endothelial cells (HUVECs). Statistical differences between two groups were determined with t-test. In multiple comparisons, one-way ANOVA was followed by Bonferroni post-hoc correction.

Tnmd was expressed in a temporal manner in OAF and to very low extent in NP. Tnmd −/− mice exhibited more rapid progression of age-related IVD degeneration. These signs included smaller collagen fibril diameter, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis and inflamatory cell infiltration in OAF as well as more hypertrophic-like chondrocytes in the NP. In addition, Tnmd−/− Chm1 −/− mice displayes not only accelerated IVD phenotpye, but also ectopic bone formation in the IVD. Lastly, the abscence of Tnmd in OAF-derived cells significantly promoted HUVECs migratory capacity.

These findings provide clear evidence that Tnmd plays a critical role in IVD homeostasis.


Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 18 - 18
1 Jul 2020
Pattappa G Koch M Weber J Lang S Bohrer A Johnstone B Docheva D Zellner J Angele P Krueckel J Franke D
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Osteoarthritis (OA) is one of the most prevalent joint diseases involving progressive and degenerative changes to cartilage resulting from a variety of etiologies including post-traumatic incident or aging. OA lesions can be treated at its early stages through cell-based tissue engineering therapies using Mesenchymal Stem Cells (MSCs). In vivo models for evaluating these strategies, have described both chondral (impaction) and osteochondral (biopsy punch) defects. The aim of the investigation was to develop a compact and reproducible defect inducing post-traumatic degenerative changes mimicking early OA. Additionally, a pilot study to evaluate the efficacy of MSC-hydrogel treatment was also assessed.

Surgery was performed on New Zealand white rabbits (male, 5–8 months old) with defects created on medial femoral condyle. For developing an appropriate defect, three approaches were used for evaluation: a biopsy punch (n = three at six and twelve weeks), an impaction device1 (n = three at six and twelve weeks) and a dental drill model (n = six at six and twelve weeks). At stated time points, condyles were harvested and decalcified in 10% EDTA, then embedded in Tissue-Tek and sectioned using a cryostat. Upon identification of region of interest, sections were stained with Safranin-O/Fast green and scored using OARSI scoring system by two blinded observers2. For the pilot study, autologous bone marrow was harvested from rabbits and used to isolate and expand MSCs. The Dental drill model was applied to both knee condyles, left untreated for six weeks at which stage, PKH26 fluorescently labelled MSCs were seeded into a hyaluronic acid hydrogel (TETEC). Repair tissue was removed from both condyles and MSC-hydrogel was injected into the left knee, whilst right knee was left empty. Rabbits were sacrificed at one (n = 1), six (n = 3) and twelve (n = 3) weeks post-treatment, processed as previously described and cartilage regeneration evaluated using Sellers score3.

Impacted condyles exhibited no observed changes histologically (Mean OARSI score = 1 + 1), whereas biopsy punched and dental drilled defects demonstrated equal signs of cartilage erosion (OARSI score = 3 + 1) at assessed time points. However, biopsy punched condyles formed a diffusive defect, whereas dental drilled condyles showed a more defined, compact and reproducible defect. In the pilot study, PKH-labelled MSCs were observed at one and six weeks post-implantation within the defect space where hydrogel was injected. Tissue regeneration assessment indicated no difference between empty (Mean Sellers score = 14 + 2) and MSC treated defects (Sellers score = 16 + 5) at six weeks post-injection. At twelve weeks, MSC treated defects showed improved tissue regeneration with substantial subchondral bone restoration and good integration of regenerative cartilage with surrounding intact tissue (Sellers score = 10 + 1), whereas untreated defects showed no change in regeneration compared to six weeks (Sellers score = 16 + 2).

Dental drill model was found to be the appropriate strategy for investigating early OA progression and treatment. Application of MSCs in defects showed good cartilage regeneration after twelve weeks application, indicating their promise in the treatment of early OA defects.