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Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_15 | Pages 38 - 38
1 Nov 2018
Yin H Popov C Schieker M Nerlich M Docheva D
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Background: The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established a 3D collagen gel system and studied the remodeling capacity of two different TSPC lines: young, Y-TSPC and aged/degenerative, A-TSPC. We specifically investigated the involvement of integrin receptors in the remodeling process. Methods: Y- and A-TSPC were derived from human Achilles tendon. RT-PCR was used to assess the expression of collagen-binding integrins. Integrins a1 and a11 were silenced by lentiviral delivery of shRNA in the Y-TSPC. Control-shRNA, a1-shRNA and a11-shRNA virus was given for 24h and then cells were selected with zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, time-lapse recording of gel contraction of Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC were performed. Results: Integrin a1 and a11 were significantly downregulated in A-TSPC. Therefore, to mimic the A-TSPC we carried out a1 and a11 KD in Y-TSPC. PCR and western blot validated very efficient KD. Analyses of collagen contraction revealed that Y-TSPC+a11KD had significant reduction in collagen contractibility comparable to A-TSPC phenotype. Regarding integrin a1, we found that this receptor had no effect on the contraction rate of TSPC. Thus, to our knowledge we have now identified for the first time a novel role of a11 integrin in tendon matrix remodeling, and a follow up analyses of the exact downstream cascade are on the way.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_4 | Pages 21 - 21
1 Apr 2018
Keppler A Neuerburg C Westphal I Aszodi A Böcker W Schieker M
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Introduction

Osteoarthritis (OA) is a slow progressive disease and a huge economic burden. A new target for therapy could be a growth factor treatment to prevent the loss of cartilage following injuries to the joint. BMP-7 is a promising candidate for such a novel therapy based on growth factors. In this study we combined the chondroprotective effects of BMP-7 with a novel thermosensitive hydrogel to prevent cartilage degeneration in a murine OA model.

M&M

A BDI based thermosensitive hydrogel (Pluronic 123 with Butandiisyocyanate (BDI); LivImplant GmbH, Germany) was augmented with BMP-7 (rh-BMP-7, Olympus Biotech, France; 0.2 µg BMP-7/10µg Hydroge). To investigate the effects on OA progression we used the murine DMM (Destabilization of the medial meniscus) model for OA induction. Animal testing was approved by the Government Commitee of Upper Bavaria (file reference: 55.2-1-54-2532-150-13). A total of 38 C57BL/6 mice were included in this study. Immediately after the DMM surgery and wound closure BMP-7 mixed with BDI Hydrogel or only the BDI Hydrogel was administered via intraarticular injection. The following groups were examined: A) BMP-7 augmented BDI hydrogel B) only BDI hydrogel C) no injection following surgery D) control, healthy contralateral knee joint. After 4 (n=4 per group) and 8 (n=8) weeks mice were euthanized and knees were compared histologically.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_4 | Pages 17 - 17
1 Apr 2018
Daumer M Fürmetz J Keppler A Höfling H Müller A Hariry S Schieker M Grassi M Greese B Nuritdinow T Aigner G Lederer C Böcker W
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Mobility plays an important role, in particular for patients with osteoporosis and after trauma surgery, both as an outcome and as treatment. Mobility is closely linked to the patient”s quality of life and exercise is a powerful additional treatment option. In order to be able to generate an evidence base to evaluate various surgical and non-surgical treatment options, objective measurements of patient mobility and exercise over a certain time period are needed. Wearables are a promising candidate, with obvious advantages compared to questionnaires and/or PROs. However, when extracting parameters with wearables, one often faces the problem of algorithms not performing well enough for special cases like slow gait speeds or impaired gait, as they typically appear in this patient group. We plan to further extend the applicability of the actibelt system (3D accelerometer, 100Hz), in particular to improve the measurement precision of real-world walking speed in slow and impaired walking. We are using a special measurement wheel including a rotating 3D accelerometer that allows to capture high quality real-world walking speed and distance measurements, and a mobile high resolution camera system. In a first block 20 patients with osteoporosis were included in the study at the Ludwigs-Maximilians-University”s Department of General, Trauma and Reconstructive Surgery in Munich, Germany and equipped with an actibelt. Patients were asked to walk as “normal” as possible, while wearing their usual apparel, in the building and outside the building. They climbed stairs and had to deal with all unexpected “stop and go” events that appear in real-world walking. Various gait parameters will be extracted from the recorded data and compared to the gold standard. We will then tune the existing algorithms as well as new algorithms (e.g. step detection based on continuous wavelet transformation) to explore potential improvements of both step detection and speed estimation algorithms. Further refinement and validation using real world data is warranted.


Orthopaedic Proceedings
Vol. 92-B, Issue SUPP_IV | Pages 505 - 505
1 Oct 2010
Mayer S Büttner A Jansson V Mayer W Müller P Schieker M Schiergens T Sievers B
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Background: In regenerative medicine the autologous cartilage implantation (ACI) has been used for the repair of cartilage defects. As modification of ACI, the matrix assisted ACI is used nowadays with varying results. There is a general discussion about whether supporting scaffolds should be used or whether a scaffold-free cartilage repair is the method of choice. The major problem of scaffold-free regenerates is how to keep the cells in place after transplantation. Aim of this study was to examine a new scaffold-free diffusion-culture model, which uses a mega-congregate of chondrocytes cultured at an air-medium interface. This scaffold-free high-density diffusion culture could be used to repair cartilage defects.

Material and methods: Human chondrocytes from passage 1–7 were expanded in monolayer and transferred to pellet-culture or diffusion-culture. After one week cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for the mRNAs of cartilage markers.

Results: Positive alcian blue staining was detectable in diffusion-culture for human chondrocytes up to passage 7. Within passages the amount of proteoglycan production in relationship to the number of cells increased. There was a positive signal for Collagen type II in diffusion-cultures up to passage 7.

In qRT-PCR a redifferentiation of human chondrocytes was shown by the transfer into diffusion-culture. Within passage 1 to 3 human chondrocytes which were cultured in monolayer lost the ability to express Collagen Type II but could regain it if they were transferred to diffusion-culture. At diffusion-culture chondrocytes showed the highest expression of Collagen type II at passage 1 when compared to monolayer or to pellet-culture.

Conclusion: It could be shown that the cultivation in a scaffold-free diffusion-culture can lead to redifferentiation of human chondrocytes Chondrocytes in diffusion-cultures tend to form their own matrix and produce Collagen type II at higher amounts than in monolayer or in normal pellet-cultures. Therefore diffusion-culture congregates might be an appropriate tool to be used for a new scaffold-free cartilage regeneration approach.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_III | Pages 446 - 446
1 Sep 2009
Pietschmann MF Frankewycz B Docheva D Shakibaei M Jansson V Schieker M Müller PE
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Irreparable tendon ruptures constitute a grave clinical problem. Especially for large rotator cuff tears, there often is no primary causal therapy available. As a sad result, the development of a rotator cuff tear arthropathy is more often than not inevitable. Our study investigates the effects of scaffold based tendon regeneration with special focus on mesenchymal stem cells in a rat model.

We used ‘native’ bone marrow stromal cells and cultivated mesenchymal stem cells from male rats that were implanted into female rats. As scaffolds polyglycol acid (PGA) and a collagen I were used. A full-thickness-defect of 2–3 mm in the middle third of the rats achilles tendon was created, which was then filled, with either cell-seeded or not cell-seeded scaffolds and, due to the low primary stability of the scaffolds, fixed with a 4-0 suture. After 12 weeks, a DNA PCR was conducted to verify the existence of male Y-chromosomes in the female regenerated tissue. We determined the maximum tensile load of the regenerated tissue and also did a histological evaluation.

Macroscopically the regenerated tendons were much bigger in diameter, much firmer and also much less elastic than a normal tendon. In the ‘mesenchymal stem cells’ group the implanted cells could be clearly identified after 12 weeks by DNA PCR. The collagen I scaffold yielded better results in the biomechanical study than the PGA scaffold. No evidence of positive influence of the cells on the mechanical stability of the regenerated tissue was found. Collagen I and the use of BMSC histologically lead to increased ossification of the regenerated tissue. In the PGA scaffold group a significant inflammatory reaction was found.

Both scaffold/cell combination seem to be unsuitable for tendon replacement. in-vitro studies on the influence of scaffold material on cell differentiation needs to be done.


Orthopaedic Proceedings
Vol. 88-B, Issue SUPP_I | Pages 34 - 34
1 Mar 2006
Seitz S Horvath GG Guelkan H Regauer M Neth P Mutschler W Schieker M
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In tissue engineering, scaffolds are vitalized by cells in vitro. Human mesenchymal stem cells (hMSC) are very interesting because of their ability to differentiate towards the osteogenic lineage and their self renewing capacity. Yet, it is important that implanted cells do not disseminate and exhibit unwanted cell growth outside the implantation site. Therefore the aim of this study was to detect migrated cells in organs of mice after implantation of a composite (cell-scaffold) substitute.

HMSC (Cambrex, USA) were inoculated on a clinically approved 3D scaffold (Tutobone(TM), Tutogen, Germany). One composite and one scaffold without cells were implanted subcutanously, left and right paravertebrally in athymic nude mice (nu/nu). After 2, 4, 8 and 12 weeks constructs were explanted and organs (liver, spleen, lungs, kidney, heart, testicles, brain and blood) were harvested. The entire organs were homogenized and genomic DNA was isolated for qualitative and quantitative PCR.

Human DNA was found in all explanted composites at all examined time points. No human DNA could be detected in control scaffolds. Moreover we did not detect human DNA in all explanted organs at any time point. As internal controls we could detect 1 single hMSC in a pool of 106 mouse cells.

In conclusion, we could proof that cells of implanted composite substitutes do not migrate to other organs. Furthermore, this study showed that implanted hMSC seeded on 3D scaffolds survive over time frames up to 12 weeks.