Martin Russlies

Characteristics of human chondrocytes, osteoblasts and fibroblasts seeded onto a type I/III collagen sponge under different culture conditions. A light, scanning and transmission electron microscopy study.

Hyaline cartilage has only a limited capacity of regeneration, thus, lesions of articular cartilage can lead to early osteoarthrosis. Current concepts in conservative orthopedic therapy do not always lead to satisfying results. As one new attempt to facilitate cartilage repair, autologous transplantation of articular chondrocytes is investigated in different assays. This study was designed to create a resistible and stable cell-matrix-biocomposite with viable and biosynthetically active human chondrocytes, osteoblasts or fibroblasts. This biocomposite might serve as an implant to treat deep osteochondral defects in the knee. We collected cartilage, spongiosa and skin probes from healthy patients undergoing hip-surgery and enzymatically liberated the chondrocytes, seeded them into culture flasks and cultured them until confluent. The spongiosa and the skin samples were also placed in culture flasks and cells cultured until confluent. After 4-6 weeks, cells were trypsinized and grown on a type I/III collagen matrix (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland) for 7 days in standard Petri dishes and in a special perfusion chamber culture system. As controls, cells were seeded onto plastic surfaces. Then scaffolds were fixed and embedded for light microscopy and electron microscopy by routine methods. Light microscopically, chondrocytes grown on the surface of the scaffold form clusters or a dense layer of sometimes rather fibroblast-like and sometimes roundish, chondrocyte-like cells. Only a few cells grow deeper into the matrix. In transmission electron microscopy, the cells have a rather chondrocyte-like morphology which emphasizes the matrix-induced redifferentiation after dedifferentiation of chondrocytes in monolayer-culture in culture flasks. Chondrocytes on plastic surfaces have a spinocellular aspect with little signs of differentiation. Grown on Chondrogide, cells are more roundish and adhere firmly to the collagen fibrils of the scaffold. Osteoblasts grown on the collagen scaffold and examined by light microscopy form a thin cell-layer on the surface of the matrix with a reticular layer of dendritic cells underneath this sheet. Transmission electron micrographs show spinocellular and flat cells on the collagen fibrils. Scanning electron micrographs show large dendritic osteoblasts on plastic and a confluent layer of flattened, dendritic cells on the collagen scaffold. Fibroblasts form a thick multi-layer of typical spinocellular cells on the collagen matrix. Fibroblasts grown on plastic surfaces and examined by scanning electron microscopy also show a dense layer of fibroblast-like cells. For all three different types of cells no morphological differences could be seen when comparing cultivation in the perfusion culture system to cultivation in standard Petri dishes, although mechanical stress is believed to induce differentiation of chondrocytes. Especially the observed partially differentiated chondrocyte-matrix biocomposite might serve as an implant to treat deep cartilage defects, whereas osteoblasts and fibroblasts seem to be less suited.

A cell-seeded biocomposite for cartilage repair*

Chondrocytes in monolayer cultures lose their phenotype and capability to express type-II collagen, they dedifferentiate into a fibroblastic cell type. Using three-dimensional culture systems a redifferentiation of these cells may occur. In the present study we investigated the morphology and biosynthetic activity of human articular chondrocytes seeded on porous matrices of type I/III collagen (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland). Microscopical examinations showed that chondrocytes adhere firmly to a collagen-I/III-membrane exhibiting their characteristic spherical cell shape. Cell numbers after enzymatic digestion of the membrane showed a 93% recovery of seeded cells. Immunohistological examination revealed positive staining for type-II collagen in some areas. The generated biocomposite withstands mechanical stress, keeps its size and design and does not shrink in culture. It is therefore easy to handle, can be sutured, glued or fixed with pins. This study shows, that in vitro production of autologous cartilage-like tissue could be established using a bilayer collagen type I/III fleece. This biocomposite carries active chondrocytes and is currently being evaluated in vivo in a sheep model as well as in a clinical trial for the repair of localized cartilage defects in the knee.

Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge.

The regenerative capacity of hyaline articular cartilage is limited. Thus, lesions of this tissue are a proarthrotic factor, and up to now the conservative treatment of cartilage lesions and arthrosis does not yield satisfying results. Therefore, autologous transplantation of articular chondrocytes is being investigated in a variety of different assays. The aim of our study was to create a mechanically stable cell-matrix implant with viable and active chondrocytes which could serve to fill out articular lesions created in the knees of sheep. For this purpose, articular cartilage was collected from knee lesions, chondrocytes were liberated enzymatically and seeded in culture flasks and cultured till confluency. Cells were then trypsinized and grown on a type I/III collagen matrix (Chondro-Gide, Geistlich Biomaterials, Wolhusen, Switzerland) for 3, 6 and 10 days before being fixed and embedded for electron microscopy by routine methods. Scanning electron microscopy was performed after dehydration in acetone, critical point drying and sputter-coating with gold-paladium. Light microscopically, clusters of chondrocytes can be seen on the surface of the matrix with a few cells growing into the matrix. Transmission electron microscopic photographs yield a rather differentiated chondrocyte-like appearance, which is evidence of a matrix-induced redifferentiation after dedifferentiation during the growth period in the culture flasks. Scanning electron microscopic results show large, flattened chondrocytes without signs of differentiation on plastic, whereas chondrocytes grown on the Chondro-Gide sponge show a more roundish aspect wrapping firmly around the collagen fibrils, exhibiting numerous contacts with the matrix. This cell-matrix biocomposite can now serve to fill out articular cartilage lesions created in the knees of sheep.

Effects of hyaluronic acid on the morphology and proliferation of human chondrocytes in primary cell culture

Hyaline articular cartilage is a specialised connective tissue with weight bearing and adsorbing functions. Injury or loss of which often leads to impaired joint function and severe pain. Since the self-renewing abilities of hyaline articular cartilage are limited, there is major interest in the development of bioengineered cartilaginous implants. A cell-matrix-biocomposite composed of a collagen I/III scaffold seeded with autologous chondrocytes is currently being used in clinical trials; however, in order to optimise culture conditions, we cultured human condrocytes and seeded them on type I/III collagen membranes and on Thermanox plastic coverslips with media containing 0 to 500 microg/ml Hyaluronic Acid. After 4 days, the cells were either fixed or BrdU incorporation procedures begun. HE staining clearly demonstrated that cells grown in HA form three dimensional clusters and produce secretory vesicles as opposed to the monolayer control cells with noticeably fewer secretory vesicles. BrdU incorporation revealed a noticeable increase in cell proliferation in cells grown in 100 microg/ml; however, no comparable increase in 500 micorg/ml but rather a slight depression in proliferation. Immunohistochemistry for collagen II and aggrecan revealed an obvious increase in deposition of these two substances with increased HA administration as compared to the control; however, again, the higher concentration of HA, 500 microg/ml, did not result in a further increase in production. These results suggest that HA at 100 microg/ml not only influences chondrocytes to differentiate and produce more Collagen II and aggrecan, but also increases proliferation. We, therefore, propose that the addition of HA at low to middle dosages in condrocyte culturing might help improve condrocyte redifferentation and thus, the bioengineered cartilage.

Cell-Laden and Cell-Free Matrix-Induced Chondrogenesis versus Microfracture for the Treatment of Articular Cartilage Defects: A Histological and Biomechanical Study in Sheep.

Objective: The aim of this study was to evaluate the regenerative potential of cell-laden and cell-free collagen matrices in comparison to microfracture treatment applied to full-thickness chondral defects in an ovine model. Methods: Animals (n = 30) were randomized into 5 treatment groups, and 7-mm full-cartilage-thickness defects were set at the trochlea and medial condyle of both knee joints and treated as follows: 2 scaffolds in comparison (collagen I/III, Chondro-Gide®; collagen II, Chondrocell®) for covering microfractured defects (autologous matrix-induced chondrogenesis), both scaffolds colonized in vitro with autologous chondrocytes (matrix-associated chondrocyte transplantation), or scaffold-free microfracture technique. One year after surgery, cartilage lesions were biomechanically (indentation test), histologically (O’Driscoll score), and immunohistochemically (collagen type I and II staining) evaluated. Results: All treatment groups of the animal model induced more repair tissue and showed better histological scores and biomechanical properties compared to controls. The average thickness of the repair tissue was significantly greater when a scaffold was used, especially the collagen I/III membrane. However, none of the index procedures surpassed the others from a biomechanical point of view or based on the histological scoring. Collagen type II expression was better in condylar defects compared to the trochlea, especially in those treated with collagen I/III membranes. Conclusion: Covering of defects with suitable matrices promotes repair tissue formation and is suggested to be a promising treatment option for cartilage defects. However, it failed to improve the biomechanical and histological properties of regenerated articular cartilage compared to microfracture alone in an ovine model under the given circumstances.

Apoptotic chondrocyte death in cell-matrix biocomposites used in autologous chondrocyte transplantation

Tissue engineering may be a promising approach for the treatment of focal articular cartilage defects. Programmed cell death (apoptosis) plays an important role in multiple degenerative processes of cartilage (e.g. osteoarthritis). It is known that matrix provides a trophic signal for the cells and an altered matrix may influence the availability of factors that regulate apoptosis. In this study we investigate the viability of chondrocytes seeded on a Chondrogide scaffold (Geistlich Biomaterials, CH), which we use in matrix-induced autologous chondrocyte transplantation (MACT). By now, we have studied material from 29 patients treated for localized articular cartilage defects in the knee. Our results indicate that light microscopy (Mayers hematoxylin-eosin, Masson-Goldner, Trypan-blue and TUNEL method) and electron microscopy can be used to investigate for apoptotic cells grown on a Chondrogide resorbable scaffold. Neither the handling of the cell-matrix biocomposite nor the procedures for fixation could destroy the scaffold or the cell sheet adhering firmly to the matrix. Apoptotic cells were revealed in all samples and with all techniques used. Mayers hematoxylin-eosin and Masson-Goldner staining show cells with a condensed, pycnotic nucleus and shrunken cytoplasm. In electron microscopy we observed cells with chromatin condensation and volume shrinkage consistent with apoptosis. The results of the Trypan-blue staining show a mean viability of 92.1 +/- 9.8% (range 57-100%). The TUNEL method revealed 44.6 +/- 20.4% positive cells. Our results indicate that apoptosis plays an important role in chondrocytes grown on a scaffold. An optimal scaffold will determine the growth, morphology and phenotype of the chondrocytes by its physical and chemical characteristics.

Magnetic resonance imaging and correlative gross anatomy of the ligamentum semicirculare humeri (rotator cable).

The purpose of this study has been to demonstrate macroscopic and MRI anatomy of the so‐called rotator cable, otherwise known as the ligamentum semicirculare humeri (LSCH) of the superior shoulder joint capsule. Twelve shoulder joints from eight cadavers were dissected; seven of which, from four of the cadavers, were studied using MR arthrography (1.5‐Tesla device Somatom Symphony®, Siemens, Erlangen, Germany) prior to dissection. The MRI protocol included T1WI, PDWI, and DESS 3D WI standard sequences. The results of MRI were compared with gross anatomic dissection findings. The macroscopically recognizable capsular bundle of LSCH fibers was identified by anatomic dissection in all specimens. On MRI, the entire ligament or parts of it could be identified in six of seven cases. It was best visualized on axial images. In the evaluation of magnetic resonance images of superior shoulder joint structures, additional knowledge on the anatomy of the LSCH can be used by the radiologist to facilitate detailed interpretation of the shoulder MRI. Clin. Anat. 21:420–426, 2008.

The anterior glenohumeral joint capsule: macroscopic and MRI anatomy of the fasciculus obliquus or so-called ligamentum glenohumerale spirale

The purpose of this study was to demonstrate the macroscopic and MRI anatomy of the fasciculus obliquus, otherwise known as the ligamentum glenohumerale spirale or spiral GHL of the anterior shoulder joint capsule. Conventional and MR arthrography (1.5-T device Somatom Symphony, Siemens with shoulder coil) images in standard planes were compared with gross anatomic dissection findings in six fresh shoulder specimens from three cadavers. The MR imaging protocol included T1, PD and DESS 3D WI sequences. The macroscopically recognisable band—the spiral GHL—was identified by anatomic dissection and MRI in all the specimens. It was best visualised by MR arthrography on axial and oblique sagittal planes (T1; PD WI) and appeared as a low signal intensity stripe within the superficial layer of the anterior joint capsule. The absence of the variable middle glenohumeral ligament did not influence the anatomic properties and the MR imaging of the spiral GHL. Diagnostic visualisation of the normal anatomic structures is a prerequisite to distinguish between normal and pathologic conditions. Anatomy of the spiral GHL can be used by radiologists for more detailed interpretation of the anterior shoulder joint capsule ligaments on MR images.

Successful treatment of extensively drug-resistant Pseudomonas aeruginosa osteomyelitis using a colistin- and tobramycin-impregnated PMMA spacer

Discovered in 1949, the antibiotic colistin was initially used for therapeutic purposes. Parenteral use of colistin was gradually abandoned because of its side-effect profile, especially its nephrotoxicity and neurotoxicity. Despite the risk of these potentially serious adverse effects, increasing resistance of Gram-negative bacteria has led to a renaissance of intravenous use of colistin in the last few years. Local administration of colistin is an alternative method to minimise the risk of systemic toxicity. We present a case of extensively drug-resistant Pseudomonas aeruginosa osteomyelitis treated successfully with high-dose colistin- and tobramycin-impregnated bone cement as a drug delivery vehicle. For the first time, local colistin concentrations in drainage and synovial fluid were quantified in order to determine the optimal dose and to minimise serious side effects. Insertion of a bone cement spacer loaded with a high dose of tobramycin and colistin resulted in local colistin levels at the infection site that exceeded the minimum inhibitory concentration (MIC) of colistin against the isolated P. aeruginosa five-fold on Day 4. Thus, the treatment may be expected to exert a prolonged effect. Whereas systemic administration of colistin alone was not sufficient to treat the infection, combined local and parenteral therapy led to eradication of P. aeruginosa in this patient. Plasma colistin levels remained in the therapeutic range, which confirms the systemic safety of the method.

Investigating the effects of bone cement, cyanoacrylate glue and marine mussel adhesive protein from Mytilus edulis on human osteoblasts and fibroblasts in vitro.

Bone cement is a widely used standard fixation substance in Orthopaedic Surgery. Cyanoacrylate glue is available for wound closure to supplement suturing. The mussel adhesive protein extracted from Mytilus edulis (Cell-Tak, BD Biosciences, Heidelberg, Germany) is an experimental fixation device used for in vitro purposes of cell adhesion. The aim of this study is to introduce a cell culture model investigating the effects of commonly applied and experimental glues on human fibroblasts and osteoblasts in vitro. Cells cultured without additives served as a control group. Microscopic examination was performed to evaluate the morphologic changes. An apoptosis test (Apo-Tag, Chemicon International, Temecula, CA, U. S. A.) was applied to determine the rate of natural cell death at the end of the study. It could be demonstrated that morphological changes in bone cement are different in fibroblasts and osteoblasts. Osteoblasts seem to grow on bone cement and develop an orderly formation. Fibroblasts grow in a confluent monolayer around bone cement but do not adhere to the cement itself. This is a desirable effect since most Orthopaedic applications aim at osteointegration as opposed to fibrous tissue overgrowth. Apoptosis attributed to bone cement is comparable to the respective natural rate of apoptosis. Cyanoacrylate glue and the mussel adhesive protein lead to an almost complete apoptosis in the investigated cells. Their routine application should be avoided. The developed cell culture model seems appropriate for performing further investigations.