[Clinical trials as a central part of patient-centered clinical research]. / Klinische Studien als zentraler Bestandteil patientenorientierter Forschung.

As the continuation and implementation of findings from basic (pre­)clinical research, clinical trials make a significant contribution to medical research. They form the central building block of translational medicine and thus make a decisive contribution to bringing medical knowledge into general care. This helps to make possible a healthcare system that is aligned to the needs of patients and functions efficiently in the long term. Based on the specific objective, clinical trials must comply with national, but increasingly also with European and international regulatory requirements. In academia in particular, expertise in a variety of fields is required in order to make investigator-driven clinical trials a success. This expertise can be provided by a clinical trial center based within the institution conducting the trial.

[Clinical trials as a central part of patient-centered clinical research]. / Klinische Studien als zentraler Bestandteil patientenorientierter Forschung.

As the continuation and implementation of findings from basic (pre-)clinical research, clinical trials make a significant contribution to medical research. They form the central building block of translational medicine and thus make a decisive contribution to bringing medical knowledge into general care. This helps to make possible a healthcare system that is aligned to the needs of patients and functions efficiently in the long term. Based on the specific objective, clinical trials must comply with national, but increasingly also with European and international regulatory requirements. In academia in particular, expertise in a variety of fields is required in order to make investigator-driven clinical trials a success. This expertise can be provided by a clinical trial center based within the institution conducting the trial.

Challenges for academic investigator-initiated pediatric trials for rare diseases.

BACKGROUND: Clinical trials require great effort, time, expertise, and money. For clinicians at university hospitals with their full work load of teaching and medical care, the planning of an investigator-initiated clinical trial seems almost unthinkable. Despite their expertise in distinct diseases, university clinicians lack the time necessary to organize the funding and to initiate and conduct Phase III clinical trials in adults or in children. OBJECTIVE: We sought to determine whether the difficulties faced by a clinician conducting a pediatric clinical trial can be overcome by passionate motivation and external support. METHODS: Critical aspects of the application process of the world's first clinical trial in children with the rare hereditary kidney disease Alport syndrome treated with an angiotensin-converting enzyme inhibitor (Early Prospective Therapy Trial to Delay Renal Failure in Children With Alport Syndrome [EARLY PRO-TECT Alport]; http://www.clinicaltrials.gov NCT01485978; EudraCT 2010-024300-10) are described. RESULTS: The following crucial factors enabled the investigator to complete this trial: (1) support through clinical trial, biometrician, and regulatory experts (Institute for Applied Research and Clinical Studies [IFS], Göttingen, Germany); (2) advice from the university's ethics committee (University Medicine Göttingen, Göttingen, Germany); (3) public funding (€1 million from the German Federal Ministry of Education and Research); (4) support from the respective medical society, aiming at the resolution of an important clinical problem (German Society of Pediatric Nephrology); and (5) support from the investigator's university as the official sponsor of the trial, providing long-term commitment and covering financial risks (University Medical Center Göttingen, Göttingen, Germany). CONCLUSIONS: The study could pave the way for approval of ramipril as a drug to treat children with Alport syndrome. Even though the study might not result in label changes, the EARLY PRO-TECT Alport trial provides the basis of an educational campaign to sensitize physicians, especially pediatricians, general practitioners, and nephrologists, to pay special attention to the early detection of kidney diseases in children, which could improve medical care for all children with kidney diseases.

Cell-free repair of small cartilage defects in the Goettinger minipig: the effects of BMP-7 continuously released by poly(lactic-co-glycolid acid) microspheres.

OBJECTIVE: Cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed after treatment with cell-free collagen type-I gel with or without additional BMP-7 loaded poly(lactic-co-glycolid acid) microspheres. METHODS: Two full-thickness chondral defects were created in the trochlear groove of one hind leg knee in six Goettinger minipigs. Six defects were treated with a cell-free collagen type-I gel plug of 10 mm, the corresponding six defects were treated with a cell-free collagen type-I plug with poly(lactic-co-glycolid acid) microspheres loaded with recombinant BMP-7 (100 ng/ml gel). After 1 year, the animals were sacrificed. Immediately after recovery, non-destructive biomechanical testing was performed. The repair tissue quality was evaluated by immunohistochemistry and the O'Driscoll score was calculated. RESULTS: After 1 year, a robust cellular migration into the cell-free collagen gel plugs occurred and a hyaline-like repair tissue was found. Collagen type-II production and cellular organisation were higher in the BMP-7 microsphere group. The determination of the E-modulus, creep and relaxation revealed that mechanical properties of the BMP-7 microsphere group in summary were closer to control hyaline cartilage. CONCLUSIONS: While all specimens revealed a homogeneous cellular distribution, ECM production, cellular organisation and mechanical properties were enhanced by continuous BMP-7 release.

Safety and Efficacy of the ACE-Inhibitor Ramipril in Alport Syndrome: The Double-Blind, Randomized, Placebo-Controlled, Multicenter Phase III EARLY PRO-TECT Alport Trial in Pediatric Patients.

Introduction. Retrospective observational data show that ACE-inhibitor therapy delays renal failure and improves life expectancy in Alport patients with proteinuria. The EARLY PRO-TECT Alport trial assesses the safety and efficacy of early therapy onset with ramipril in pediatric Alport patients. Methods and analysis. This double-blind, randomized, placebo-controlled, multicenter phase III trial (NCT01485978; EudraCT-number 2010-024300-10) includes 120 pediatric patients aged 24 months to 18 years with early stages of Alport syndrome (isolated hematuria or microalbuminuria). From March 2012, up to 80 patients will be randomized 1:1 to ramipril or placebo. In the event of disease progression during 3-year treatment, patients are unblinded and ramipril is initiated, if applicable. Approximately 40 patients receive open-label ramipril contributing to the safety database. Primary end-points are "time to progression to next disease level" and "incidence of adverse drug events before disease progression." Treatment effect estimates from the randomized comparison and Alport registry data will be combined in supportive analyses to maximize evidence. Conclusion. Without this trial, ACE inhibitors may become standard off-label treatment in Alport syndrome without satisfactory evidence base. The results are expected to be of relevance for therapy of all pediatric patients with kidney disease, and the trial protocol might serve as a model for other rare pediatric glomerulopathies.

Simultaneous anabolic and catabolic responses of human chondrocytes seeded in collagen hydrogels to long-term continuous dynamic compression.

Cartilage repair strategies increasingly focus on the in vitro development of cartilaginous tissues that mimic the biological and mechanical properties of native articular cartilage. However, current approaches still face problems in the reproducible and standardized generation of cartilaginous tissues that are both biomechanically adequate for joint integration and biochemically rich in extracellular matrix constituents. In this regard, the present study investigated whether long-term continuous compressive loading would enhance the mechanical and biological properties of such tissues. Human chondrocytes were harvested from 8 knee joints (n=8) of patients having undergone total knee replacement and seeded into a collagen type I hydrogel at low density of 2×10(5)cells/ml gel. Cell-seeded hydrogels were cut to disks and subjected to mechanical stimulation for 28 days with 10% continuous cyclic compressive loading at a frequency of 0.3 Hz. Histological and histomorphometric evaluation revealed long-term mechanical stimulation to significantly increase collagen type II and proteoglycan staining homogenously throughout the samples as compared to unstimulated controls. Gene expression analyses revealed a significant increase in collagen type II, collagen type I and MMP-13 gene expression under stimulation conditions, while aggrecan gene expression was decreased and no significant changes were observed in the collagen type II/collagen type I mRNA ratio. Mechanical propertywise, the average value of elastic stiffness increased in the stimulated samples. In conclusion, long-term mechanical preconditioning of human chondrocytes seeded in collagen type I hydrogels considerably improves biological and biomechanical properties of the constructs, corroborating the clinical potential of mechanical stimulation in matrix-associated autologous chondrocyte transplantation (MACT) procedures.

Effects of low concentration BMP-7 on human osteoarthritic chondrocytes: comparison of different applications.

While BMP-7 (OP-1) is one of the most potent growth factors in cartilage tissue engineering, the effects of exogenous low concentration BMP-7 on osteoarthritic chondrocytes are still unknown. Human osteoarthritic chondrocytes obtained from the femoral condyles of 10 patients were grown either in monolayer or in 3D collagen type-I gel culture in vitro. The growth factor was either given as a single dose of 50 ng/mL, a repeated dose, or continuously released from PGLA microspheres. Matrix formation was monitored by immunohistochemical staining and real-time PCR. In contrast to monolayer culture, the differentiated phenotype was prevailed in 3D culture. Collagen type-II protein production in the 3D group with a continuous BMP-7 release was enhanced in comparison to all other groups. Gene expression of collagen type-II and aggrecan was elevated in all treatment groups, with the highest extent in the BMP-7 microsphere group. In summary, treatment of articular chondrocytes with a low dose of BMP-7 leads to an elevated production of extracellular matrix components. This effect is further increased when BMP-7 is given repeatedly or continuously, which proved to be the most effective form of application.

Bioreactor cultivation and remodelling simulation for cartilage replacement material.

For the development of articular cartilage replacement material, it is essential to study the dependence between mechanical stimulation and cell activity in cellular specimens. Bioreactor cultivation is widely used for this purpose, however, it is hardly possible to obtain a quantitative relationship between collagen type II production and applied loading history. For this reason, a bioreactor system is developed, measuring applied forces and number of loading cycles by means of a load cell and a forked light barrier, respectively. Parallel to the experimental study, a numerical model by means of the finite element method is proposed to simulate the evolution of material properties during cyclic stimulation. In this way, a numerical model can be developed for arbitrary deformation cases.

A comparative study of 3 different cartilage repair techniques.

PURPOSE: The value of cell-free techniques in the treatment of cartilage defects remains under debate. In this study, cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed by treatment with a cell-free collagen type-I gel or a collagen type-I gel seeded with autologous chondrocytes. As a control, abrasion arthroplasty was included. METHODS: In 18 adult Goettinger minipigs, three full-thickness chondral defects were created in one knee of the hind leg. They were either treated with a cell-free collagen gel, a collagen gel seeded with 2 × 10(5)/ml chondrocytes, or left untreated. All animals were allowed unlimited weight bearing. At 6, 12, and 52 weeks, 6 animals were sacrificed. Immediately after recovery, a non-destructive biomechanical testing was performed. The repair tissue quality was evaluated histologically, and the O'Driscoll score was calculated. RESULTS: After 6 weeks, a high number of cells migrated into the initially cell-free collagen gel. After 1 year, a hyaline-like repair tissue in both groups has been created. As assessed by O'Driscoll scoring and col-II staining, repair tissue quality of the initially cell-free gel was equal to defects treated by cell-seeded collagen gel implantation after 1 year. All untreated control defects displayed a fibrous repair tissue. The mechanical properties represented by the e-modulus were inconsistent in the course of the study. CONCLUSIONS: The implantation of a cell-free collagen type-I gel can lead to a high-quality repair tissue in the Goettinger minipig that equals a cell-based procedure after 1 year postoperatively. This study demonstrates the high chondrogenic potential of the applied collagen gel, which might help to overcome the disadvantages inherent in conventional cartilage tissue engineering methods.

Continuous cyclic compressive loading modulates biological and mechanical properties of collagen hydrogels seeded with human chondrocytes.

PURPOSE: This study investigated the potential of cyclic compressive loading in the generation of in vitro engineered cartilaginous tissue with the aim of contributing to a better understanding of mechanical preconditioning and its possible role in further optimizing existing matrix-associated cartilage replacement procedures. METHODS: Human chondrocytes were harvested from 12 osteoarthritic knee joints and seeded into a type I collagen (col-I) hydrogel at low density (2 × 10(5) cells/ml gel). The cell-seeded hydrogel was condensed and cultivated under continuous cyclic compressive loading (frequency: 0.3 Hz; strain: 10%) for 14 days under standardized conditions. After retrieval, specimens were subject to staining, histomorphometric evaluation, gene expression analysis and biomechanical testing. RESULTS: Cellular morphology was altered by both stimulation and control conditions as was staining for collagen II (col-II). Gene expression measurements revealed a significant increase for col-II under either cultivation condition. No significant differences in col-I, aggrecan and MMP-13 gene expression profiles were found. The col-II/col-I mRNA ratio significantly increased under stimulation, whereas the biomechanical properties deteriorated under either cultivation method. CONCLUSIONS: Although the effects observed are small, mechanical preconditioning has demonstrated its potential to modulate biological properties of collagen hydrogels seeded with human chondrocytes.

Effects of controlled released BMP-7 on markers of inflammation and degradation during the cultivation of human osteoarthritic chondrocytes.

The aim of the present study is to investigate the effects of BMP-7 released from polylactide microspheres on the appearance of various catabolic and inflammatory cytokines secreted by osteoarthritic chondrocytes cultivated in a collagen gel. Articular chondrocytes of 15 patients suffering from osteoarthritis are transferred to a collagen type-I gel. Additionally, BMP-7 encapsulated into polylactide microspheres (50 ng BMP-7/mL gel) is added. After 14 days, gene expression and protein appearance of various genes involved in matrix turnover and inflammation are investigated by immunohistochemical staining and RT-PCR and compared to untreated controls. TNF-α, MMP-13, IL-6, IL-1ß, and VEGF gene expressions are decreased in the treatment group. In contrast, BMP-7-induced matrix synthesis is not affected, leaving collagen type-II (Col-II) gene expression to be elevated, while collagen type-I (Col-I) is decreased. In summary, controlled release of low concentrated BMP-7 from polylactide microspheres leads to a decrease in gene expression of the investigated inflammation and matrix degradation markers whereas matrix synthesis is induced.

Millicurrent stimulation of human articular chondrocytes cultivated in a collagen type-I gel and of human osteochondral explants.

BACKGROUND: Here we investigate the effect of millicurrent treatment on human chondrocytes cultivated in a collagen gel matrix and on human osteochondral explants. METHODS: Human chondrocytes from osteoarthritic knee joints were enzymatically released and transferred into a collagen type-I gel. Osteochondral explants and cell-seeded gel samples were cultivated in-vitro for three weeks. Samples of the verum groups were stimulated every two days by millicurrent treatment (3 mA, sinusoidal signal of 312 Hz amplitude modulated by two super-imposed signals of 0.28 Hz), while control samples remained unaffected. After recovery, collagen type-I, type-II, aggrecan, interleukin-1beta, IL-6, TNFalpha and MMP13 were examined by immunohistochemistry and by real time PCR. RESULTS: With regard to the immunostainings 3 D gel samples and osteochondral explants did not show any differences between treatment and control group. The expression of all investigated genes of the 3 D gel samples was elevated following millicurrent treatment. While osteochondral explant gene expression of col-I, col-II and Il-1beta was nearly unaffected, aggrecan gene expression was elevated. Following millicurrent treatment, IL-6, TNFalpha, and MMP13 gene expression decreased. In general, the standard deviations of the gene expression data were high, resulting in rarely significant results. CONCLUSIONS: We conclude that millicurrent stimulation of human osteoarthritic chondrocytes cultivated in a 3 D collagen gel and of osteochondral explants directly influences cell metabolism.

Effects of low concentrated BMP-7 administered by co-cultivation or plasmid transfection on human osteoarthritic chondrocytes.

INTRODUCTION: While BMP-7 has proven to be one of the most potent growth factors in cartilage tissue engineering, protein concentration and route of administration remain a matter of debate. Here we investigated the effects of a low concentration of BMP-7 on human osteoarthritic chondrocytes administered by protein co-cultivation and plasmid transfection. METHODS: Freshly released (P0) or in vitro propagated chondrocytes (P2) were cultivated in a collagen type-I gel for 3 weeks in vitro or in nude mice. Seeded chondrocytes were treated with 50 ng/mL BMP-7 directly added to the medium or were subject to transient BMP-7 plasmid transfection prior to gel cultivation. Untreated specimens served as a control. After recovery, samples were investigated by histological and immunohistochemical staining and real-time PCR. RESULTS: In vitro, collagen type-II protein production was enhanced, and it was stored mainly pericellularly. Collagen type-II and aggrecan gene expression were enhanced in both treatment groups. After nude mouse cultivation, col-II protein production was further enhanced, but specimens of the BMP-7 transfection group revealed a clustering of col-II positive cells. Gene expression was strongly upregulated, chondrocyte number was increased and the differentiated phenotype prevailed. In general, freshly released chondrocytes (P0) proved to be superior to chondrocytes pre-amplified in vitro (P2). CONCLUSIONS: Both BMP-7 co-cultivation and plasmid transfection of human osteoarthritic chondrocytes led to improved cartilage repair tissue. Nevertheless, the col-II distribution following BMP-7 co-cultivation was homogeneous, while samples produced by transient transfection revealed a col-II clustering.

BMP-7-loaded PGLA microspheres as a new delivery system for the cultivation of human chondrocytes in a collagen type I gel: the common nude mouse model.

PURPOSE: Bone morphogenic protein 7 (BMP-7) released from polylactide (PLGA) microspheres has proven to be a potent system in cartilage tissue engineering in vitro. However, in vivo data are still lacking. The aim of this study was to investigate this BMP-7 release system utilizing the nude mouse as a small animal model. METHODS: Human osteoarthritic chondrocytes of 10 patients were enzymatically released and transferred into a collagen type-I gel. A concentration of 2x10(5) cells/mL was used. BMP-7 encapsulated in PGLA microspheres was added at an initial concentration of 500 ng BMP-7/mL gel. Untreated specimens and specimens with empty microspheres served as control. Samples were cultivated subcutaneously in nude mice for 6 weeks. RESULTS: After recovery, chondrocytes of all groups displayed a spheroid morphology without signs of dedifferentiation. The proteoglycan and collagen type II content of the control groups was restricted to the immediate pericellular region, whereas treatment group samples showed enhanced collagen type II production. Collagen type II and aggrecan gene expression was enhanced in treatment group samples with respect to the two control groups (mean +/- SD: 0.268 +/- 0.450 to 0.152 +/- 0.129 and 0.155 +/- 0.216 ng/ng beta-actin for collagen type II; 0.535 +/- 0.731 to 0.367 +/- 0.651 and 0.405 +/- 0.326 ng/ng beta-actin for aggrecan), whereas collagen type I gene expression decreased by a factor of 10. Relative protein quantification of collagen type II, collagen type I and proteoglycan was in accordance. CONCLUSIONS: Our data suggest that BMP-7 release from PGLA microspheres led to an improved tissue-engineered cartilage analogue in vivo with an increase in hyaline-cartilage-specific components.

A cell-free collagen type I device for the treatment of focal cartilage defects.

The purpose of this study was to evaluate the potential value of a cell-free collagen type I gel plug for the treatment of focal cartilage defects. Cellular migration and proliferation was addressed in vitro, and the formation of repair tissue in a nude mouse-based defect model. A cell-free plug made of collagen type I was placed in the center of an incubation plate. Surrounding space was filled with a collagen type I gel (Arthro Kinetics, Esslingen, Germany) seeded with 2 x 10(5) human articular chondrocytes/mL gel. After cultivation for up to 6 weeks in vitro, samples were subject to histological and immunohistochemical staining and gene expression analysis. Subsequently, chondral defects of human osteochondral blocks were treated with the plug, and specimens were cultivated subcutaneously in nude mice for 6 weeks. The repair tissue was evaluated macroscopically, and collagen type II production was investigated immunohistochemically. In vitro, morphology of immigrated cells did not show any differences, as did collagen type II gene expression. After 4 weeks, the plug was homogeneously inhabited. After 6 weeks of cultivation in nude mice, collagen gel plug treatment led to a macroscopically excellent repair tissue. Histological staining revealed a tight bonding, and the collagen gel plug started to be remodeled. We conclude that the novel collagen gel plug device offers an environment favorable for the migration of articular chondrocytes and leads to a good-quality repair tissue in the nude mouse model. The arthroscopic transplantation of a collagen gel plug may be one option in the treatment of focal cartilage defects.

Condensed cellular seeded collagen gel as an improved biomaterial for tissue engineering of articular cartilage.

Three-dimensional autologous chondrocyte implantation based on collagen gel as matrix scaffold has become a clinically applied treatment for focal defects of articular cartilage. However, the low biomechanical properties of collagen gel makes intraoperative handling difficult and creates the risk of early damages to the vulnerable implant. The aim of the study was to create a stabilized form of collagen gel and to evaluate its biomechanical and biochemical properties.Collagen type-I gel was seeded with human articular chondrocytes. 20 samples were subject to condensation which was achieved mechanically by compression and filtration. Control samples were left uncondensed. From both types of gels 10 samples were used for initial biomechanical evaluation by means of unconfined compression and 10 samples were cultivated under standard conditions in vitro. Following cultivation the samples were evaluated by conventional histology and immunohistochemistry. The proliferation rate was calculated and matrix gene expression was quantified by real-time PCR.The biomechanical tests revealed a higher force carrying capacity of the condensed specimens. Strain rate dependency and relaxation was seen in both types of collagen gel representing viscoelastic material properties. Cells embedded within the condensed collagen gel were able to produce extracellular matrix proteins and showed proliferation.Condensed collagen gel represents a mechanically improved type of biomaterial which is suitable for three-dimensional autologous chondrocyte implantation.

Ablation of articular cartilage with an erbium:YAG laser: an ex vivo study using porcine models under real conditions-ablation measurement and histological examination.

BACKGROUND AND OBJECTIVES: The use of an erbium:YAG laser in arthroscopic surgery has the advantage of a precise treatment of soft tissue. Due to the high absorption in water, the laser energy is perfectly matched to smoothing the hydrous, fibrillated articular cartilage surface. In minimal invasive surgery, the workspace is filled with aqueous liquids for enlargement. This appears contrary to the absorption characteristics of erbium:YAG laser radiation in water. The purpose of this study was to evaluate the ablated volume per pulse of cartilage lesions and the potential side effects including thermal damage and tissue necrosis. STUDY DESIGN/MATERIALS AND METHODS: Twenty-four osteochondral specimens of porcine knee joints were irradiated with an Er:YAG laser completely submerged in water, with distances to the cartilage surface of 1, 3 and 5 mm and pulse durations of 75 and 100 microseconds. To keep a constant peak power of approximately 6 kW, pulse energies of 450 and 580 mJ were used at a pulse repetition rate of 15 Hz. After a histological preparation, ablated volumes, depths, and widths of the cuts were investigated. Additionally, laser protocols were correlated with different markers of cartilage tissue damage and apoptosis. RESULTS: Ablation could be observed for every measurement. The influence of the distance showed a statistical significance (P < 0.001) for the volume, depth, and width of the cuts. For the pulse duration, statistical significance (P < 0.001) was found only for the volume and the depth. We observed no loss of proteoglycan or collagen type II. The total cell number, cell morphology, and number of apoptotic cells in an area close to the cutting edge and in a corresponding unaffected area of the same specimens revealed no differences regardless of the applied protocol. CONCLUSION: The use of an Er:YAG laser demonstrates the successful application in liquid environments for cartilage removal without any damage of the surrounding tissue.

Effects of pulsed and sinusoid electromagnetic fields on human chondrocytes cultivated in a collagen matrix.

Although several effects of electromagnetic fields (EMFs) on articular cartilage have been reported in recent studies, the use of EMFs to treat osteoarthritis remains a matter of debate. In an in vitro study, human chondrocytes harvested from osteoarthritic knee joints were released from their surrounding matrix and transferred in defined concentration into a 3D matrix (type-I collagen gel). The cultivation, performed under standard conditions, lasted up to 14 days. During this time, treatment groups were continuously exposed to either sinusoid or pulsed electromagnetic fields (PEMFs). The PEMFs revealed the following characteristics: maximum magnetic flux density of 2 mT, frequency of the bursts of 16.7 Hz with each burst consisting of 20 pulses. Similarly, the sinusoid EMFs also induced a maximum flux density of 2 mT with a frequency of 50 Hz. Control groups consisting of equal number of samples were not exposed to EMF. Immunohistological examinations of formalin-fixed, paraffin-embedded samples revealed positive staining for type-II collagen and proteoglycans in the immediate pericellular region with no differences between the two different treatment groups and the control groups. With increasing cultivation time, both type-II collagen and aggrecan gene expression declined, but no significant differences in gene expression were found between the treatment and control groups. In conclusion, using our in vitro setting, we were unable to detect any effects of pulsed and sinusoidal magnetic fields on human adult osteoarthritic chondrocytes.