[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
While all specimens revealed a homogeneous cellular distribution, ECM production, cellular organisation and mechanical properties were enhanced by continuous BMP-7 release.
Full-text · Article · Jun 2013 · Journal of Biomaterials Applications
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Feb 2012 · Annals of anatomy = Anatomischer Anzeiger: official organ of the Anatomische Gesellschaft
[Show abstract][Hide abstract]ABSTRACT: Cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed by treatment with cell-free collagen type-I gel plugs of three different sizes.
In 6 adult Goettinger minipigs, three full-thickness chondral defects were created in the trochlear groove of one knee of the hind leg. These defects were treated with a cell-free collagen type-I gel plug of 8, 10, or 12 mm diameter. All animals were allowed unlimited weight bearing. After 1 year, the animals were killed. Immediately after recovery, a non-destructive biomechanical testing was performed. The repair tissue quality was evaluated immunohistologically, collagen type-II protein was quantified, and a semiquantitative score (O'Driscoll score) was calculated.
After 1 year, a high number of cells migrated into the initially cell-free collagen gel plugs and a hyaline-like repair tissue had been created. The O'Driscoll scores were: 8 mm, 21.2 (SD, 2.8); 10 mm, 21.5 (SD, 1.6); and 12 mm, 22.3 (SD, 1.0). The determination of the e-modulus, creep and relaxation revealed that mechanical properties of the two smaller defects were closer to unaffected hyaline cartilage.
As cell-free collagen type-I gel plugs of all three different sizes created hyaline-like repair tissue, this system seems suitable for the treatment of even larger defects.
No preview · Article · Dec 2011 · Knee Surgery Sports Traumatology Arthroscopy
[Show abstract][Hide abstract]ABSTRACT: 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-1β, 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.
No preview · Article · Nov 2011 · Journal of Biomaterials Applications
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Jul 2011 · Medical Engineering & Physics
[Show abstract][Hide abstract]ABSTRACT: For more than 30 years the potential effects of electrical stimulation on bone healing have been investigated. Up to now this therapy is controversial and not established as a standardised treatment option. This systematic review and metaanalysis focuses on the potential effects of electromagnetic fields and high-frequency electric fields on bony healing. In a systematic literature search randomised clinical trials were identified and analysed. Those studies with the primary endpoint "rate of bony healing" were combined in a metaanalysis which was performed with the "random effects" model. We found a total of 14 randomised clinical trials. These studies included a total of 915 patients. The majority of these studies used pulsed electromagnetic fields (PEMF). Out of the 14 studies nine were suitable for the metaanalysis which revealed a cumulative odds ratio of 3.5 and a 95 % confidence interval of 1.94-6.3. When performing a subgroup analysis a statistically significant result could not be confirmed by the studies with a higher methodological quality. In view of the heterogeneous physical parameters with different frequencies, time course, flux densities and in view of the methodological deficits, a general conclusion seems difficult. Recommendations or standards of therapy are so far not available.
No preview · Article · Jun 2011 · Zeitschrift fur Orthopadie und Unfallchirurgie
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
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.
No preview · Article · Mar 2011 · Knee Surgery Sports Traumatology Arthroscopy
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
Although the effects observed are small, mechanical preconditioning has demonstrated its potential to modulate biological properties of collagen hydrogels seeded with human chondrocytes.
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Dec 2010 · Journal of Biomaterials Applications
[Show abstract][Hide abstract]ABSTRACT: The treatment of cartilage defects remains a major problem in orthopaedics. With regard to cartilage tissue engineering, the reimplantation of pre-cultivated chondrocytes in the form of a chondrocyte graft is a promising alternative to conventional methods. Clinical practice requires this MACT procedure (matrix-associated autologous chondrocyte transplantation) to produce a biocompatible replacement tissue with adequate mechanical properties. Mechanical stimulation has the capacity to improve the quality of these cell-seeded biomaterials. By altering chondrocytes' cellular activities, the biological and biomechanical properties of cartilage replacement tissue can be modulated. Different systems are used for this purpose, e.g. shear, perfusion, hydrostatic pressure or compression. The mechanisms, biological effects, chances and problems of the techniques are presented and assessed. Among the stimulating techniques considered are systems that apply indirect and direct shear forces such as spinner flasks, rotating-wall bioreactors, direct tissue shear and perfusion culture systems. The application of hydrostatic pressure or compression may be brought about by either static or dynamic loading systems. Compressive loading is considered in the light of both its short- and long-term effects; additionally two exemplified systems are discussed in detail. However, despite promising approaches and seemingly favourable tissue characteristics, the in vitro culturing of functional cartilage replacement tissue with cartilage-like mechanical and biological characteristics still remains elusive. Furthermore, controlling, monitoring and regulating culturing conditions are general biotechnological requirements of a standardised in vitro cultivation. Among these, different aspects such as aseptic operation, media supplementation, nutrient and gas exchange, temperature and humidity control are considered.
No preview · Article · Oct 2010 · Zeitschrift fur Orthopadie und Unfallchirurgie
[Show abstract][Hide abstract]ABSTRACT: Here we investigate the effect of millicurrent treatment on human chondrocytes cultivated in a collagen gel matrix and on human osteochondral explants.
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.
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.
We conclude that millicurrent stimulation of human osteoarthritic chondrocytes cultivated in a 3 D collagen gel and of osteochondral explants directly influences cell metabolism.
Full-text · Article · Aug 2010 · BMC Complementary and Alternative Medicine
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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).
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.
No preview · Article · Jun 2010 · The International journal of artificial organs
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Jan 2010 · Artificial Organs
[Show abstract][Hide abstract]ABSTRACT: 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.
No preview · Article · Jan 2010 · Bio-medical materials and engineering
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
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.
Full-text · Article · Jan 2010 · The International journal of artificial organs
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
The use of an Er:YAG laser demonstrates the successful application in liquid environments for cartilage removal without any damage of the surrounding tissue.
No preview · Article · Nov 2009 · Lasers in Surgery and Medicine