The use of fibrin beads for tissue engineering and subsequential transplantation.

New biological technologies such as tissue engineering procedures require the transplantation of functionally active cells within supportive carrier matrices. This paper describes a sequential culture procedure for different types of cells. The technique includes the initial preparation of a mixed alginate-fibrin vehicle that guaranteed an initial cell proliferation and differentiation to establish a stable matrix structure, and the subsequent removal of the alginate component prior to transplantation to circumvent the problem of missing bioresorbability. The resulting biodegradable carrier is mechanically stable and promotes further tissue maturation. Chondrocytes, periosteal-derived cells, as well as nucleus pulposus cells were entrapped in fibrin-alginate beads and in fibrin beads. The results indicate a promising technical approach to create stable transplants for reconstructive surgery of cartilage and bone.

Semiquantitative analysis of types I and III collagen from tendons and ligaments in a rabbit model.

The present study presents a simple and reliable micro-method for the semiquantitative analysis of types I and III collagen in tendons and ligaments in a rabbit model. After pretreatment of the analytical material by homogenization, a double cyanogen bromide cleavage was performed and the peptide fragments were visualized by polyacrylamide gel electrophoresis and silvcr staining. On the basis of this procedure, the method presented here can be used to analyze very small amounts of sample material (less than 10microg) by electrophoresis. The results of the study showed that type I collagen is predominant in the ligaments and the tendons of the knee, e.g., medial and lateral collateral ligaments. anterior and posterior cruciate ligaments. patellar tendon, achilles tendon, and semitendinosus tendon. However, a markedly higher proportion of type III collagen was detected in the ligaments (approximately 10%) than in the tendons (approximately 5%). The ligaments differ markedly from the tendons in biochemical mapping; the ligaments are functionally and metabolically the more active tissue and have a higher adaptation potential.

The influence of transforming growth factor beta1 on mesenchymal cell repair of full-thickness cartilage defects.

To repair full-thickness articular cartilage defects in rabbit knees, we transplanted periosteal cells in a fibrin gel and determined the influence of transforming growth factor beta (TGF-beta) in vitro. Alginate served as a temporary supportive matrix component and was removed prior to transplantation. The defects were analyzed macroscopically, histologically, and electron microscopically, and evaluated with a semi-quantitative score system. Periosteal cell transplants showed a chondrogenic differentiation, which results in the development of embryonic-like cartilage tissue after 4 weeks and complete resurfacing of the patellar groove after 12 weeks. In the control groups, no repair was observed. Under the influence of TGF-beta1 we observed a reduction of the cartilage layer, whereas the osteochondral integration and the zonal architecture were improved. Periosteal cell-beads are stable cartilage transplants and have stiffness and elasticity enough for easy and sufficient transplant fixation. Further investigations are necessary to optimize the application of TGF-beta1 for cartilage repair.

Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits.

The biological bone healing depends on the presence of osteochondral progenitors and their ability for proliferation. Isolated periosteal cells were seeded into biodegradable PGLA polymer fleece or fibrin beads and cultivated for 14 days after prior monolayer culture. On 12 New Zealand white rabbits 8 mm metadiaphyseal ulna defects were created bilaterally and subsequently filled with cell-fibrin beads, with polymers seeded with cells compared to controls with fibrin beads and polymers alone and untreated defects. A semiquantitative grading score was applied for histomorphological and radiological analysis after 28 days. Histologically intense bone formation was observed in both experimental groups with cell transplants only. The histological and radiological scoring was superior for both experimental groups. Control groups revealed only poor healing indices and untreated defects did not heal. The highest histological score was noted in the group with polymer fleeces containing periosteal cells. Applying the radiographic score system we determined a significant difference between experimental groups and controls without cells. The radiographic and histological scores for both experimental groups containing periosteal cells differed not significantly. The results strongly encourage the approach of the transplantation of pluripotent mesenchymal cells within a suitable carrier structure for the reconstruction of critical size bone defects.

Joint cartilage repair with transplantation of embryonic chondrocytes embedded in collagen-fibrin matrices.

OBJECTIVE: The objective of this study was to assess the feasibility of transplanting embryonic chondrogenic cells within a collagen-fibrin substrate for the reconstitution of full-thickness cartilage defects in chicken knee joints. METHODS: Full-thickness cartilage defects were created mechanically on the weight-bearing surface of the tibial condyle in 45 adult chickens and subsequently filled with chondrocytes embedded in a chondrocyte-collagen-fibrin gel. The transplants were compared to untreated defects and collagen-fibrin transplants without cells. The results were analyzed using histochemical and morphometrical methods after 3, 12 and 24 weeks. A semiquantitative histological grading system was applied to evaluate the transplant integration and the newly formed cartilage architecture. RESULTS: Chondrocyte-gel grafts developed to hyaline-like cartilage without any granulation tissue in the interface after 3 weeks. After 12 weeks the defects in the experimental group were filled completely with hyaline cartilage. The defects in the control groups in all cases healed with fibrous repair tissue. CONCLUSION: Fibrin-collagen gel allowed stable graft fixation and provided an adequate microenvironment for embryonic chondrocytes to generate hyaline-like neocartilage in a full-thickness cartilage defect.

Cultivation of porcine cells from the nucleus pulposus in a fibrin/hyaluronic acid matrix.

Use of multiplied viable cells from the nucleus pulposus in altered discs, following in vitro cultivation, may be a promising therapy for degenerative disc disease. Up till now, alginate has been used as a three-dimensional cell carrier to cultivate nucleus pulposus cells. However, the biocompatibility of the alginate, which depends on the composition and purity of alginate materials used, has not been considered for in vivo application so far. In this study, we cultured porcine cells from the nucleus pulposus in a mixture of fibrin and hyaluronic acid (HA). The DNA content and proteoglycan synthesis were compared to those measured in an alginate matrix. Although the increase in DNA content was 2.5-fold higher in the alginate culture after 3 weeks, the proteoglycan synthesis in relation to the DNA content was significantly higher in the fibrin/HA matrix. We found that the fibrin/hyaluronic acid matrix can be used as a substrate for in vitro cultivation.

Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants.

For cartilage engineering a variety of biomaterials were applied for 3-dimensional chondrocyte embedding and transplantation. In order to find a suitable carrier for the in vitro culture of chondrocytes and the subsequent preparation of cartilage transplants we investigated the feasibility of a combination of the well-established matrices fibrin and alginate. In this work human articular chondrocytes were embedded and cultured either in alginate, a mixture of alginate and fibrin, or in a fibrin gel after the extraction of the alginate component (porous fibrin gel) over a period of 30 days. Histomorphological analysis, electron microscopy, and immunohistochemistry were performed to evaluate the phenotypic changes of the chondrocytes, as well as the quality of the newly formed cartilaginous matrix. Our experiments showed that a mixture of 0.6% alginate with 4.5% fibrin promoted sufficient chondrocyte proliferation and differentiation, resulting in the formation of a specific cartilage matrix. Alginate served as a temporary supportive matrix component during in vitro culture and can be easily removed prior to transplantation. The presented tissue engineering method on the basis of a mixed alginate-fibrin carrier offers the opportunity to create stable cartilage transplants for reconstructive surgery.

Retention of hyaluronic acid in alginate beads: aspects for in vitro cartilage engineering.

Alginate has been used successfully for three-dimensional chondrocyte cultures and may be important for cartilage transplant formation. However, alginate is not a natural component of the cartilage matrix. The aim of this study was (a) to supplement alginate with the extracellular matrix component hyaluronic acid; and (b) to analyze the hyaluronic acid retention in different alginate gels. Hyaluronan is assumed to improve proteoglycan retention and may be important for in vitro matrix formation, tissue turgor, and biomechanical quality. Alginate and hyaluronan were mixed with chondrocytes and polymerized as were alginate, hyaluronan, and fibrinogen. [3H]hyaluronan was used to quantitate the leakage of hyaluronan from the gel beads. After 28 days in culture, 1.2% alginate beads supplemented with 0.26% hyaluronan contained only 9% of the initial amount of hyaluronan whereas 2.4% alginate beads still contained about 55% of the initial 0.22% hyaluronan. Release of hyaluronan from the beads was significantly lower if the beads additionally contained fibrin. Alginate beads supplemented with hyaluronan or fibrin showed increased chondrocyte proliferation compared to controls. Supplemented hyaluronan greatly diffuses out of alginate gels of lower densities. It must be assumed also that most of the hyaluronan newly synthesized by chondrocytes in these cells diffuses into the surrounding culture medium. The in vitro development of a sufficiently hygroscopic cartilage ground substance therefore may be very limited. Sufficient hyaluronic acid retention can be achieved in alginate gels with concentrations above 1.2% or by addition of fibrin.

Elimination of tritium-labelled hyaluronic acid from normal and osteoarthritic rabbit knee joints.

The half-life of [3H]hyaluronic acid in rabbit knee joints was estimated using two methods: (i) by following the [3H]hyaluronan content of the synovial fluid after intra-articular injection and (ii) by following the 3H2O radioactivity of plasma after intra-articular injection of [3H]hyaluronan. For normal rabbits we obtained a half-life of 15.8 hours (method I) and 17.5 +/- 1.0 hours (mean +/- SEM, method II), respectively. The second method was used to estimate the kinetics of the hyaluronan elimination from normal, sham-operated, as well as from osteoarthritic rabbit knee joints (Colombo model of osteoarthritis). Four weeks after injury, during the developing phase of osteoarthritis, the half-life of hyaluronan rose significantly to 23.5 +/- 2.1 hours and returned to normal levels (17.4 +/- 2.7 hours) 12 weeks after the operation (osteoarthritis developed). At the stage of developed osteoarthritis, the clearance rates were considerably higher than in normal rabbits.

[Experimental studies of mechanically-induced articular cartilage defects following implantation of allogeneic embryonal chondrocytes in a collagen-fibrin gel in chickens]. / Experimentelle Untersuchungen mechanisch induzierter Gelenkknorpeldefekte nach Implantation allogener embryonaler Chondrozyten in einem Kollagen-Fibrin-Gel beim Huhn.

Full thickness defects (diameter 1,7 mm; depth 2,5 mm) were created mechanically in articular cartilage and subchondral bone of the condyles of tibiotarsal joints of 9-month old chickens. This full-thickness defects were repaired with cultured allogenic embryonic chick epiphyseal chondrocytes from the tibiae and femura of 10-days-old chicken embryos. The cells were embedded in a collagen-fibrinogen-matrix. Controls were similarly operated, but received either no treatment or implants the delivery substance only. Healing of the defects was observed macroscopically, histologically, histochemically and histomorphometrically after 3, 12 and 24 weeks. This graft was successfully transplanted in mechanically induced defects in 80%. The resulting hyaline cartilage was structurally reorganized according to the host pattern and under the influence of environmental conditions. The articular zone preserved it's cartilaginous phenotype, whereas the subchondral regions were transformed into bone. 12 weeks after the operation the defects in the experimental group were completely filled. In all instances in this group, there was an initial extreme increase of mitotic rate and cell number. After 24 weeks normal and subnormal values were founded. The defects in the control groups healed with fibrocartilage. Our results showed, that only the defects in the experimental group were completely filled with reparative hyaline cartilage tissue. In the present study the mixture of cultured allogenic embryonic chondrocytes and a collagen-fibrinogen-matrix was used successfully as a transplant for repairing defects in articular cartilage of chickens. Thus allogenic transplantation of cultured embryonal chondrocytes appears to be one of the most promising methods for the restoration of articular cartilage.

[Synovial volume of healthy and arthrotic human knee joints]. / Das Synovia-Volumen gesunder und arthrotischer menschlicher Kniegelenke.

The volume of synovial fluid in human knee joints was determined by the dilution method through the intra-articular injection of 35 ml 0.5% hydroxyethyl starch in saline. In 15 control knees the mean synovial volume was 6.7 +/- 2.3 ml (mean +/- SD). In patients with latent gonarthrosis we found a mean intra-articular volume of 13.6 +/- 7.4 ml (n = 21) and in activated gonarthrosis 24.2 +/- 16.3 ml (n = 34). All 3 groups differ significantly. After entire aspiration of the effusion, we found yet 13.4 +/- 4.4 ml (n = 32) synovial fluid in the knee joint. Using the dilution method, we obtained a punctate from each knee joint. The punctates can be used for biochemical investigations. Since the dilution factors of the punctates are known, the concentrations of HWM parameters in the original synovial fluid can be calculated.

[Analysis of hyaluronic acid in punctates in biochemical functional tests of joints]. / Bestimmung von Hyaluronsäure in Punktaten des biochemischen Gelenkfunktionstests.

The punctates of the biochemical joint function test contain hydroxyethyl starch which disturbs the assay of uronic acid and glucosamine for the estimation of hyaluronate. Therefore hyaluronate (0.03-0.75 g/l) was estimated by precipitation with Alcian blue on cellulose acetate membranes and measuring of the absorbance at 678 nm after dissolution in aqueous SDS (75 g/l). The gel-filtration HPLC separates the high-molecular-mass hyaluronate from low-molecular-mass impurities of biological samples. By comparing with a standard curve (0.5-2.5 g/l hyaluronate) the concentration can be determined. The results obtained by the two methods were significantly correlated (p less than 0.05). The Alcian blue technique is useful for rapid screening, the HPLC technique to confirm these values.

[Plasminogen activator activity of the synovial fluid as an indicator of activation phenomena in degenerative joint diseases]. / Die Plasminogenaktivator-Aktivität der Synovialflüssigkeit als Indikator für Aktivierungsphänomene bei degenerativen Gelenkerkrankungen.

Activity of plasminogen activator in synovial fluids of patients with osteoarthritis was determined by a radial diffusion assay. Synovial fluid from human knee joints was obtained by joint lavage with 35 ml physiological saline containing 0.5% hydroxyethyl starch. Synovial fluids from contralateral, healthy knee joints served as controls. The activity of plasminogen activator in synovial fluid from activated (painful) osteoarthritis is significantly higher than in cases of latent osteoarthritis, whereas the activity of controls is significantly lower as in latent osteoarthritis. The findings are helpful for the discovery of patients with enhanced plasminogen activator levels in synovia which should be treated therapeutically.

The influence of mechanical loading on the knee-joint. Animal-experimental examinations.

A physiological maximal loading of the knee-joint of sheep is simulated by a 4-hour peak overloading of 24.5 N and a frequency of 42 impulses per minute. The reactions of articular cartilage, synovia and periarticular muscular tissue to the load were examined histologically, histochemically and biochemically. 20 hours after the loading we could observe a leucocytic influx into the joint and a metabolic hyperactivity in the examined tissue. The changes found during this acute phase are reversible as 180 days after peak overloading the parameters detected to be changed had returned to normal again.

[Monoiodoacetic acid-induced arthropathy of the rabbit knee--a contribution to the pathogenesis of arthrosis]. / Die Monojodazetat-induzierte Arthropathie am Kaninchenknie--Ein Beitrag zur Pathogenese der Arthrose.

A chemically-induced joint degeneration was produced by an one-time intraarticular injection of the glycolysis inhibitor monoiodic acetate into the rabbit knee joint (32 joints of 16 rabbits + 4 control animals). The investigation is founded on Kalbhen's animal model of osteoarthrosis and on Salter's and Moskowitz' experiments. Histomorphological examinations were done in intervals of 1 day to 12 weeks. The results raise several open questions in experimental as well as clinical aspects. On that account these results were discussed pathogenetically in the view of the own pathophysiological and biocybernetical conception of osteoarthrosis development.

[Inhibition of prednisolone osteopathy by diphosphonate (EHDP) in an animal experiment]. / Inhibierung der Prednisolon-Osteopathie durch Diphosphonat (EHDP) im Tierexperiment.

The application of a low dose of prednisolone (0.4 mg per kg body weigh and day) in young hamsters leads to a loss of bone mass. This effect is inhibited by the administration of the diphosphonate EHDP (6.2 mg per kg body weight and day). The total volume of the limb bones is increased. The application of a high dose of prednisolone (40 mg per kg body weight and day) induces a severe osteopathy. These pathological bone changes couldn't be prevented by EHDP, but there was a significant shift of the investigated parameters into the direction of normalization.

Low molecular mass trypsin inhibitors in normal and osteoarthritic human articular cartilage.

A modified radial diffusion assay was used for the direct semiquantitative determination of low molecular mass trypsin inhibitors in small samples of human cartilage. The low molecular mass trypsin inhibitor in articular cartilage of normal human femoral heads is not distributed evenly but occurs in areas of low, medium and high content. The weight-bearing area of the femoral head belongs among the regions with low inhibitor content. The results obtained with osteoarthritic femoral heads showed that the inhibitor content in osteoarthritic cartilage is significantly lower than that in normal articular cartilage (p less than 0.1%).

[Risk of hemorrhage in the knee joint following intraarticular injections]. / Das Blutungsrisiko in das Kniegelenk nach intraartikulären Injektionen.

The relative and absolute blood contents in the knee joint after intraarticular injections were determined using a biochemical joint function test. Normally, only minimal amounts of blood leak into the joint cavity, but in a few cases greater quantities were found, unnoticed at the time of injection. Intraarticular injections should be done only by a specialist.

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay.

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.

[Detection of low molecular weight trypsin inhibitors in small samples of cartilage, cartilage extracts and synovial fluid by gel diffusion]. / Nachweis von niedermolekularen Trypsininhibitoren in kleinen Knorpelproben, in Knorpelextrakten und Gelenkflüssigkeit mittels Geldiffusion.

The radial diffusion assay is a very useful method for detection of low amounts of proteinase inhibitors in biological materials. The determination of low molecular weight (LMW) inhibitors in the presence of high molecular weight inhibitors is possible by the combination of radial diffusion and ultrafiltration. Using this method LMW trypsin inhibitors could be demonstrated in human articular cartilage, but not in human synovial fluid. In cow, pig and sheep a LMW trypsin inhibitor could be found in both the articular cartilage and in the synovial fluid. On the other hand, a LMW trypsin inhibitor could not be found neither in the canine cartilage nor in the canine synovial fluid. The method allows also the direct determination of LMW trypsin inhibitors in cartilage extracts in the presence of 4 M guanidinium hydrochloride or 6 M urea. Therefore, the method is recommended for direct determination of LMW inhibitors by column chromatographic separations of inhibitors.