Klaus Lindenhayn

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.

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.

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.

In vitro-cultivation of human periosteum derived cells in bioresorbable polymer-TCP-composites.

Bone replacement materials for reconstruction of bone defects must be biocompatible and biodegradable and must have osteoconductive or even osteogenic potential. Ideally, their shape should also be adaptable to the defect and they should possess long-term adaptability to the biomechanical situation at the implantation site. Human mesenchymal stem cells of the cambium layer of the periosteum were cultivated, placed in a fibrin suspension on a preformed carrier structure (PGLA polymer + beta-TCP), and cultivated under conditions of osteogenic differentiation. After 10, 20, 30, and 40 days, histological examination was performed, alkaline phosphatase activity and levels of osteocalcin, DNA, and collagen were determined, and the influence of addition of TGF-beta1 at a concentration of 5 ng/ml to the culture medium was investigated. Demonstration of bone-specific marker proteins indicated that the in vitro combination of mesenchymal stem cells, PGLA polymer, beta-TCP, and fibrin resulted in de-novo synthesis of human preosseous tissue, while addition of TGF-beta1 resulted in greater new bone formation with significantly higher concentrations of marker proteins. Histological examination showed the presence of newly formed bone at the surface of the implant. As compared with the use of structured TCP or hydroxyapatite implants as in earlier works, use of a combination of autologous cell material, PGLA polymer, and beta-TCP results in a malleable, vital implant that is adaptable to the bone defect. This combination thus may represent a new option for the treatment of bone defects.

The influence of transforming growth factor β1 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.

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.

Human intervertebral disc cell culture for disc disorders.

Repair of degenerated intervertebral discs by engineered tissue is a clinical challenge in spinal surgery. Prerequisites are cultivation of intervertebral disc cells and determination of their biologic properties. The influence of disc damage in different spinal disorders on the outcome of disc cell cultures has not been discussed previously. This study showed the feasibility of cultivation of cells from damaged human intervertebral discs and the dependence of cellular culture properties on the underlying disc disorder. Human intervertebral disc cells were isolated from disc tissue obtained during surgical procedures for scoliosis, osteochondrosis, and disc herniation. After proliferation in monolayer culture, cells were embedded in a mixed matrix composed of fibrin and hyaluronic acid. Deoxyribonucleic acid content, hydroxyproline content, and proteoglycan synthesis were determined on Days 7, 14, and 21. In a three-dimensional environment only cells obtained from scoliotic and osteochondrotic discs showed significant deoxyribonucleic acid and proteoglycan synthesis. However, hydroxyproline content increased only in cells from scoliotic discs. The results of this study show that the formation of extracellular matrix components under three-dimensional culture conditions is dependent on the nature of intervertebral disc damage of the tissue processed.

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%).

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.

Chondrocytes and Fibrin Glue

Growth, morphology and differentiation of cells in vitro depend on the composition of the cellular environment. Therefore, a variety of matrices have been studied to provide optimal conditions for chondrocyte culture. The introduction of alginate as a supporting matrix into cell culture was a blessing for cells that need to be cultured in a three-dimensional matrix. Alginate, a linear polysaccharide, is a co-polymer of two uronic acids: L-guluronic and D-mannuronic acid linked by 1 to 4 glycosidic bonds. The polymer undergoes instantaneous ionotrophic gelation in the presence of divalent cations, e.g. calcium. Compared to other gel matrices, alginate beads offer a simple way of dissolution to the monomer subunits using chelating agents, such as sodium citrate, releasing the entrapped cells together with their synthesized high molecular matrix.