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.

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.

Tissue engineered cartilage repair using cryopreserved and noncryopreserved chondrocytes.

The objective of this study was to reconstruct full thickness cartilage defects in rabbit knees with in vitro engineered cartilage tissue based on noncryopreserved and cryopreserved chondrocytes in polymer fleece scaffolds. Osteochondral defects in rabbits were filled with polymer cylinders with noncryopreserved or cryopreserved allogeneic chondrocytes and compared with empty defects and defects filled with polymers alone. The defects were evaluated macroscopically and histologically 4 and 12 weeks after surgery. Transplant samples were graded using a semiquantitative score system. Successful healing was defined as complete integration of a hyalinelike and structurally intact cartilage into the defect and occurred in 71% of the group with noncryopreserved chondrocytes after 4 weeks and 100% of the rabbit knees after 12 weeks, whereas hyalinelike cartilage was seen in 71% of the group with cryopreserved chondrocytes after 4 weeks, and in 85% after 12 weeks. No newly formed cancellous bone was present in the subchondral bone. In the control groups, no cartilagelike tissue was seen. Transplantation of chondrocytes in polymer fleece constructs is a suitable approach for joint cartilage repair. Noncryopreserved chondrocytes are preferred to cryopreserved chondrocytes because of their regenerative potential. In vitro engineered cartilage offers broad opportunities for optimization of cartilage transplantation based on the controlled use of morphogenic and biologically active factors such as transforming growth factor-beta and bone morphogenetic proteins.

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.

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.