Risultati finali
Results description:
As a first step, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (Hyaff-11®), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGF3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution.
In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.
The second step consisted in demonstrating that differences in the local composition of bi-zonal fibrocartilaginous tissues resulted in different local biomechanical properties in compression and tension. Bovine articular chondrocytes were loaded into hyaluronan-based meshes (HYAFF®-11) and cultured for 4 weeks in mixed flask, a Rotary Cell Culture System (RCCS), or statically.
Resulting tissues were assessed histologically, immunohistochemically, by scanning electron microscopy and mechanically in different regions. Local mechanical analyses in compression and tension were performed respectively by indentation-type scanning force microscopy and by tensile tests on punched out concentric rings.
Tissues cultured in mixed flask or RCCS displayed an outer region positively stained for versican and type I collagen, and an inner region positively stained for glycosaminoglycans and types I and II collagen. The outer fibrocartilaginous capsule included bundles (up to 2m diameter) of collagen fibers and was stiffer in tension (up to 3.6-fold higher elastic modulus), whereas the inner region was stiffer in compression (up to 3.8-fold higher elastic modulus). Instead, molecule distribution and mechanical properties were similar in the outer and inner regions of statically grown tissues.
In conclusion, exposure of articular chondrocyte-based constructs to hydrodynamic flow generated tissues with locally different composition and mechanical properties, resembling some aspects of the complex structure and function of the outer and inner zones of native meniscus.
Dissemination of the results:
The results described above have been published into an international peer-reviewed journal, under the following titles:
Bi-zonal cartilaginous tissues engineered in a rotary cell culture system.
A.Marsano, D.Wendt, TM. Quinn, TJ. Sims, J. Farhadi, M.Jakob, M. Heberer I. Martin. Biorheology (2006) 43, 553-60.
Use of hydrodynamic forces to engineer cartilaginous tissues resembling the non-uniform structure and function of meniscus.
A.Marsano, D.Wendt, R. Raiteri, R. Gottardi, M. Stolz, D. Wirz, AU. Daniels, D. Salter, M.Jakob, T. Quinn, I. Martin. Biomaterials (2006), 27: 5927-5934.
Use of the results:
The culture of meniscus shaped constructs was performed in mixed flasks in order to engineer meniscus grafts based on autologous chondrocytes for implantation in a sheep model. The medical university of Wien, Austria, and the Istituto Ortopedico Rizzoli, Bologna, Italy, have tested the capacity of engineered grafts to repair a critical meniscus defect.
Expected benefits:
A translation of these results to the clinical treatment of meniscus lesions in human beings is expected. The grafts engineered using the identified prototype bioreactor should be beneficial for the treatment of menisectomies.
Design and preparation of an innovative meniscus device and scaffolds based on the natural structure of the natural meniscus.
The device was prepared by using the lamination technique to obtain a composite structure with appropriate mechanical and functional properties. Blends of Hyaff11 and PCL was used as matrix while PLA and or PGA fibres as reinforcement.
Assessment of the mechanical an processing properties of the device.
The complete characterization included the analysis of the mechanical properties (static compression dynamic and creep), water absorption. This properties were also analysed as function of sterilization methods (Gamma ray and ETO and after implantation in vivo).
Processing has been optimised by the definition of systematic organisation of the different phases of the preparation by using microtomography imaging technique and mould design by 3D systems.
Results description:
Objective. To identify an appropriate cell source for the generation of meniscus substitutes, among those which would be available by arthroscopy of injured knee joints.
Methods. Human inner meniscus cells, fat pad cells, synovial membrane cells and articular chondrocytes were expanded with or without specific growth factors (Transforming Growth Factor-beta1, TGF-?1, Fibroblast Growth Factor-2, FGF-2 and Platelet-Derived Growth Factor bb, PDGF-bb, TFP) and then induced to form 3D cartilaginous tissues in pellet cultures, or using a hyaluronan-based scaffold (Hyaff®-11), in culture or in nude mice. Human native menisci were assessed as reference.
Results. Cell expansion with TFP enhanced glycosaminoglycan (GAG) deposition by all cell types (up to 4.1-fold) and mRNA expression of collagen type II by fat pad and synovial membrane cells (up to 472-fold) following pellet culture. In all models, tissues generated by articular chondrocytes contained the highest fractions of GAG (up to 1.9% of wet weight) and were positively stained for collagen type II (specific of the inner avascular region of meniscus), type IV (mainly present in the outer vascularised region of meniscus) and types I, III and VI (common to both meniscus regions). Instead, inner meniscus, fat pad and synovial membrane cells developed tissues containing negligible GAG and no detectable collagen type II protein. Tissues generated by articular chondrocytes remained biochemically and phenotypically stable upon ectopic implantation.
Conclusions. Under our experimental conditions, only articular chondrocytes generated tissues containing relevant amounts of GAG and with cell phenotypes compatible with those of the inner and outer meniscus regions. Instead, the other investigated cell sources formed tissues resembling only the outer region of meniscus. It remains to be determined whether grafts based on articular chondrocytes will have the ability to reach the complex spatial organization typical of meniscus tissue.
Dissemination of the results:
The results described above have been published into an international peer-reviewed journal, under the following title: Differential cartilaginous tissue formation by human synovial membrane, fat pad, meniscus cells and articular chondrocytes.
A.Marsano, SJ. Millward-Sadler, DM. Salter, A. Adesida, T. Hardingham., E. Tognana, E. Kon, C. Chiari-Grisar., S. Nehrer, M.Jakob, I. Martin. Osteoarthritis and Cartilage (2007) 15, 48-58.
Use of the results:
The selected cell source, articular chondrocytes, has been used for in vivo studies of meniscus repair in a sheep model. The medical university of Wien, Austria, and the Istituto Ortopedico Rizzoli, Bologna, Italy, have tested the capacity of sheep autologous articular chondrocytes to repair a critical meniscus defect.
Expected benefits:
A translation of these results to the clinical treatment of meniscus lesions in human beings is expected. The grafts engineered using the identified cell source should be beneficial for the treatment of menisectomies.
As a first step a pilot study in 8 sheep was performed. In this study with an evaluation point after 6 weeks it was shown that the surgical handling of the biomaterial was feasible, that the implants stayed in place and were integrated to the capsule. Tissue formation, vascularization and cellular infiltration (foreign body cells, fibroblasts) were present. No animals were lost, no signs of allergic or inflammatory responses to the biomaterial were noticed.
In the following large scale study a total of 64 sheep were included. 26 sheep were evaluated after 4 and 38 sheep after 12 months respectively. Two different surgical techniques were developed and compared. In one model the implant was sutured to the joint capsule and the horns, in the second model additional transosseous sutures were used.
Two types of implants were used one group received cellfree scaffolds, the other group received scaffolds seeded with articular chondrocytes, which had been harvested from the contralateral stifle joints, expanded in monolayer culture and dynamically seeded on the scaffolds. The evaluation was done on a macrosopically using specific scores. The Gross Assessment of Implant Score, which had been designed specially for this study, includes nine different categories implant integration, implant position, horn position, implant shape, presence of tears, implant surface, tissue quality and condition of the synovia. Joint status was assessed using the Gross Assessment of Joint changes Score, which scores twelve different areas of the joint, including the femoral and tibial cartilage, the patella, its femoral groove, the menisci and the femoral junction.
The implants were evaluated histologically, blocks of meniscal implant were assessed for the presence of residual scaffold, cellularity, foreign body response, inflammatory cell (lymphocytes, plasma cells and neutrophils) infiltrate, blood vessel in-growth, fibrous and cartilaginous matrix and integration of the implant with the joint capsule.
A high incidence of tears, clefts and implant extrusions, especially in cases with a trans-tibial rigid fixation of the horns was detected indicating that the mechanical properties of the scaffold were not sufficient in the current model. As such the biomechanical properties of the material need to be improved for future experimentation, possibly by insertion of a stronger resorbable net inside the scaffold. The degree of damage seen in the meniscal implants appear to be related to the implant fixation technique used. Worse results were observed in cases where trans-tibial, rigid fixation, of the horns was utilized It is likely that too rigid horn fixation in this weight-bearing, large animal model, resulted in overly high mechanical stresses on the implant leading to a higher incidence of graft tears.
The implants were well tolerated immunologically by the sheep with no evidence macroscopically or microscopically of a significant synovitis or lympho-plasmacytic infiltrate. There was however a prominent foreign body giant cell response to the implant, presumably part of the physiological resorption process, which was similar in cell-seeded and cell-free constructs.
Histological analysis of the implant material confirmed the gross findings of tissue formation within and upon the implant with bonding to the capsule. The tissue in growth consisted of a fibro-vascular connective tissue with a foreign body response to the implant material. Collagen appeared as a fine fibrillar network without orientation. Blood vessels were present throughout the implants without suggestion of formation of vascular and avascular areas as might be expected in normal menisci. The impact of the giant cell reaction is difficult to estimate. Neither the synovial biopsies nor the smears revealed acute inflammatory cells.
Although no significant difference was shown between cell-seeded and cell-free implants macroscopically, histological analysis did demonstrate deposition of a cartilaginous matrix only in cell-seeded implants, which became more evident after 12 months compared to the 4 months results. As the matrix is seen predominantly in the cell seeded constructs, it is likely that the implanted cells are involved in its production. Moreover, the distribution of the cartilaginous matrix deposition on the edges and the tip of the implant are directly related to the distribution of cells and matrix in the scaffold at the time of grafting.
The results support the idea that the HA-PCL scaffold has the potential for total meniscal substitution as it is tolerated immunologically and induces tissue in-growth. There are however some limitations with regards to the mechanical properties of the scaffold. Less rigid fixation technique of total meniscus implant is considered preferable in sheep model and recommended for further animal studies. Seeding of the scaffolds with autologous articular chondrocytes provides some benefit with more fibrocartilaginous tissue.
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