Articular cartilage, also called shock absorber, is a complex living soft tissue that covers gliding surfaces of joint and enables the joint to withstand weight bearing from human. Since there is no direct blood supply in the articular cartilage, it is generally hard to be repaired itself when it is injured. Although there have been several approaches to the repair of injured articular cartilage, current medical treatment is not able to give patients satisfactory treatment. Ultrasound has been used as one of physical therapy tools. Recently, there have been frequent reports that ultrasound has beneficial effect on the repair of bone fracture and soft tissue healing including articular cartilage. Although there have been appreciation of beneficial effect of ultrasound therapeutically, its mechanism is not fully understood and under investigation. From literature review, several researches tried to find optimal conditions of ultrasound such as intensity, frequency and duration on the repair of articular cartilage and it was reported that more effective ultrasound dose was found. However, different reports have different optimized ultrasound dose. It might be due to the variations of the type of ultrasound wave, intensity, frequency and duration as well as the different condition of experimental samples. Therefore, low intensity pulsed ultrasound (LIPUS) was investigated on the repair of articular cartilage and chondrocyte migration from this study. Also, optimal conditions of LIPUS dose on chondrocyte migration were investigated for the repair of articular cartilage.

Extrinsic repair techniques for the treatment of acute chondral injuries continue to yield suboptimal repair. The inability of these techniques to produce hyaline cartilage underscores the limitations in our understanding of basic chondrocyte biology. Conversely, intrinsic repair tissue has not been extensively studied despite the fact that it can yield hyaline-like cartilage and is commonly observed in osteoarthritis. Attempts at extrinsic repair could therefore benefit from a better understanding of the successes and failures inherent in the intrinsic repair process. Chondrocyte culture has typically been conducted under non-physiologic conditions whereby chondrocytes readily dedifferentiate. Consequently, much of the knowledge gained about chondrocytes has been misleading thus hindering advancements in chondrocyte biology and attempts at extrinsic articular cartilage (AC) repair. Hypoxic culture conditions, which are beneficial towards the preservation of the chondrocyte phenotype, remain insufficient due to elevated collagen I gene expression. As such, an appropriate model system does not yet exist in which to study physiologically-relevant chondrocyte biology. The presence and prevalence of collagen I in both degenerate and de novo osteoartritic tissue was examined immunohistochemically. Collagen I deposition during osteoarthritic progression was compared against IHC staining for collagen II and aggrecan. A novel model system was also evaluated for chondrocytic phenotype retention. To this end, hypoxic, high-density-monolayer-chondrocyte (HDMC) cultures were compared to freshly isolated chondrocytes for their ability to maintain a chondrocytic extracellular matrix (ECM) gene expression profile. HDMC culture conditions prevented the severe loss of the phenotype typically associated with conventional monolayer culture. Moreover, prolonged HDMC culture resulted in the formation of a complex ECM and a marked suppression of collagen I expression. This study also demonstrated that collagen I deposition occurs in osteoarthritic AC at the onset of structural damage and increases in response to increasing structural damage. Collagen I deposition was also found in different types of de novo cartilage associated with osteoarthritic joints and suggests that it plays an important role in intrinsic cartilage repair. Taken together, this work demonstrates that collagen I is a common feature in the ECM of structurally immature and structurally damaged AC and hence may play a role in tissue stabilization. / Experimental Surgery

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名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成6年3月25日 服部宇氏の博士論文として提出された

No description available.

Tissue engineering approaches have attempted to address some of the problems associated with articular cartilage defect repair, but grafts with sufficient functional properties have yet to reach clinical practice. Mechanical loads are properly controlled in the body to maintain the functional properties of articular cartilage. This inspires the inclusion of mechanical stimulation in any in vitro production of tissue engineered constructs for defect repair. This mechanical stimulation must improve the functional properties (both biochemical and structural) of engineered articular cartilage tissue. Only a few studies have applied more than two loading types to mimic the complex in vivo load/flow conditions. The general hypothesis of the present thesis proposes that the generation of functional articular cartilage substitute tissue in vitro benefits from load and fluid flow conditions similar to those occurring in vivo. It is specifically hypothesized that application of compression, shear and perfusion on chondrocyte-seeded constructs will improve their properties. It is also hypothesized that protein production of the cell-seeded constructs can be improved in a depth-dependent manner with some loading combinations. Thus, a hydromechanical stimulator system was developed that was capable of simultaneously applying compression, shear and perfusion. Functionality of system was tested by series of short-term pilot studies to optimize some of the system parameters. In these studies, agarose-chondrocytes constructs were stimulated for 2 weeks. Then, longer-term (21- 31 days) studies were performed to examine the effects of both mechanical (compression and dynamic shear) and hydromechanical (compression, dynamic shear and fluid flow) stimulation on glycosaminoglycan and collagen production. The effects of these loading conditions were also investigated for three layers of construct to find out if protein could be localized differently depth-wise. In one of the longer-term studies, the chosen mechanical and hydromechanical stimulation conditions increased total collagen production, with higher amount of collagen for hydromechanical compared with mechanical loading condition. However, their effectiveness in increasing total glycosaminoglycan production was inconclusive with the current loading regimes. The hydromechanically stimulated construct could localize higher collagen production to the top layer compared with middle and bottom layers. Some effectiveness of hydromechanical stimulation was demonstrated in this thesis. Future studies will be directed towards further optimization of parameters such as stimulation frequency and duration as well as fluid perfusion rate to produce constructs with more glycosaminoglycan and collagen.

Articular cartilage disorders are a leading cause of human disability in many countries around the world. In this work, new techniques and strategies were developed to improve the quality of cartilage produced in vitro by methods of tissue engineering. Chondrocytes were isolated from the hip and knee joints of aborted human foetuses. The cells were expanded and seeded into scaffolds and the seeded scaffolds were cultured in perfusion bioreactors. The quality of the final cartilage constructs was assessed biochemically by measuring their content of glycosaminoglycan (GAG), total collagen and collagen type II and histologically by staining cross-sections of the constructs for GAG, collagen type I and collagen type II. The amount of proteoglycan released in the culture medium was also measured at regular intervals. Proteoglycans from tissue-engineered cartilage and spent culture medium were compared and analysed for degradation and capability of aggregation. During monolayer expansion, the chondrocyte differentiation indices decreased, the cell size increased and the percentage of cells present in G2/S??M phase decreased with the greatest changes occurring during the first passage. Expanding chondrocytes in PGA or PGA??alginate scaffolds produced cells with a higher level of differentiation than monolayer-expanded cells. However, PGA and PGA??alginate could not be justified as suitable systems for the routine expansion of chondrocytes mainly because of the relatively low cell proliferation obtained. Two new methods for seeding of cells into scaffolds were investigated using PGA and PGA??alginate as scaffold materials. Both methods produced high seeding efficiencies and homogeneous distribution of cells. When seeded PGA??alginate scaffolds were cultured in perfusion bioreactors, they produced good quality constructs with higher concentrations of extracellular matrix (ECM) components compared with previously described methods. However, when seeded PGA scaffolds were cultured in perfusion bioreactors, they produced small constructs of poor quality. Investigation of the effect of medium flow rate on the PGA scaffolds showed that a low flow rate was needed at the beginning of the culture to enable the cells to form a framework onto which other synthesised elements could deposit. Applying a gradual increase in medium flow rate to PGA scaffolds cultured in perfusion bioreactors solved the shrinkage problem and produced constructs with quality similar to those produced using PGA??alginate scaffolds. A novel compression bioreactor that mimicked the physiological stimulation of cartilage by joint movement was constructed. Using this bioreactor, compressed constructs showed significantly higher wet weight and higher concentrations of GAG, total collagen and collagen type II compared with non-compressed constructs.

Articular cartilage disorders are a leading cause of human disability in many countries around the world. In this work, new techniques and strategies were developed to improve the quality of cartilage produced in vitro by methods of tissue engineering. Chondrocytes were isolated from the hip and knee joints of aborted human foetuses. The cells were expanded and seeded into scaffolds and the seeded scaffolds were cultured in perfusion bioreactors. The quality of the final cartilage constructs was assessed biochemically by measuring their content of glycosaminoglycan (GAG), total collagen and collagen type II and histologically by staining cross-sections of the constructs for GAG, collagen type I and collagen type II. The amount of proteoglycan released in the culture medium was also measured at regular intervals. Proteoglycans from tissue-engineered cartilage and spent culture medium were compared and analysed for degradation and capability of aggregation. During monolayer expansion, the chondrocyte differentiation indices decreased, the cell size increased and the percentage of cells present in G2/S??M phase decreased with the greatest changes occurring during the first passage. Expanding chondrocytes in PGA or PGA??alginate scaffolds produced cells with a higher level of differentiation than monolayer-expanded cells. However, PGA and PGA??alginate could not be justified as suitable systems for the routine expansion of chondrocytes mainly because of the relatively low cell proliferation obtained. Two new methods for seeding of cells into scaffolds were investigated using PGA and PGA??alginate as scaffold materials. Both methods produced high seeding efficiencies and homogeneous distribution of cells. When seeded PGA??alginate scaffolds were cultured in perfusion bioreactors, they produced good quality constructs with higher concentrations of extracellular matrix (ECM) components compared with previously described methods. However, when seeded PGA scaffolds were cultured in perfusion bioreactors, they produced small constructs of poor quality. Investigation of the effect of medium flow rate on the PGA scaffolds showed that a low flow rate was needed at the beginning of the culture to enable the cells to form a framework onto which other synthesised elements could deposit. Applying a gradual increase in medium flow rate to PGA scaffolds cultured in perfusion bioreactors solved the shrinkage problem and produced constructs with quality similar to those produced using PGA??alginate scaffolds. A novel compression bioreactor that mimicked the physiological stimulation of cartilage by joint movement was constructed. Using this bioreactor, compressed constructs showed significantly higher wet weight and higher concentrations of GAG, total collagen and collagen type II compared with non-compressed constructs.

Disorders affecting articular cartilage are amongst the most common problems in orthopaedics. Osteoarthritis, the end stage of the disease of articular cartilage, reduces the quality of life for tens of millions of people in the world, and has a profound impact on the economics of industrialized countries. Despite progress in articular cartilage research, the problem is still far from being defeated. Various models e.g. in vitro cartilage explants and in vivo animal models, have been established for cartilage research, but each has its own limitations. Thus, a novel ex vivo isolated joint organ culture model was developed. Bovine metatarsophalangeal joints were chosen as a suitable synovial joint because it consists of a hinge-type joint that is similar to the human knee joint, and has a large cartilage surface that provides enough space for multiple sampling in the same joint. The joints were isolated aseptically and placed into culture media. The viability of chondrocytes, glycosaminoglycan (GAG) content of cartilage matrix, cartilage morphology and water content of matrix were evaluated under different culture conditions, i.e. static, static with flowing media, and dynamic with different durations of the movement period. The model was used to investigate the effect on the sharp scalpel cartilage injury of adding serum to the culture medium by culturing the whole joint explants in serum-supplied or serum-free media. The feasibility of investigating the early phases of chondrocyte implantation in this model was also studied: circular holes of 2.5 mm diameter were created by making a pilot hole with a 2.0 mm drill followed by using a fresh 2.5 mm biopsy punch. Allogeneic isolated chondrocytes at different passages were aggregated as cell pellets and implanted in the holes to evaluate their integration ability and the response from the recipient cartilage. Results from the static model showed that, after 28 days culture, the chondrocytes were still alive with 66.5%, 80.9% and 46.9% viability in the superficial, middle and deep zones, respectively. The GAG content of the static model decreased 19.2% after the first week of culture and then lost another 15.0% during the third week. Paradoxically, at end of the 4th week the GAG level rebounded to some extent and increased 19.0% relative to the previous week. Interestingly, the cell viability of all three zones improved if the culture fluid was flowing as seen with the experiments carried out with stirred media or dynamic movement of the articular surfaces. (e.g. for the stirred media after 28 days of culture the chondrocyte viability was 80.6%, 92.4% and 70.4% for the superficial, middle and deep zones respectively.) The GAG content was maintained at a constant level in the contact area of the dynamic model, but decreased as in the media-stirred model and non-contact area of the dynamic model to a similar extent to that observed with the static model. In the injury model, the GAG content fell approximately 10.8% straight after the scalpel cut, but no further loss was observed if the joint was cultured in the serum-supplied media. In contrast, if the injured joint was cultured in the serum-free media, the GAG content continued to fall week by week and finally dropped by 41.7% at the end of the 4th week. In the chondrocyte implantation model, the majority of the host chondrocytes around the circular defect were alive (78.5 % viability). Viewed from the surface, the dead cells were all within 20 μm from the cut edge. The implanted chondrocytes, which were aggregated as cell pellets, began to transform their shapes and spread to the surrounding surface of the recipient cartilage, but did not appear to integrate with the host tissue during the first 2 weeks of culture. The results supported the validity of this ex vivo joint model and demonstrated that the chondrocytes subjected to flow of the media or dynamic loads survived well over a 4 week period. Of importance was the finding that there was no measured loss of the matrix GAG content when the joints were under dynamic load compared to all of the non-loaded conditions. This whole joint model could be of value in providing a more natural and controllable platform where research involving the normal processes or pathologic mechanisms of articular cartilage can be investigated, as well as the early response to newly developed pharmacological agents and cartilage tissue engineering constructs.

Au cours de l'arthrose (OA), une différenciation hypertrophique des chondrocytes, une minéralisation du cartilage, une angiogenèse ostéochondrale et un remodelage de l'os sous-chondral sont observés dans l'articulation. L'angiogenèse ostéochondrale pourrait être impliquée dans l'OA mais les mécanismes moléculaires qui la gouvernent restent inconnus. Nous supposons qu'au cours de l'OA, la différenciation hypertrophique des chondrocytes jouerait un rôle clé dans le remodelage de la jonction ostéochondrale et notamment dans l'angiogenèse à travers un déséquilibre entre la production de facteurs angiogéniques et angiostatiques. Un lien entre la différenciation hypertrophique, la vascularisation ostéochondrale et la progression de l'OA a été confirmé dans des cartilages humains. Le potentiel angiogénique des chondrocytes hypertrophiques a été étudié dans un modèle de différenciation hypertrophique de chondrocytes articulaires murins en culture primaire. Les chondrocytes articulaires expriment les marqueurs chondrocytaires (Sox9, Acan, Col2a1), tandis que l'expression des marqueurs de l'hypertrophie (Runx2, OC, Osx) augmente avec la différenciation hypertrophique. Les chondrocytes hypertrophiques sont capables de minéraliser leur matrice. L'expression/production de facteurs angiogéniques (VEGF, bFGF¿) augmentent avec la différenciation hypertrophique alors que celles des facteurs angiostatiques (TSP-1...) diminuent. Une analyse microarray a été réalisée afin d'identifier des cibles innovantes. La différenciation hypertrophique des chondrocytes pourrait participer aux mécanismes physiopathologiques de l'OA en favorisant la vascularisation et la dégradation du cartilage articulaire / Osteochondral angiogenesis is an important step in the remodeling of the cartilage/subchondral bone junction in osteoarthritis (OA). Cellular and molecular stimuli of this angiogenesis are largely unknown. We hypothesize that osteochondral angiogenesis in OA is controlled by hypertrophic chondrocyte differentiation, as it occurs during development and growth (endochondral ossification process). Chondrocyte hypertrophy is detected by osteocalcin immunostaining in human OA knee tissues. OA is evaluated by modified Mankin score and osteochondral angiogenesis by the vascular channels number reaching the articular cartilage. An original model of hypertrophic differentiation of mouse articular chondrocytes in primary culture has been developed in order to study the angiogenic potential of hypertrophic chondrocytes compared to articular ones. Hypertrophic chondrocytes and osteochondral angiogenesis are positively correlated and linked to OA progression. The expression of chondrocyte markers (Sox9, Acan, Col2a1) decreases with hypertrophic differentiation in vitro, whereas Runx2, Osteocalcin and Osterix mRNAs levels significantly increase. Hypertrophic chondrocytes are characterized by strong matrix calcifications. Hypertrophic differentiation stimulates the angiogenic factors expression (VEGF, bFGF…) whereas angiostatic factors (TSP-1, chondromodulin 1…) undergo a decreased expression level. A microarray analysis has been realized in order to identify innovative molecular targets. These results suggest a key role of chondrocyte hypertrophy in osteochondral angiogenesis and thus in the remodeling of the cartilage/subchondral bone junction in OA, leading to cartilage degradation.