Introduction Autologous chondrocyte cell-sheet transplantation is a scaffold-free regenerative strategy that may augment joint-preserving surgery for knee osteoarthritis, but durability beyond early follow-up has remained unclear. Case presentation Eight patients with 2 distinct clinical entities---focal posttraumatic cartilage lesions in anterior cruciate ligament (ACL)-deficient knees, or generalized medial compartment osteoarthritis---underwent autologous chondrocyte cell-sheet transplantation combined with ACL reconstruction or medial open-wedge high tibial osteotomy, respectively. Seven patients completed minimum 10-year follow-up were analyzed. Lysholm Knee Score improved from 50.7 ± 16.1 preoperatively to 98.1 ± 2.2 at 3 years and remained higher than baseline at 10 years (82.3 ± 18.4; P = .005), although it declined versus 3 years (P = .043). Knee injury and Osteoarthritis Outcome Score domains improved from baseline; only the Pain subscale declined significantly between 3 and 10 years (P = .038). Kellgren-Lawrence grade progressed in 2 knees. Conclusion In this small, uncontrolled case series, a combined joint-preserving procedure that included autologous chondrocyte cell-sheet transplantation with ACL reconstruction or open-wedge high tibial osteotomy was associated with maintained clinical improvement and native-knee preservation in most selected patients at minimum 10-year follow-up. Because the independent contribution of the cell sheet cannot be separated from concomitant biomechanical correction, debridement, marrow stimulation, rehabilitation, and patient selection, these findings should be regarded as hypothesis-generating. The observation that late deterioration occurred mainly after intercurrent trauma or serious systemic illness suggests, but does not prove, that long-term outcome may depend on preserved joint-level biomechanics in addition to local cartilage repair.

Allogeneic cell therapies are not fully effective in treating osteoarthritis of the knee (OAK). We recently reported that transplantation of autologous chondrocyte cell-sheets along with open-wedge high tibial osteotomy promoted hyaline cartilage repair in humans. Here we describe our regenerative therapy for OAK using polydactyly-derived allogeneic chondrocyte cell-sheets (PD sheets) and temperature-responsive culture inserts. Ten patients with OAK and cartilage defects categorized arthroscopically as Outerbridge grade III or IV received the therapy. Cartilage viscoelasticity and thickness were assessed before and after transplantation. Arthroscopic biopsies obtained 12 months after transplantation were analyzed histologically. Gene expression was analyzed to evaluate the PD sheets. In this small initial longitudinal series, PD sheet transplantation was effective in treating OAK, as indicated by changes in cartilage properties. Gene marker sets in PD sheets may predict outcomes after therapy and provide markers for the selection of donor cells. This combined surgery may be an ideal regenerative therapy with disease-modifying effects in OAK patients.

In anatomic anterior cruciate ligament (ACL) reconstruction, several pitfalls in creating the femoral bone tunnels at the correct position are of great concern. Our new method, the tibia rotational (TR) technique, may contribute to resolving these. The purpose of this study is to describe further details about the TR technique in anatomic double-bundle ACL reconstruction. Both anteromedial and posterolateral femoral bone tunnels were drilled through a posterolateral tibial bone tunnel using tibial rotation without deep knee flexion. When it is difficult to reach the mark with the rigid guide pin, the narrow curved TR technique guide and the flexible drill system allow drilling femoral bone tunnels in the correct position. The TR technique offers the technical ease required for widespread acceptance while prioritizing the fundamental goals of an anatomic double-bundle ACL reconstruction.

Background: Like all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span.Methods: We transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo.Results: Our results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT-or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components.Conclusions: Focal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

Background Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage. Results The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets. Conclusion The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.

Some treatments for full thickness defects of the articular cartilage, such as the transplantation of cultured chondrocytes have already been performed. However, in order to overcome osteoarthritis, we must further study the partial thickness defects of articular cartilage. It is much more difficult to repair a partial thickness defect because few repair cells can address such injured sites. We herein show that bioengineered and layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for the repair of partial thickness defects. We also show that a chondrocyte-plate using a rotational culture system without the use of a scaffold may also be useful as a core cartilage of an articular cartilageous defect. We evaluated the properties of these sheets and plates using histological findings, scanning electrical microscopy, and photoacoustic measurement methods, which we developed to evaluate the biomechanical properties of tissue-engineered cartilage. In conclusion, the layered chondrocyte sheets and chondrocyte-plates were able to maintain the cartilageous phenotype, thus suggesting that they could be a new and potentially effective therapeutic product when attached to the sites of cartilage defects.

The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here, we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal extracellular environment using an HCPS we originally developed, and an atelocollagen honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1 within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS generated an extracellular environment similar to that of heparan sulfate proteoglycan within the scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.

There is a demand in the field of regenerative medicine for measurement technology that enables determination of functions and characterizations of engineered tissue. Regenerative medicine involving the articular cartilage in particular requires measurement of viscoelastic properties and characterization of the extracellular matrix, which plays a major role in articular cartilage. To meet this demand, we previously proposed a noninvasive method for determination of the viscoelasticity using laser-induced thermoelastic wave(1,2). We also proposed a method for characterization of the extracellular matrix using time-resolved autofluorescence spectroscopy, which could be performed simultaneously with laser-induced thermoelastic wave measurement(3). The purpose of this study was to verify the usefulness and limitation of these methods for evaluation of actual engineered cartilage. 3rd Q-SW Nd:YAG laser pulses, which are delivered through optical fiber, were used for the light source. Laser-induced thermoelastic waves were detected by a sensor consisting of a piezoelectric transducer, which was designed for use in arthroscopy(4). The time-resolved fluorescence spectroscopy was measured by a photonic multichannel analyzer with 4ch digital signal generator. Various tissue-engineered cartilages were developed as samples. Only a limited range of sample thickness could be measured, however, the measured viscoelastic parameters had a positive correlation with culture time, that is, the degree of formation of extracellular matrix(5,6). There were significant differences in the fluorescent parameters among the phenotypic expressions of cartilage because chondrocyte produces specific extracellular matrix as in collagen types depending on its phenotype.

The extracellular matrix (ECM) of articular cartilage has several functions that are unique to joints. Although a technique for transplanting cultured chondrocytes has already been introduced, it is difficult to collect intact ECM when using enzymes to harvest samples. Temperature-responsive culture dishes have already been clinically applied in the fields of myocardial and corneal transplantation. Earlier studies have shown that a sheet of cultured cells with intact ECM and adhesive factors can be harvested using such culture dishes, which allow the surface properties of the dish to be reversibly altered by changing the temperature. Human chondrocytes were subjected to enzymatic digestion and then were seeded in temperature-responsive culture dishes. A sheet of chondrocytes was harvested by only reducing the temperature after the cultured cells reached confluency. A real-time PCR analysis of the chondrocyte sheets confirmed that type II collagen, aggrecan, and fibronectin were present. These results suggested that, although chondrocytes undergo dedifferentiation in a monolayer culture, multilayer chondrocyte sheets grown in a similar environment to that of three-dimensional culture may be able to maintain a normal phenotype. A histological examination suggested that multilayer chondrocyte sheets could thus prevent the loss of proteoglycans because the area covered by the sheets was well stained by safranin-O. The present experiments suggested that temperature-responsive culture dishes are useful for obtaining cultured chondrocytes, which may then be clinically employed as a substitute for periosteal patches because such sheets can be applied without a scaffold.

Some treatments for full thickness defects of articular cartilage, such as cultured chondrocyte transplantation, have already been done. However, to overcome osteoarthritis, we must further study the partial thickness defect of articular cartilage. It is much more difficult to repair a partial thickness defect because few repairing cells can address such injured sites. We herein show that bioengineered layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for partial thickness defects. We evaluated the property of these sheets using real-time PCR and histological findings, and allografted these sheets to evaluate the effect of treatment using a rabbit partial model. In conclusion, layered chondrocyte sheets were able to maintain the cartilageous phenotype, and could be attached to the sites of cartilage damage which acted as a barrier to prevent a loss of proteoglycan from these sites and to protect them from catabolic factors in the joint.

Background and Objectives: We demonstrated that photoacoustic measurement enables viscoelastic characterization of biological tissue. The purpose of this study was to develop a practical photoacoustic measurement system for diagnosis of osteoarthritis (OA) by viscoelastic characterization of articular cartilage.Study Design/Materials and Methods: The portable system consists of a commercially available 3rd harmonic Q-switched Nd:YAG laser as a light source and a transducer, which is arranged coaxially with an optical fiber. Cell proliferation tests were performed to study the effect of laser irradiation on chondrocytes. Photoacoustic measurements were performed using enzymatically treated cartilage as a model of OA.Results: There was no significant damage of chondrocytes caused by laser irradiation (100 mu J/mm(2), 5 Hz, 30 shots). The change in relaxation times measured by the photoacoustic measurement had a positive correlation with time of enzymatic treatment, that is, the degree of cartilage degeneration.Conclusions: We have developed a noninvasive photoacoustic measurement system designed for arthroscopic use and have demonstrated the applicability of this system to the diagnosis of OA-like cartilage degeneration.

We demonstrated the capability of a photoacoustic method for viscoelastic measurement. The measurement method has already proved to be useful for evaluation of regenerative medicine of articular cartilage. However, characterization of the extracellular matrix as well as determination of the viscoelastic property should be carried out for evaluation of regenerative medicine because the extracellular matrix plays an important role. We therefore developed a method for characterization of the extracellular matrix that can be performed Simultaneously with the photoacoustic measurement. Since collagen molecules, which are the major contents of the cartilage extracellular matrix, are well known as endogenous fluorescent molecules, it is possible that fluorescence measurement will enable characterization of the extracellular matrix. Third harmonic Q-switched Nd:YAG laser pulses were used as an excitation light source. The time-resolved fluorescence spectroscopy was obtained by using a photonic multi-channel analyzer. Tissue-engineered cartilages cultured under different conditions for various periods were used as samples. Different culture conditions resulted in different extracellular matrix formations. There were significant differences in the measured fluorescent parameters among, the culture conditions of cartilage because chondrocytes produce a specific extracellular matrix depending on its Culture condition. The specific extracellular matrix contained a specific type of collagen Such as collagen type I or type II, which each have specific fluorescent features. Thus, the fluorescent parameters enabled characterization of synthesis of cartilage-associated extracellular matrix. Therefore,, the combination of fluorescence and photoacoustic measurement is expected to become a useful evaluation method in regenerative medicine.

We demonstrated the capability of photoacoustic measurement for viscoelastic characterization. Since tissue viscoelasticity affects the propagation and attenuation of photoacoustic waves generated in the tissue, the relaxation times of the photoacoustic waves give the viscosity-elasticity ratio of the tissue. The relaxation times of photoacoustic waves of articular cartilage tissues engineered under various culture conditions were closely correlated with intrinsic viscosity-elasticity ratios measured by using a conventional viscoelastic analyzer (R > 0.98). In order to apply the photoacoustic measurement method to evaluation of the regeneration of articular cartilage as a method to validate the surgery, the method should enable not only evaluation of engineered tissue during cultivation in vitro but also evaluation after transplantation of engineered tissue in vivo. The aim of this study was to verify the usefulness of the photoacoustic method for repeated measurement of viscoelastic properties in order to evaluate the process of regeneration of a full-thickness defect in rabbit articular cartilage using allografted tissue-engineered cartilage. Photoacoustic waves were induced by 266- and 355-nm, 5-7 ns, light pulses delivered through an optical silica fiber from an Q-switched Nd:YAG laser and were detected by a piezoelectric transducer, which we had designed. About a 40% difference between the viscosity-elasticity ratio of allografted cartilage that of tissue surrounding the defect was shown just after surgery. The difference was significantly reduced at 4 and 12 postoperative weeks. Therefore, since the photoacoustic measurement method enables assessment of the progress of restoration of the viscoelasticity of articular cartilage, its main function, this method would be useful as an evaluation method in regenerative medicine.