The purpose of this study is to evaluate the feasibility of human amniotic membrane (HAM) as a chondrocyte carrier by assessing cell proliferation and maintenance of phenotype in vitro and cartilage regeneration in vivo. Intact HAM was treated with 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA) for 15 min and the epithelial cells removed to make a denuded HAM. Rabbit articular chondrocytes were then seeded on three different HAM substrates: the epithelial side of intact HAM (IHE), basement side of denuded HAM (DHB), and stromal side of denuded HAM (DHS). These cell-substrate specimens were cultured for up to 4 weeks, and cell proliferation rate and phenotypic stability were examined at weeks 1 and 4. While chondrocytes grew in monolayer fashion on the surface of IHE and DHB substrates, the cells seeded in DHS penetrated and spread into the whole thickness of the stromal layer. The proliferating activity of chondrocytes in DHB was continuously up-regulated. A similar proliferating activity was observed in DHS in the first week, which remained stable for up to 4 weeks. The expression of type II collagen gradually increased with time in the DHS group, while it gradually decreased in the DHB group or was not detected at all in the IHE group. These results suggested that denuded HAM was able to support chondrocyte proliferation and maintenance of phenotype in vitro, seemingly more favorable when DHS was used. Based on this data, the DHS with chondrocytes was used to cover rabbit osteochondral defect with the stromal side facing in. The defect area was successfully regenerated with hyaline cartilage in the Safranin-O stain and International Cartilage Repair Society (ICRS) scoring after 8 weeks of implantation. In conclusion, our findings suggest that denuded HAM could be one of the ideal cell carrier matrices for cartilage regeneration.

Abstract:We have observed in our previous study that a cell-derived extracellular matrix (ECM) scaffold could assure the growth of a cartilage tissue construct in vitro.The purpose of the present study was to evaluate the feasibility of a chondrocyte-seeded cell-derived ECM scaffold by implanting it in vivo in nude mouse. A porous cell-derived ECM scaffold was prepared with a freeze-drying protocol using porcine chondrocytes. Rabbit articular chondrocytes were seeded onto the scaffold and cultured for 2 days in vitro, and then implanted into the nude mouse subcutaneously. They were retrieved at 1, 2, and 3 weeks postimplantation. Under macroscopic analysis, the cartilage-like tissue formation matured with time and developed a smooth, white surface.Contrary to the control (in which no cells were seeded), the size of the neocartilage tissue increased slightly by the third week and remained more stable. Total glycosaminoglycan (GAG) content and the GAG/DNA ratio increased significantly with time in the chemical analysis. The histology exhibited a sustained accumulation of newly synthesized sulfated proteoglycans. Immunohistochemistry, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) clearly identified type II collagen at all time points. Compressive strength of in vivo neocartilage increased from 0.45 Ϯ 0.06 MPa at 1 week to 1.18 Ϯ 0.17 MPa at 3 weeks. In conclusion, this study demonstrated that the cell-derived ECM scaffold could provide chondrocytes with favorable in vivo environment to produce a hyaline-like cartilage tissue.

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PurposePatellofemoral instability is a common cause of anterior knee pain in adolescents and young adults. Most normal and pathological values for diagnosing patellofemoral instability are based on Western literature. We conducted this radiological study to determine normal values for different patellofemoral parameters in a Korean population and to evaluate their usefulness in diagnosis.Materials and MethodsWe retrospectively reviewed the rotational profile computerized tomography (CT) scans of the patellar dislocation and control groups. Trochlear, patellar, rotational profile, and trochleo-patellar alignment parameters were compared between the groups. Receiver operating characteristic curves were drawn for significant parameters, and sensitivity and specificity were calculated for the cut-off values.ResultsThere were 48 patients in the patellar dislocation group and 87 patients in the control group. In the control group and patellar dislocation group, the mean sulcus angle was 132.5° and 143.3°, respectively, trochlear depth was 6.04 mm and 3.6 mm, bisect offset was 56.4% and 99.9%, lateral patellar tilting was 9.8° and 19.2°, patellar facet asymmetry was 63.5% and 45.16%, and the tibial tuberosity-trochlear groove (TT-TG) distance was 10.91 mm and 27.16 mm, respectively.ConclusionsThe trochlear depth, bisect offset, patella tilting, and TT-TG distance were parameters that significantly contributed to patellar instability. Rotational profile CT can be considered a good diagnostic tool to assess all these parameters that help to identify anatomical aberration resulting in patellofemoral instability, thereby helping in formulating the most effective treatment plan.

Bone marrow stimulation techniques (BSTs) are widely used in clinics to treat cartilage defects, but yet have a critical limitation from the loss of blood clots. In this work, a novel cartilage extracellular matrix (CECM) membrane is developed to protect blood clots after BSTs. The CECM membrane was made of ECM fabricated naturally by cultured porcine chondrocytes, and then decellularized and multi-layered to confer optimal mechanical strength. Highly compatible with cells, the CECM membrane did not show any cytotoxicity or immune responses in vivo. The CECM membrane was very thin (30-60 µ m thick) and bendable, but had good tensile strength (85.64 N), suitable for protecting blood clots from leakage in rabbit cartilage defect. Moreover, the CECM membrane showed low but enough diffusion coeffi cient to allow delivery of small proteins in synovial fl uid into the repaired tissue. In a beagle model, covering the cartilage defect with the CECM membrane after BST generated more hyaline cartilage-like tissues than the BST alone in histology and chemical analyses at 18 weeks. Its ICRS score was approximately 2.5 times higher than that of the BST alone. Therefore, the CECM membrane is proposed as a useful tool that can improve the outcome of BSTs to treat cartilage defects.

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Graphene/hexagonal boron nitride (h-BN) heterostructures have attracted a great deal of attention in recent years due to their unique and complementary properties for use in a wide range of potential applications. However, it still remains a challenge to synthesize large-area high quality samples by a scalable growth method. In this work, we present the synthesis of both in-plane and stacked graphene/h-BN heterostructures on Cu foils by sequentially depositing h-BN via ion beam sputtering deposition (IBSD) and graphene with chemical vapor deposition (CVD). Due to a significant difference in the growth rate of graphene on h-BN and Cu, the in-plane graphene/h-BN heterostructures were rapidly formed on h-BN domain/Cu substrates. The large-area vertically stacked graphene/h-BN heterostructures were obtained by using the continuous h-BN film as a substrate. Furthermore, the well-designed sub-bilayered h-BN substrates provide direct evidence that the monolayered h-BN on Cu exhibits higher catalytic activity than the bilayered h-BN on Cu. The growth method applied here may have great potential in the scalable preparation of large-area high-quality graphene/h-BN heterostructures.

Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant cutaneous disorder characterized by a mixture of hyperpigmented and hypopigmented macules of various sizes on the extremities and caused by the mutations of adenosine deaminase acting on RNA1 (ADAR1) gene. We screened 14 unrelated families or sporadic cases for mutation in the full coding sequence of this gene. Eight novel heterozygous mutations of ADAR1 and four known mutations were identified, including four missense mutations (p.R26K, p.Y1192D, p.R916Q, p.R1155W), six frameshift mutations (p.N205fsX217, p.V211fsX217, p.V404fsX417, p.I914fsX927, p.L1053fsX1076, p.L1070fs1092), and two nonsense mutations (p.R474X, p.R1096X). Interestingly, we failed to detect any mutations of ADAR1 in one family. Including our data, there are now 93 different mutations reported in 105 independent patients that we have tabulated. From the review of clinical features in these reports, we found that the same mutation could lead to different phenotypes even in the same family and did not establish a clear correlation between genotypes and phenotypes. Finally this study is useful for functional studies of the protein and to define a diagnostic strategy for mutation screening of the ADAR1 gene.