组织工程软骨修复全层大面积关节软骨损伤的实验研究

目的观察由软骨细胞体外培养的人工软骨组织对家兔膝关节软骨全层缺损修复的可行性.方法分离收集兔软骨细胞(4周龄),用培养液悬浮,经离心管体外培养成组织工程软骨,然后植入兔膝关节股骨负重面的缺损(5 mm×4 mm)部位,观察2,4,8,12周软骨缺损的修复情况,并进行大体和组织学观察.结果移植后第8周,移植部位填充物与宿主已完全整合,缺损处表面光滑;组织学显示,缺损部位新生软骨与所移植软骨有较好的连续性,并且缺损底部有软骨下骨及海绵状骨生成;对照组(未移植组),从组织学观察到缺损部位呈纤维样修复,未见有由新生软骨细胞形成软骨组织.结论用体外培养的组织工程软骨做移植物修复家兔关节软骨组织深层缺损具有较好的疗效.     

组织工程软骨修复兔膝关节软骨大面积深层缺损的实验研究

目的　将培养的软骨细胞埋在胶原凝胶中,观察其对家兔漆关节软骨大面积深层缺损的修复作用。方法体外培养软骨细胞,然后与胶原以一定比例混合培养,植入免膝关节股骨负重面的缺损（５ｍｍ ×４ｍｍ）部位,观察 ２、４、８．１２周软骨缺损的修复情况,并进行大体和组织学观察。结果移植后第 ８周,所修复组织的边缘变得模糊,与周围宿主整合得很好,缺损边缘不清晰。组织学观察,缺损处表面光滑,边缘与宿主完全整合,缺损处呈典型的关节软骨样组织,并且底部有新形成的软骨及骨小梁生成。对照组（未移植组）组织学观察缺损部位主要为纤维组织所填充,未见有由新生软骨细胞形成的软骨组织。结论埋在胶原凝胶中软骨细胞对家兔膝关节软骨深层缺损的修复作用。     

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

OBJECTIVE: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. METHODS: Bovine calf articular cartilage discs or isolated cells were used in 1 of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. RESULTS: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from approximately 25 microg/cm(2)/day on days 0-1 to approximately 3 microg/cm(2)/day on days 5-9), while increasing in monolayer culture (from approximately 1 pg/cell/day on days 0-1 to approximately 7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching approximately 2 microg/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. CONCLUSION: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.     

COX-2, NO, and cartilage damage and repair

The production of nitric oxide (NO) and prostaglandin E2 (PGE₂) is increased in human osteoarthritis-affected cartilage. These and other inflammatory mediators are spontaneously released by OA cartilage explants ex vivo. The excessive production of nitric oxide inhibits matrix synthesis, and promotes its degradation. Furthermore, by reacting with oxidants such as superoxide anion, nitric oxide promotes cellular injury, and renders the chondrocyte susceptible to cytokine-induced apoptosis. PGE₂ exerts both anabolic and catabolic effects on chondrocytes, depending on the microenvironment and physiological condition. Thus, NO and PGE₂, produced by activated chondrocytes in diseased cartilage, may modulate disease rogression in osteoarthritis, and should therefore be considered potential targets for therapeutic intervention.     

Aging,articular cartilage chondrocyte senescence and osteoarthritis

The incidence of osteoarthritis (OA), the disease characterized by joint pain and loss of joint form and function due to articular cartilage degeneration, is directly correlated with age. The strong association between age and increasing incidence of osteoarthritis (OA) marks OA as an age related disease. Yet, like many other age related diseases, OA is not an inevitable consequence of aging; instead, aging increases the risk of OA. Articular cartilage aging changes that may lead to articular cartilage degeneration include fraying and softening of the articular surface, decreased size and aggregation of proteoglycan aggrecans and loss of matrix tensile strength and stiffness. These changes most likely are the result of an age related decrease in the ability of chondrocytes to maintain and repair the tissue manifested by decreased mitotic and synthetic activity, decreased responsiveness to anabolic growth factors and synthesis of smaller less uniform aggrecans and less functional link proteins. Our recent work suggests that progressive chondrocyte senescence marked by expression of the senescence associated enzyme beta-galactosidase, erosion of chondrocyte telomere length and mitochondrial degeneration due to oxidative damage causes the age related loss of chondrocyte function. New efforts to prevent the development and progression of OA might include strategies that slow the progression of chondrocyte senescence or replace senescent cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Extracellular nucleotides,cartilage stress,and calcium crystal formation

Nucleotides are released by chondrocytes at rest and in response to mechanical stimulation. Extracellular nucleotides are metabolized by a variety of ectoenzymes, producing free phosphate (Pi) or pyrophosphate (PPi) and promoting matrix mineralization. Ectoenzymes are differentially localized in cartilage and may be co-released with nucleotides during mechanical stimulation. Extracellular nucleotides can also serve as substrates and/or modulators of enzymes such as tissue transglutaminase and ecto-protein kinases that modify matrix proteins and regulate crystal deposition or growth. Understanding the evolution of osteoarthritis and calcium crystal deposition diseases will require clearer knowledge of the functions of nucleotides and ectoenzymes in the cartilage extracellular matrix.     

Phytomitogen-induced,lymphokine-mediated cartilage proteoglycan degradation

The significance of T-cell activation in the immunopathogenesis of articular disease has been studied in an in vitro model to assess the capacity of phytomitogen-induced lymphokines to degrade cartilage matrix proteoglycans. Supernatants derived from T-cell activation of normal human peripheral blood lymphocytes were shown to contain a monocyte-dependent, serum-inhibitable, dialyzable factor or factors capable of inducing significant proteoglycan degradation, as assessed by release of macroprecipitable ³⁵S-labeled proteoglycan from heterologous cartilage substrates.     

Clonal precursor of bone,cartilage, and hematopoietic niche stromal cells

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.     

S-100 protein in normal,osteoarthrotic, and arthritic cartilage

Summary The occurrence of the S-100 protein was studied in normal and pathological cartilage from patients with osteoarthrosis and rheumatoid arthritis. In normal cartilage all cells of the different zones exhibit immunoreactivity for this protein. An identical distribution is observed in cases of osteoarthrosis and rheumatoid arthritis. Cellular pannus tissue usually is devoid of the S-100 protein. However, if chondroid metaplasia occurs in this tissue the cells become positive for the S-100 protein. From these results it is concluded that a chondroid metaplasia not only leads to a matrix comparable to cartilage but also contains cells that gain further characteristics of original chondrocytes.     

Articular cartilage preservation and storage. II. Mechanical indentation testing of viable, stored articular cartilage.

Mature rabbit articular cartilage in the form of distal femoral condyles, composite osteoarticular structures, were incubated in the presence of alpha-tocopherol (200 micrograms/ml) over a period of time. Indentation testing and 35S uptake indicate preservation of sustained load carrying capacity and viability, respectively, in the presence of alpha-tocopherol for up to 30 days in organ culture. Condylar cartilage stored in the absence of alpha-tocopherol as well as frozen cartilage demonstrated progressive inability to resist sustained loading over time. Nonoptimal synthetic function apparently occurred in these latter two groups when compared to alpha-tocopherol stored material.     

Osteoarthritic cartilage contains increased calcium, magnesium and phosphorus

Using plasma emission spectroscopy, we measured the calcium (Ca), phosphorus (P), magnesium (Mg), and sulphur (S) concentration in femoral head cartilage from 7 patients with osteoarthritis (OA) and 22 normal patients. We found that in OA cartilage Ca = 271.9 +/- 32.7 mmol/kg dry weight compared to Ca = 113.2 +/- 12.6 mmol/kg dry weight in normal controls. The Ca:P molar ratio in OA cartilage was 1.93 compared to Ca:P = 2.27 in normals, and Ca:P = 1.67 in apatite crystals. We conclude that OA cartilage has a greater binding capacity for Ca, Mg, and P than does normal cartilage. Apatite crystals, if present, comprise maximally 0.4% wet weight of OA cartilage.     

Phonoarthrography,musculoskeletal ultrasonography,and biochemical biomarkers for the evaluation of knee cartilage in osteoarthritis

The aim of this study was to examine the relationship among three different parameters used to assess cartilage in osteoarthritis (OA) of the knee. These parameters are phonoarthrography (Phono-A), musculoskeletal ultrasonography (MSUS) from the 4 condyles, and biochemical markers; notably, matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of proteinase (TIMP-1). A total of 100 knees with chronic idiopathic OA diagnosed according to the American College of Rheumatology (ACR) criteria were studied, together with 50 normal knees. The knee sounds were recorded by Phono-A and the cartilage thickness was measured by MSUS. All patients and controls had MMP-3 and TIMP-1 measured in a blood sample, using an enzyme-linked immunosorbent assay (ELISA). Conventional knee X-rays were obtained for diagnosis and for Kellgren–Lawrence (K-L) grading purposes. The results showed that Phono-A values were inversely correlated with cartilage thickness, both of these being sensitive parameters for cartilage degeneration. Phono-A values were higher in patients than in controls, denoting more degeneration of cartilage, and the cartilage thickness of all 4 condyles showed significant reductions in patients compared with normal controls. Most of the patients were categorized as grade 2 (36%) and grade 3 (30%) of the K-L classification. Mean levels of MMP-3 and TIMP-1 were significantly elevated in both groups but they were not correlated with each other. MMP-3 continued to rise with increasing radiological grades until grade 4, where it fell unexpectedly. In conclusion, Phono-A and cartilage thickness measured by MSUS seem to support each other. They can be used as parameters for following up cartilage in OA of the knees. The first deals with the roughness of the cartilage surface and the second with its thickness, complementing each other. MMP-3 continues to rise in early and middle grades of OA, denoting cartilage destruction.     

Phytomitogen-induced, lymphokine-mediated cartilage proteoglycan degradation.

The significance of T-cell activation in the immunopathogenesis of articular disease has been studied in an in vitro model to assess the capacity of phytomitogen-induced lymphokines to degrade cartilage matrix proteoglycans. Supernatants derived from T-cell activation of normal human peripheral blood lymphocytes were shown to contain a monocyte-dependent, serum-inhibitable, dialyzable factor or factors capable of inducing significant proteoglycan degradation, as assessed by release of macroprecipitable 35S-labeled proteoglycan from heterologous cartilage substrates.