Gene and Protein Expression During Differentiation and Matrix Mineralization in a Chondrocyte Cell Culture System

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10⁵ cell/cm² and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and β-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro. Received: 12 December 1996 / Accepted: 26 June 1997     

Biochemical identification and immunolocalizaton of aggrecan,ADAMTS5 and inter‐alpha‐trypsin–inhibitor in equine degenerative suspensory ligament desmitis

We describe analysis of suspensory ligaments from horses with advanced degenerative suspensory ligament desmitis (DSLD) to identify the major proteoglycans (PGs), ADAMTS‐aggrecanases and inter‐alpha‐trypsin inhibitor (IαI) components associated with ligament degeneration. Specific anatomical regions of suspensory ligaments from two normal horses and four diagnosed with DSLD were analyzed by Western blot and immunohistochemistry for the following: aggrecan, aggrecan fragments, decorin, ADAMTS4, ADAMTS5, and IαI components. When compared to normal, DSLD ligaments showed about a 15‐fold increase (P < 0.0014) in aggrecan levels and markedly enhanced staining with Safranin O. The aggrecan was composed of two distinct high molecular weight core protein species. The largest species was found only in DSLD samples and it co‐migrated with aggrecan synthesized by equine mesenchymal stem cells (MSC). Many of the DSLD samples also contained abnormally high concentrations of ADAMTS4, ADAMTS5, and IαI. Notably, the ADAMTS5 in DSLD samples, but not normals, was present largely as a high molecular weight complex. We conclude that ligament degeneration in DSLD is associated with matrix changes characteristic of an inflammatory nonhealing wound, specifically containing chondrogenic progenitor cells. Since aggrecan accumulation is a major feature of incomplete healing in tendon and skin of the ADAMTS5 knockout mouse, we propose that ligament failure in DSLD results from a process involving tissue inflammation and the complexation of ADAMTS5. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:900–906     

In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure

This study tested the effects of hydrostatic pressure (10 MPa) on adult articular chondrocyte mRNA and extracellular matrix synthesis in vitro. High density primary cultures of bovine chondrocytes were exposed to hydrostatic pressure applied intermittently at 1 Hz or constantly for 4 hours in serum-free medium or in medium containing 1% fetal bovine serum, mRNAs for aggrecan, types I and II collagen, and β-actin were analyzed by Northern blots and quantified by slot blots. Proteoglycan synthesis was quantified by ³⁵SO₄ uptake into cetylpyridinium chloride-precipitable glycosaminoglycans, and cell-associated aggrecan and type-II collagen were detected by immunohistochemical techniques. In serum-free medium, intermittent pressure increased aggrecan mRNA signal by 14% and constant pressure decreased type-II collagen mRNA signal by 16% (p < 0.05). In the presence of 1% fetal bovine serum, intermittent pressure increased aggrecan and type-II collagen mRNA signals by 31% (p < 0.01) and 36% (p < 0.001), respectively, whereas constant pressure had no effect on either mRNA. Intermittent and constant pressure stimulated glycosaminoglycan synthesis 65% (p < 0.001) and 32% (p < 0.05), respectively. Immunohistochemical detection of cell-associated aggrecan and type-II collagen was increased in response to both intermittent and constant pressure. These data support the hypothesis that physiologic hydrostatic pressure directly influences the extracellular matrix metabolism of articular chondrocytes.     

Topographic variation in biglycan and decorin synthesis by articular cartilage in the early stages of osteoarthritis: An experimental study in sheep

Articular cartilage contains large molecular weight proteoglycans that aggregate with hyaluronic acid (aggrecan) and small species, particularly biglycan (dermatan sulphate proteoglycan-1) and decorin (dermatan sulphate proteoglycan-2). that do not. Mechanical stresses have been shown to profoundly influence the metabolism of aggrecan by articular chondrocytes; however, there are limited corresponding data on the metabolism of dermatan sulphate proteoglycans 1 and 2. The objective of this study was to examine the metabolism of aggrecan, biglycan, and decorin in articular cartilage from different weight-bearing areas of normal ovine stifle joints and in joints 6 months after meniscectomy, a procedure that has been shown to induce early osteoarthritic changes. [³⁵S]proteoglycans synthesised by cartilage explants from eight different weight-bearing regions of unoperated and meniscectomised ovine stifle joints during 48 hours of culture were separated by size-exclusion chromatography, hydrophobic chromatography, and sodium dodecyl sulfatepolyacrylamide gel electrophoresis and were quantitated by phosphor-screen autoradiography. The synthesis and degradation of the proteoglycans were expressed relative to the DNA content of the explants. In control joints, the cartilage exposed to high contact stress synthesised significantly less proteoglycan overall and more decorin than joint regions bearing less stress. Explants from high stress regions also released significantly greater amounts of resident proteoglycans (dimethylmethylene blue positive) into media during culture. After lateral meniscectomy, the lateral tibial and femoral cartilages showed elevated biosynthesis of both ³⁵S-dermatan sulphate proteoglycans 1 and 2. This chondrocyte biosynthetic response was accompanied by increased catabolism of aggrecan and the release of its degradation products into culture media. These experiments revealed, in normal joints, a topographic variation in proteoglycan synthesis by articular cartilage that was related to the mechanical stress to which the tissues were subjected in vivo. This biosynthetic pattern changed when the load distribution of the joint was altered by unilateral meniscectomy. These data suggest that an altered chondrocyte phenotypic expression of proteoglycans in response to abnormal mechanical loading is an early event in osteoarthritis.     

Association of serum levels of aggrecan ARGS,NITEGE fragments and radiologic knee osteoarthritis in Tunisian patients

ObjectivesProteolytic degradation of aggrecan is a hallmark of the pathology of osteoarthritis. The aim of this study was to develop enzyme-linked immunosorbent assay (ELISA) to quantify the serum levels of specific aggrecan fragments generated by aggrecanases-mediated cleavage. We investigated the relationships between these two aggrecan degradations fragments and urinary CTX-II levels.MethodsThe competitive ELISAs employ a polyclonal antibody raised against the aggrecan fragments containing two neoepitopes NITEGE³⁷³ and ³⁷⁴ARGSVI. We measured serum levels of ARGSV and NITEGE in 125 women with knee osteoarthritis (mean ± SD age of 53.6 ± 7.6 years, mean ± SD disease duration of 3.6 ± 3.8 years), and 57 women age-matched controls.ResultsAggrecan neoepitopes assays showed an intra- and inter-assay imprecision (CV) lower than 20% for both tests and good linearity. Median serum ARGSVI (by 18%; P = 0.002), and NITEGE (36.4%; P < 0.001) levels were significantly decreased in patients with knee osteoarthritis compared with controls. Minimal joint space width was negatively correlated with ARGSVI (r = –0.368, P = 0.04) and NITEGE (r = –0.274, P = 0.038) in knee osteoarthritis patients. Median urinary CTX-II levels were significantly increased by 39.5% (P = 0.001) in knee OA patients compared with controls.ConclusionMarkers of degradation aggrecan were analyzed for the first time in an African osteoarthritis population. These markers can be used to monitor aggrecanase activity in human joint disease. Their combination with CTX-II can improve clinical investigation of patients with osteoarthritis patients.     

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.     

A simple technique for studying struvite crystal growth in vitro

Summary Struvite urolithiasis forms as a consequence of a urinary tract infection by urease-producing species of bacteria such as Proteus mirabilis. Ammonia, produced by the enzymatic hydrolysis of urea, elevates urine pH causing a supersaturation and precipitation of Mg⁺⁺ as struvite (NH₄MgPO₄). Calcium often precipitates as well, forming the mineral carbonate-apatite (Ca₁₀(PO₄)₆CO₃). We have developed a procedure based on direct observation by light microscopy whereby struvite crystal growth can be quickly monitored in response to chemical changes in urine. As struvite crystals assume a characteristic shape or crystal habit based on their growth rate, the effect of urine chemistry and the action of various crystallization or urease inhibitors on struvite formation can be quickly shown. In addition preliminary effects of alkaline pH, or the presence of toxic compounds on bacteria can also be shown through their loss of motility.     

Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments

OBJECTIVE: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. METHODS: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. RESULTS: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. CONCLUSION: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.     

Progenitor cells are responsible for formation primary epithelial cultures in the prostate epithelial model

The adult stem cells (ASC) are supposed to regenerate epithelium. We hypothesized prostate epithelial CD133-positive ASC to be responsible for establishing the primary cell culture. The prostate epithelial stem cells were isolated using anti-CD133 microbeads in order to form different cell populations. The morphology of cultures developed from CD133⁺ and CD133⁻ prostate epithelial cells were compared with prostate epithelium cell culture obtained after simple isolation procedure. Four 8-week-old Wistar rats were used in the experiment and six cultures were obtained. Double CD133⁺ and CD133⁻ cultures from two rats were established after enzymatic digestion and positive selection by SuperMACS device, and two non-selected CD133⁺/CD133⁻ cultures were developed by simple prostate epithelial cell isolation from two other rats. The epithelial nature was confirmed by anti-cytokeratine antibodies. It was observed that growth of the CD133⁺/CD133⁻ and CD133⁺culture resembled epithelial-like prostate cell culture. It was not possible to establish epithelial-like culture from CD133⁻ cell population. The primary epithelial cell culture collapsed in a few days after the CD133-positive ASC were removed. We concluded that the epithelial progenitor cells are responsible for establishing primary prostate epithelial cultures in vitro.     

Gene and protein expressions of bone marrow mesenchymal stem cells in a bone tunnel for tendon-bone healing

Purpose

Bone marrow mesenchymal stem cells (BMSCs) injected around tendon grafts can enhance tendon-bone healing and promote fibrocartilage formation. To understand gene and protein expressions of cells during tendon-bone healing, auto-BMSCs were implanted into a bone tunnel in anterior cruciate ligament reconstruction in a rabbit model.

Methods

BMSCs were harvested from New Zealand white rabbits. By an anterior cruciate ligament reconstruction model, 1 × 10⁶ BMSCs in 0.35 mL of fibrin glue was injected into bone tunnel as Fibrin-BMSC group. Only fibrin glue (Fibrin group) was injected as control. Three chondrogenesis genes and proteins, including Sox 9, collagen Type II (COII), aggrecan, and three osteogenesis genes and proteins, including Runx2, collagen type I (COI), and osteocalcin, between Fibrin-BMSC and Fibrin group were compared by real-time polymerase chain reaction assay and immunohistochemical assay postoperation.

Results

In real-time polymerase chain reaction assay, Sox9, COII, aggrecan, COI, and osteocalcin expressions upregulated and Runx2 downregulated were determined in Fibrin-BMSC group at 1 week. COII, aggrecan upregulated, and Runx2 and osteocalcin downregulated were determined at 4 weeks. In immunohistochemical assay, only Sox9, COII, and aggrecan proteins in only Fibrin-BMSC group were observed at 4 weeks. The protein expression as same as gene expression was obtained in a bone tunnel.

Conclusion

Auto-BMSCs promoted COII and aggrecan expression and reduced Runx2 and osteocalcin expression in a bone tunnel. It demonstrated that these cells could enhance fibrocartilage formation because of higher chondrogenesis expression during tendon-bone healing.     

Intracellular free calcium mediates glioma cell detachment and cytotoxicity after photodynamic therapy

Photofrin photodynamic therapy (PDT) caused a dose-dependent decrease of enzymatic cell detachment by trypsin/ethylenediamine tetra-acetic acid (EDTA) in human glioma U251n and U87 cells. This happened coincidently with the increase of intracellular free calcium ([Ca²⁺]_i). Thapsigargin, which increased [Ca²⁺]_i, induced further decrease in enzymatic cell detachment and increased cytotoxicity. Opposite effects were observed when 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid tetrakis, an intracellular Ca²⁺ chelator, was used. PDT-induced changes in [Ca²⁺]_i and cell detachment were not blocked by calcium channel antagonists nickel (Ni²⁺) or nimodipine, nor were they altered when cells were irradiated in a buffer free from Ca²⁺ and magnesium (Mg²⁺), suggesting that [Ca²⁺]_i is derived from the internal calcium stores. Decreased cell migration was observed after PDT, as assessed by chemotactic and wound-healing assays. Our findings indicated that internal calcium store-derived [Ca²⁺]_i plays an important role in PDT-induced enzymatic cell detachment decrease and cytotoxicity. Cell migration may be affected by these changes.     

Statin stimulates bone morphogenetic protein-2, aggrecan,and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and ³⁵S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2µM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.     

Precipitation of hydroxyapatite from dilute solutions upon seeding

Summary The kinetics of precipitation of hydroxyapatite, HA, was studied by seeding dilute supersaturated solutions with well characterized HA crystals. In solutions having initial degrees of supersaturation comparable to those present in human serum, the precipitation rates were related to the thermodynamic driving force (degree of supersaturation with respect to HA) and not to the solution composition. The following relationshipR ₀=KA(DS) ⁿ , whereR ₀=initial precipitation rate, A=amount of seeds, DS=degree of supersaturation, and K andn are parameters obtained from the experimental data, was found to apply over a DS range of 6.6×10¹⁰ to 3.3×10⁶. These observations are consistent with the occurrence of a simple growth process on the HA seeds. No evidence for the formation of discrete calcium phosphates other than HA was detected. The Ca/P molar ratio of the precipitating phase, calculated from solution compositions, was invariably higher than that expected for HA; this result is shown to be consistent with an initial adsorption phenomenon. Anomalous kinetic behavior was observed at low seeding levels and may relate to the surface phenomenon described. It is concluded that, most probably, under physiological conditions, formation and remineralization of hard tissues occur through the reported crystal growth process.     

Particulate cartilage under bioreactor-induced compression and shear

Purpose

Our aim was to explore the effect of varying in vitro culture conditions on general chondrogenesis of minced cartilage (MC) fragments.

Methods

Minced, fibrin-associated, bovine articular cartilage fragments were cultured in vitro within polyurethane scaffold rings. Constructs were maintained either free swelling for two or four weeks (control), underwent direct mechanical knee-joint-specific bioreactor-induced dynamic compression and shear, or they were maintained free swelling for two weeks followed by two weeks of bioreactor stimulation. Samples were collected for glycosaminoglycan (GAG)/DNA quantification; collagen type I, collagen type II, aggrecan, cartilage oligomeric matrix protein (COMP), proteoglycan-4 (PRG-4) messenger RNA (mRNA) analysis; histology and immunohistochemistry.

Results

Cellular outgrowth and neomatrix formation was successfully accomplished among all groups. GAG/DNA and collagen type I mRNA were not different between groups; chondrogenic genes collagen type II, aggrecan and COMP revealed a significant downregulation among free-swelling constructs over time (week two through week four). Mechanical loading was able to maintain chondrogenic expression with significantly stronger expression at long-term time points (four weeks) in comparison with four-week control. Histology and immunohistochemistry revealed that bioreactor culture induced stronger cellular outgrowth than free-swelling constructs. However, weaker collagen type II and aggrecan expression with an increased collagen type I expression was noted among this outgrowth neotissue.

Conclusions

The method of MC culture is feasible under in vitro free-swelling and dynamic loading conditions, simulating in vivo posttransplantation. Mechanical stimulation significantly provokes cellular outgrowth and long-term chondrogenic maturation at the mRNA level, whereas histology depicts immature neotissue where typical cartilage matrix is expected.     

Structure of chondroitin sulfate on aggrecan isolated from bovine tibial and costochondral growth plates

The structure of chondroitin sulfate on aggrecan isolated from the rib and proximal tibial growth plates of bovine fetuses was investigated, and the previously reported increase in the hydrodynamic size of chondroitin sulfate chains between the reserve and hypertrophic zones of the rib was confirmed in the tibial growth plate. Superose 6 gel chromatography, calibrated for chondroitin sulfate chain length by monosaccharide analysis, showed that the average molecular mass of chondroitin sulfate in the reserve and maturing zones of both growth plates was 21,600 and 30,400, respectively. Determination by capillary zone electrophoresis of the disaccharide composition of chains following chondroitinase digestion showed that ΔDi-0S, ΔDi-4S, and ΔDi-6S together accounted for more than 98% of the disaccharides in the digests from all zones of both growth plates; Δdisulfated and Δtrisulfated disaccharides were not detected. Furthermore, this analysis revealed a gradient in chondroitin sulfate composition from the reserve to the hypertrophic zone, characterized by a marked increase in the content of ΔDi-6S (from about 32% to about 52%) and a marked decrease in the content of ΔDi-4S (from about 53% to about 35%). Moreover, this altered pattern of sulfation was detected on chains of all sizes in the hypertrophic zone, suggesting that a proportion of the reserve zone aggrecan might be removed and replaced with aggrecan rich in chondroitin-6-sulfate synthesized during the proliferative and maturation stages of the resident chondrocytes. These data are discussed in relation to the biosynthetic mechanisms that control chondroitin sulfate chain length and sulfation on aggrecan and their modification during chondrocyte proliferation, maturation, and hypertrophy in the growth plate.     

Activation of rat nucleus pulposus cells by coculture with whole bone marrow cells collected by the perfusion method

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor‐β in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [³⁵S]‐sulfate and [³H]‐thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF‐β secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:222–228, 2009     

三步酶消化法高效分离兔原代关节软骨细胞及体外培养观察

目的　观察设计的三步酶消化法分离原代关节软骨细胞的效果,并对体外培养不同代次细胞的生物学活性进行评价。方法　三步酶消化法设计为以培养液配制的1g/L胰蛋白酶及1g/LEDTA混合液、1g/L透明质酸酶和2g/LⅠ型胶原酶顺序消化关节软骨分离细胞,检测细胞收获效率和存活率;体外传代培养观察细胞形态、生长及胞外基质中Ⅰ型和Ⅱ型胶原、蛋白多糖聚集体等的变化。结果　(1)关节软骨经三步酶消化基质逐步解离和降解,细胞得以完全释放和分离,每克软骨的细胞收获量平均为50 3×106 个细胞,细胞存活率平均为98. 8%。(2)原代和第一代细胞附壁生长呈三角形或多角形,生长融合时呈卵圆形,Ⅱ型胶原免疫组化和甲苯胺蓝异染反应均呈阳性,原代细胞外基质有高的硫酸糖胺多糖含量为(92±10)μg/cm2;第三代后细胞逐渐变为梭形,Ⅱ型胶原免疫组化为阴性,甲苯胺蓝异染反应明显减弱,第四代细胞外基质的硫酸糖胺多糖含量为(48±12)μg/cm2。结论　(1)三步酶消化法可使关节软骨基质完全消化降解,具有高细胞收获率、高细胞存活率和操作简便等特点。(2)原代和第一代软骨细胞具有良好的生物学活性,而第三代以后的细胞生物学活性低下。