Optimization of chondrocyte expansion in culture. Effect of TGF beta-2, bFGF and L-ascorbic acid on bovine articular chondrocytes

In vitro multiplication of isolated chondrocytes is needed to repair articular cartilage defects with autologous material. In this study we used monolayer cultures of bovine articular chondrocytes. The effect of transforming growth factor beta-2, basic fibroblast growth factor or L-ascorbic acid on cell multiplication, in the presence of 10% fetal calf serum, was measured in primary culture, the third and tenth passage. TGF beta-2 stimulated the proliferation of chondrocytes in the primary culture and L-ascorbic acid stimulated in the third passage. On the basis of these results, we chose an optimal addition scheme in which TGF beta-2 was added in primary culture and first passage, followed by addition of L-ascorbic acid in the second and third passage; this resulted in a 7-fold increase in cell number compared to the control group, in about 4 weeks. Our findings stress the importance of adding the right growth factor at the right moment. Collagen type II expression was lost after the third passage, in the control as well as in the experimental condition. The ability to produce hyaline cartilage specific matrix components is essential, if multiplied cells are to be used to repair cartilage defects.     

Influence of the growth factors PDGF‐BB,TGF‐β1 and bFGF on the replicative aging of human articular chondrocytes during in vitro expansion

Decreasing replicative potential and dedifferentiation of articular chondrocytes during expansion in cell culture are essential limitations for tissue engineering and cell therapy approaches. Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. There is evidence that growth factors are involved in regulating telomerase activity. Therefore, the objective was to evaluate the effect of selected growth factors on telomere biology of serially passaged chondrocytes. Human articular chondrocytes were isolated from cartilage of three patients undergoing total knee arthroplasty. The chondrocytes were cultured in monolayer with the growth factors PDGF‐BB, TGF‐β1, and bFGF. Telomere length was measured by telomere restriction fragment length assay, and telomerase activity was determined by quantifying the gene expression of its catalytic subunit hTERT by rtPCR. Chondrocytes cultured with PDGF‐BB and TGF‐β1 showed a significantly higher proliferation rate than control cells. None of the growth factor cultures revealed an accelerated rate of telomere shortening. Telomerase was not expressed in significant amounts in any of the chondrocyte cultures. Growth factor treatment of chondrocyte cell cultures for cell therapy purposes can be regarded as safe in terms of telomere biology. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:354–360, 2010     

Lead induces an osteoarthritis‐like phenotype in articular chondrocytes through disruption of TGF‐β signaling

Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF‐β signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF‐β signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF‐β signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose‐dependent manner. Active caspase 3 and 8 were dose‐dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF‐β reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose‐dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1760–1766, 2012     

Adult human articular chondrocytes in a microcarrier‐based culture system: expansion and redifferentiation

Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer‐expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:539–546, 2011     

Inhibition of TGF‐β signaling promotes expansion of human epidermal keratinocytes in feeder cell co‐culture

Cultured epidermal autografts have been used worldwide since 1981 for patients with extensive third‐degree burn wounds and limited skin donor sites. Despite significant progress in techniques toward improving clinical outcome of skin grafts, the long in vitro preparation time of cultured autografts has remained a major factor limiting its widespread use. Here, we show that pharmacological inhibition of TGF‐β signaling promotes the expansion of human epidermal keratinocytes (HEKs) with high proliferative potential in co‐cultures with both murine 3T3‐J2 cells and human feeder cells, including dermal fibroblasts and preadipocytes. In contrast, TGF‐β signaling inhibition does not enhance the growth of HEKs in a serum‐ and feeder‐free condition, an alternative approach to propagate HEKs for subsequent autograft production. Our results have important implications for the use of TGF‐β signaling inhibition as a viable therapeutic strategy for improving Green's methodology and for more efficient production of customized skin autografts with human feeder cells.     

Regulation of the friction coefficient of articular cartilage by TGF‐β1 and IL‐1β

Articular cartilage functions to provide a low‐friction surface for joint movement for many decades of life. Superficial zone protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary lubrication. In both cell and explant cultures, TGF‐β1 and IL‐1β have been demonstrated to, respectively, upregulate and downregulate SZP protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF‐β1 or IL‐1β) and a smooth glass surface due to sliding in the boundary lubrication regime was measured with a pin‐on‐disk tribometer. SZP was quantified using an enzyme‐linked immunosorbant assay and localized by immunohistochemistry. Both TGF‐β1 and IL‐1β treatments resulted in the decrease of the friction coefficient of articular cartilage in a location‐ and time‐dependent manner. Changes in the friction coefficient due to the TGF‐β1 treatment corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL‐1β treatment were independent of SZP depth of staining. However, the changes induced by the IL‐1β treatment corresponded to changes in surface roughness, determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of articular cartilage can be modified by TGF‐β1 and IL‐1β treatment and that the friction coefficient depends on multiple factors, including SZP localization and surface roughness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:249–256, 2009     

重组PcDNA3-hBMP3转染兔关节软骨细胞和对其增殖的影响

目的研究将人骨形成蛋白３（ＢＭＰ３）的ｃＤＮＡ构建于真核,表达载体ＰｃＤＮＡ３,形成重组ＤＮＡ ＰｃＤＮＡ３－ｈＢＭＰ３,转染兔关节软骨细胞,以探讨基因转染对关节软骨细胞增殖的影响。方法利用重组ＤＮＡ和基因克隆技术构建重组ＤＮＡ;用细胞培养和基因转染技术体外转染兔关节软骨细胞;用核酸杂交和蛋白电泳检测细胞ＢＭＰ３的表达,最后通过ＦＣＭ、ＤＮＡ和葡糖醛酸含量检测以及Ⅱ型胶原探针原位杂交分析其对增殖的影响。结果软骨细胞６次传代后,Ⅱ型胶原原位杂交的灰度值降低,同样条件下转染的软骨细胞仍保持较高水平Ⅱ型胶原的表达;转染的软骨细胞经流式细胞仪的分析,软骨细胞Ｓ期细胞比例增多,说明细胞ＤＮＡ的合成增加;经ＤＮＡ和葡糖醛酸含量测定,转染的软骨细胞ＤＮＡ和葡糖醛酸含量较转染前有明显提高。结论ｈＢＭＰ３对维持软骨细胞的表型具有十分重要的作用,在脂质体的介导下,ＤＮＡ ＰｃＤＮＡ３－ｈＢＭＰ３转染兔关节软骨细胞获得成功。     

Effect of dose and release rate of CTGF and TGFβ3 on avascular meniscus healing

Meniscus tears in the avascular region rarely functionally heal due to poor intrinsic healing capacity, frequently resulting in tear propagation, followed by meniscus deterioration. Recently, we have reported that time‐controlled application of connective tissue growth factor (CTGF) and transforming tissue growth factor β3 (TGFβ3) significantly improved healing of avascular meniscus tears by inducing recruitment and step‐wise fibrocartilaginous differentiation of mesenchymal stem/progenitor cells (MSCs). In this study, we investigated effects of the dose of CTGF and the release rate of TGFβ3 on avascular meniscus healing in our existing explant model. Our hypothesis was that dose and release rate of CTGF and TGFβ3 are contributing factors for functional outcome in avascular meniscus healing by stem cell recruitment. Low (100 ng/ml) and high (1,000 ng/ml) doses of CTGF as well as fast (0.46 ± 0.2 ng/day) and slow (0.29 ± 0.1 ng/day) release rates of TGFβ3 were applied to our established meniscus explant model for meniscus tears in the inner‐third avascular region. The release rate of TGFβ3 was controlled by varying compositions of poly(lactic‐co‐glycolic acids) (PLGA) microspheres. The meniscus explants were then cultured for 8 weeks on top of mesenchymal stem/progenitor cells (MSCs). Among the tested combinations, we found that a high CTGF dose and slow TGFβ3 release are most effective for integrated healing of avascular meniscus, demonstrating improvements in alignment of collagen fibers, fibrocartilaginous matrix elaboration and mechanical properties. This study may represent an important step toward the development of a regenerative therapy to improve healing of avascular meniscus tears by stem cell recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1555–1562, 2019.     

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension.

OBJECTIVE: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. METHODS: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method. RESULTS: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures. CONCLUSION: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.     

Effects of polymethylmethacrylate on rabbit articular chondrocytes in monolayer culture

The effects of polymethylmethacrylate (PMMA) on DNA, protein, and sulfated-proteoglycan synthesis by rabbit articular chondrocytes were observed in monolayer cultures. PMMA pellets in ratios of 1:1 and 1:2 (liquid monomer:powder) significantly reduced [3H]thymidine incorporation into DNA during the first 24 h of culture and less so after 48 and 72 h. The reduction in [3H]thymidine incorporation was restricted to the cohort of chondrocytes nearest the PMMA. Consequently, cellular proliferation was unaltered by PMMA. By contrast, PMMA failed to inhibit [3H]leucine or [3H]serine/35SO4 incorporation. Both control and PMMA (1:1)-treated chondrocyte CsCl density gradient medium fraction dA1 eluted as a retarded peak on Sepharose CL-2B under associative conditions. The average partition coefficient (Kav) of PMMA-treated fraction dA1 was 0.41, as compared with 0.27 for control cultures. The Kav of medium fraction dD1 (proteoglycan monomer) was unaltered. Both control and PMMA-treated dA1 fraction elution profiles on Sepharose CL-2B were altered by incubation with Streptomyces hyaluronidase, indicating the presence of proteoglycan aggregate. The PMMA-treated cultures synthesized smaller proteoglycan aggregates. Since PMMA has been a critical factor in the success of total joint arthroplasty, defining interactions of differentiated cells with the cement is imperative for an understanding of the effects of PMMA on the biology of cartilage and bone.     

Cell attachment, collagen binding, and receptor analysis on bovine articular chondrocytes.

The purpose of this study was to investigate collagen receptors on primary bovine articular chondrocytes from full-thickness and different layers of bovine articular cartilage. Cytometric studies with antibodies showed that approximately 56% of the chondrocytes from the superficial layer and 29% of the chondrocytes from the deep layer bound anti-annexin V. A similar tendency was found for alpha5 and beta1 integrin antibodies. Flow cytometric analysis initially detected annexin V on chondrocytes following isolation; the level of detection subsequently decreased by 24 hours, whereas that of alpha5 and beta1 integrins increased. Treatment of chondrocytes with collagenase at 24 hours restored the initially high epitope recognition of annexin V, indicating masking of annexin V by newly formed collagen matrix. There was little effect on detection levels for beta1 integrin. Contrary to the specific matrix receptor expression, chondrocytes from superficial and deep layers differed little in attachment to immobilized types I and II collagens. However, the attachment was more effectively inhibited with anti-annexin V than with integrin antibodies. Competition studies with preparations of soluble collagens revealed a preferential binding of bovine type-II collagen compared with bovine type-I collagen. Anti-annexin V antibodies inhibited binding of type-II collagen more effectively than anti-alpha5 or anti-beta1 integrin antibodies. Evidently, under the in vitro conditions of this study, annexin V is the quantitatively predominant type-II collagen receptor on bovine articular chondrocytes. This opens a discussion of the possibly dualistic metabolic/mechanical annexin V-integrin receptor elements.     

Effect of transforming growth factor-β and platelet-derived growth factor-BB on articular cartilage in rats

The short-term and long-term effects on the growth zone in articular cartilage of transforming growth factor-β1 and platelet-derived growth factor-BB injected intraarticularly into the knee joint of growing rats were investigated. The changes induced by five injections of 0.5 μg of transforming growth factor-β1 included a rapid decrease in the size and number of hypertrophic cells and an enhanced subchondral bone formation. The changes were most marked in the patella but were also apparent in the tibia and femur. The proliferating cells became swollen and lost their normal organization. From the seventh day of the experiment to about 3 weeks, the matrix stained intensely with safranin O for proteoglycans. The alterations induced by transforming growth factor-β also included synovial fibroplasia and synovitis, consisting predominantly of mononuclear cells. Localised necroses in the cartilage sometimes appeared after 21 days. In long-term studies, destroyed cartilage was found in three of six rats and partial ossification of the joint cartilage was found in two after 90 and 180 days. Ossicles developed in the tendons in all six patellae. Injection of platelet-derived growth factor-BB resulted in an early and transitory minor increase in the osteogenic activity in the zone between cartilage and red bone marrow and later produced an ossicle in one of four tendons. None of the other changes noted after injection of transforming growth factor was observed.     

Hyperpolarisation of cultured human chondrocytes following cyclical pressure-induced strain: Evidence of a role for α5β1 integrin as a chondrocyte mechanoreceptor

Mechanical stimuli influence chondrocyte metabolism, inducing changes in intracellular cyclic adenosine monophosphate and proteoglycan production. We have previously demonstrated that primary monolaycr cultures of human chondrocyles have an electropysiological response after intermittent pressure-induced strain characterised by a membrane hyperpolarisation of approximately 40%. The mechanisms responsible for these changes are not fully understood but potentially involve signalling molecules such as integrins that link extracellular matrix with cytoplasmic components. The results reported in this paper demonstrate that the transduction pathways involved in the hyperpolarisation response of human articular chondrocytes in vitro after cyclical pressure-induced strain involve α5β1 integrin. We have demonstrated, using pharmacological inhibitors of a variety of intracellular signalling pathways, that the actin cytoskeleton, the phospholipase C calmodulin pathway, and both tyrosine protein kinase and protein kinase C activities are important in the transduction of the electrophysiological response. These results suggest that α5β1 is an important chondrocyte mechanoreceptor and a potential regulator of chondrocyte function.     

Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011