Molecular characterization of spontaneous and growth-factor-augmented chondrogenesis in periosteum–bone tissue transferred into a joint

Multilineage potential of progenitor cells from periosteum is well established, but conditions for differentiation within their native niche are unclear. We evaluated at cellular and molecular levels whether chondrogenesis of periosteal progenitor cells is promoted spontaneously or by growth-factor mixture (GFM) application when transferring periosteum–bone cylinders into cartilage defects. Osteochondral defects in the patellar groove of minipigs were filled with periosteum–bone cylinders and randomly supplemented with GFM. Neochondrogenesis was characterized by histology, immunohistology, and quantitative gene expression analysis. According to morphology and glycosaminoglycan accumulation, spontaneous neocartilage formation occurred in the cambium layer already at 6 weeks, increased after 12 weeks, but declined until 52 weeks, independent of GFM. Multiple cartilage differentiation markers were induced after transfer. Expression of aggrecan, COMP, decorin, and Col10a1 increased significantly within 52 weeks. Sox 9 and Col2a1 mRNA levels were elevated at 6 versus 52 weeks in the GFM group and resulted in higher collagen type II protein accumulation. Neochondrogenesis was promoted in lower periosteum layers by transfer of periosteum–bone plugs into a joint, and collagen type II protein deposition was enhanced by GFM. The final tissue subsumed typical features of periosteum and fibrocartilage but lacked an intact tide mark and features of hyaline cartilage desired for cartilage repair.     

Adhesion of tissue-engineered cartilate to native cartilage

Reconstruction of cartilaginous defects to correct both craniofacial deformities and joint surface irregularities remains a challenging and controversial clinical problem. It has been shown that tissue-engineered cartilage can be produced in a nude mouse model. Before tissue-engineered cartilage is used clinically to fill in joint defects or to reconstruct auricular or nasal cartilaginous defects, it is important to determine whether it will integrate with or adhere to the adjacent native cartilage at the recipient site. The purpose of this study was to determine whether tissue-engineered cartilage would adhere to adjacent cartilage in vivo. Tissue-engineered cartilage was produced using a fibrin glue polymer (80 mg/cc purified porcine fibrinogen polymerized with 50 U/cc bovine thrombin) mixed with fresh swine articular chondrocytes. The polymer/chondrocyte mixture was sandwiched between two 6-mm-diameter discs of fresh articular cartilage. These constructs were surgically inserted into a subcutaneous pocket on the backs of nude mice (n = 15). The constructs were harvested 6 weeks later and assessed histologically, biomechanically, and by electron microscopy. Control samples consisted of cartilage discs held together by fibrin glue alone (no chondrocytes) (n = 10). Histologic evaluation of the experimental constructs revealed a layer of neocartilage between the two native cartilage discs. The neocartilage appeared to fill all irregularities along the surface of the cartilage discs. Safranin-O and toluidine blue staining indicated the presence of glycosaminoglycans and collagen, respectively. Control samples showed no evidence of neocartilage formation. Electron microscopy of the neocartilage revealed the formation of collagen fibers similar in appearance to the normal cartilage matrix in the adjacent native cartilage discs. The interface between the neocartilage and the native cartilage demonstrated neocartilage matrix directly adjacent to the normal cartilage matrix without any gaps or intervening capsule. The mechanical properties of the experimental constructs, as calculated from stress-strain curves, differed significantly from those of the control samples. The mean modulus for the experimental group was 0.74 +/- 0.22 MPa, which was 3.5 times greater than that of the control group (p < 0.0002). The mean tensile strength of the experimental group was 0.064 +/- 0.024 MPa, which was 62.6 times greater than that of the control group (p < 0.0002). The mean failure strain of the experimental group was 0.16 +/- 0.061 percent, which was 4.3 times greater than that of the control group (p < 0.0002). Finally, the mean fracture energy of the experimental group was 0.00049 +/- 0.00032 J, which was 15.6 times greater than that of the control group. Failure occurred in all cases at the interface between neocartilage and native cartilage. This study demonstrated that tissue-engineered cartilage produced using a fibrin-based polymer does adhere to adjacent native cartilage and can be used to join two separate pieces of cartilage in the nude mouse model. Cartilage pieces joined in this way can withstand forces significantly greater than those tolerated by cartilage samplesjoined only by fibrin glue.     

Immune evasion by neocartilage-derived chondrocytes: Implications for biologic repair of joint articular cartilage

Degeneration of joint articular cartilage is a leading cause of disability worldwide, and is due in large part to the fact that adult articular cartilage is unable to undergo effective intrinsic repair. To overcome this barrier, we have developed a tissue engineering strategy which harnesses the superior anabolic activity of juvenile chondrocytes to produce a scaffold-independent, living neocartilage graft. Preclinical studies demonstrate that bioengineered neocartilage survives allogeneic and xenogeneic transplantation, suggesting the utility of universal donor-derived neocartilage for joint repair. However, the mechanism underlying neocartilage transplant tolerance remains poorly understood. We show here that neocartilage-derived chondrocytes are unable to stimulate allogeneic T cells in vitro, and they do not constitutively express cell surface molecules required for induction of T cell immune responses, including major histocompatibility complex (MHC) Class II antigens and costimulatory molecules B7-1 and B7-2. Additionally, chondrocytes suppress, in a contact-dependent manner, the proliferation of activated T cells, with suppression associated with chondrocyte expression of multiple negative regulators of immune responses, including B7 family members (B7-H1, B7-DC, B7-H2, B7-H3, and B7-H4), chondromodulin-I and indoleamine 2,3-dioxygenase. Thus, the survival of transplanted bioengineered neocartilage may depend on both passive and active mechanisms of immune evasion.     

Injectable tissue-engineered cartilage using a fibrin glue polymer.

The purpose of this study was to demonstrate the feasibility of using a fibrin glue polymer to produce injectable tissue-engineered cartilage and to determine the optimal fibrinogen and chondrocyte concentrations required to produce solid, homogeneous cartilage. The most favorable fibrinogen concentration was determined by measuring the rate of degradation of fibrin glue using varying concentrations of purified porcine fibrinogen. The fibrinogen was mixed with thrombin (50 U/cc in 40 mM calcium chloride) to produce fibrin glue. Swine chondrocytes were then suspended in the fibrinogen before the addition of thrombin. The chondrocyte/polymer constructs were injected into the subcutaneous tissue of nude mice using chondrocyte concentrations of 10, 25, and 40 million chondrocytes/cc of polymer (0.4-cc injections). At 6 and 12 weeks, the neocartilage was harvested and analyzed by histology, mass, glycosaminoglycan content, DNA content, and collagen type II content. Control groups consisted of nude mice injected with fibrin glue alone (without chondrocytes) and a separate group injected with chondrocytes suspended in saline only (40 million cells/cc in saline; 0.4-cc injections). The fibrinogen concentration with the most favorable rate of degradation was 80 mg/cc. Histologic analysis of the neocartilage showed solid, homogeneous cartilage when using 40 million chondrocytes/cc, both at 6 and 12 weeks. The 10 and 25 million chondrocytes/cc samples showed areas of cartilage separated by areas of remnant fibrin glue. The mass of the samples ranged from 0.07 to 0.12 g at 6 weeks and decreased only slightly by week 12. The glycosaminoglycan content ranged from 2.3 to 9.4 percent for all samples; normal cartilage controls had a content of 7.0 percent. DNA content ranged from 0.63 to 1.4 percent for all samples, with normal pig cartilage having a mean DNA content of 0.285 percent. The samples of fibrin glue alone produced no cartilage, and the chondrocytes alone produced neocartilage samples with a significantly smaller mass (0.47 g at 6 weeks and 0.46 g at 12 weeks) when compared with all samples produced from chondrocytes suspended in fibrin glue (p < 0.03). Gel electrophoreses demonstrated the presence of type II collagen in all sample groups. This study demonstrates that fibrin glue is a suitable polymer for the formation of injectable tissue-engineered cartilage in the nude mouse model. Forty million chondrocytes per cc yielded the best quality cartilage at 6 and 12 weeks when analyzed by histology and content of DNA, glycosaminoglycan, and type II collagen.     

Effects of cell concentration and growth period on articular and ear chondrocyte transplants for tissue engineering

This study determined the effects of chondrocyte source, cell concentration, and growth period on cartilage production when isolated porcine cells are injected subcutaneously in a nude mouse model. Chondrocytes were isolated from both ear and articular cartilage and were suspended in Ham's F-12 medium at concentrations of 10, 20, 40, and 80 million cells per cubic centimeter. Using the nude mouse model, each concentration group was injected subcutaneously in 100-microl aliquots and was allowed to incubate for 6 weeks in vivo. In addition, cells suspended at a fixed concentration of 40 million cells per cubic centimeter were injected in 100-microl aliquots and were incubated for 1, 2, 3, 4, 5, 6, 9, and 12 weeks. Each concentration or time period studied contained a total of eight mice, with four samples harvested per mouse for a final sample size of 32 constructs. All neocartilage samples were analyzed by histologic characteristics, mass, glycosaminoglycan level, and DNA content. Control groups consisted of native porcine ear and articular cartilage.Specimen mass increased with increasing concentration and incubation time. Ear neocartilage was larger than articular neocartilage at each concentration and time period. At 40 million cells per cubic centimeter, both ear and articular chondrocytes produced optimal neocartilage, without limitations in growth. Specimen mass increased with incubation time up to 6 weeks in both ear and articular samples. No significant variations in glycosaminoglycan content were found in either articular or ear neocartilage, with respect to variable chondrocyte concentration or growth period. Although articular samples demonstrated no significant trends in DNA content over time, ear specimens showed decreasing values through 6 weeks, inversely proportional to increase in specimen mass. Although both articular and ear sources of chondrocytes have been used in past tissue-engineering studies with success, this study indicates that a suspension of ear chondrocytes injected into a subcutaneous location will produce biochemical and histologic data with greater similarity to those of native cartilage. The authors believe that this phenomenon is attributable to the local environment in which isolated chondrocytes from different sources are introduced. The subcutaneous environment of native ear cartilage accommodates subcutaneously injected ear chondrocyte transplants better than articular transplants. Native structural and biochemical cues within the local environment are believed to guide the proliferation of the differentiated chondrocytes.     

Chondromalacia induced by patellar subluxation: morphological and morphometrical aspects in rabbits

The purpose of this study was to describe morphologically and quantify the changes of the articular cartilage in chondromalacia, concerning both the chondrocytes and extracellular matrix. Eight rabbits were submitted daily to patellar subluxation, causing chondromalacia after two weeks. The knee fragments obtained were processed by the standard methods. These experimental conditions caused degenerative alterations of the articular cartilage, varying from a slight decrease of proteoglycans, to fibrillations, clefts, and horizontal splitting. The results showed a significantly increase number of chondrocytes (p < 0,000139), although smaller in size (p < 0,000109). The immobilization for 2 weeks and the intermittent passive daily motion afterwards for a period of 2 weeks, was effective to cause patellar chrondomalacia in rabbits.     

Thyroid hormones enhance the biomechanical functionality of scaffold-free neocartilage

IntroductionThe aim of this study was to investigate the effects of thyroid hormones tri-iodothyronine (T3), thyroxine (T4), and parathyroid hormone (PTH) from the parathyroid glands, known to regulate the developing limb and growth plate, on articular cartilage tissue regeneration using a scaffold-free in vitro model.MethodsIn Phase 1, T3, T4, or PTH was applied during weeks 1 or 3 of a 4-week neocartilage culture. Phase 2 employed T3 during week 1, followed by PTH during week 2, 3, or weeks 2 to 4, to further enhance tissue properties. Resultant neotissues were evaluated biochemically, mechanically, and histologically.ResultsIn Phase 1, T3 and T4 treatment during week 1 resulted in significantly enhanced collagen production; 1.4- and 1.3-times untreated neocartilage. Compressive and tensile properties were also significantly increased, as compared to untreated and PTH groups. PTH treatment did not result in notable tissue changes. As T3 induces hypertrophy, in Phase 2, PTH (known to suppress hypertrophy) was applied sequentially after T3. Excitingly, sequential treatment with T3 and PTH reduced expression of hypertrophic marker collagen X, while yielding neocartilage with significantly enhanced functional properties. Specifically, in comparison to no hormone application, these hormones increased compressive and tensile moduli 4.0-fold and 3.1-fold, respectively.ConclusionsThis study demonstrated that T3, together with PTH, when applied in a scaffold-free model of cartilage formation, significantly enhanced functional properties. The novel use of these thyroid hormones generates mechanically robust neocartilage via the use of a scaffold-free tissue engineering model.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0541-5) contains supplementary material, which is available to authorized users.     

Angiogenesis after administration of basic fibroblast growth factor induces proliferation and differentiation of mesenchymal stem cells in elastic perichondrium in an in vivo model: mini review of three sequential republication-abridged reports

To date, studies on mesenchymal tissue stem cells (MSCs) in the perichondrium have focused on in vitro analysis, and the dynamics of cartilage regeneration from the perichondrium in vivo remain largely unknown. We have attempted to apply cell and tissue engineering methodology for ear reconstruction using cultured chondrocytes. We hypothesized that by inducing angiogenesis with basic fibroblast growth factor (bFGF), MSCs or cartilage precursor cells would proliferate and differentiate into cartilage in vivo and that the regenerated cartilage would maintain its morphology over an extended period. As a result of a single administration of bFGF to the perichondrium, cartilage tissue formed and proliferated while maintaining its morphology for at least 3 months. By day 3 post bFGF treatment, inflammatory cells, primarily comprising mononuclear cells, migrated to the perichondrial region, and the proliferation of matrix metalloproteinase 1 positive cells peaked. During week 1, the perichondrium thickened and proliferation of vascular endothelial cells was noted, along with an increase in the number of CD44-positive and CD90-positive cartilage MSCs/progenitor cells. Neocartilage was formed after 2 weeks, and hypertrophied mature cartilage was formed and maintained after 3 months. Proliferation of the perichondrium and cartilage was bFGF concentration-dependent and was inhibited by neutralizing antibodies. Angiogenesis induction by bFGF was blocked by the administration of an angiogenesis inhibitor, preventing perichondrium proliferation and neocartilage formation. These results suggested that angiogenesis may be important for the induction and differentiation of MSCs/cartilage precursor cells in vivo, and that morphological changes, once occurring, are maintained.     

Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test

In this study, a hydrogel composite, based on the thermo-reversible hydrogel of p(NiPAAm-co-AAc) and hyaluronic acid (HA) was used as an injectable cell and growth factor carrier for cartilage tissue engineering applications. Rabbit chondrocytes were embedded in blended hydrogel composites co-encapsulated with the transforming growth factor beta-3 (TGFbeta-3). The blended hydrogel with the embedded chondrocytes and HA co-encapsulating unloaded growth factors and those with the thermo-reversible hydrogel were used as the controls to examine the effects of TGFbeta-3 on neocartilage formation. The blended hydrogel loaded with TGFbeta-3 embedded with chondrocytes were injected subcutaneously into the nude mice. The mice were monitored for 8 weeks after the injection. Both the differentiation and level of cartilage-specific ECM production were significantly higher in the presence of HA and growth factor than in the control without the growth factor. The level of cartilage associated ECM proteins was examined by immunohistochemical staining (collagen types II and X) as well as by Safranin-O and Alcian blue (GAG) staining. The results showed the potential application of blended hydrogel mixed with the growth factor to neocartilage formation.     

Transdermal photopolymerization of poly(ethylene oxide)-based injectable hydrogels for tissue-engineered cartilage

Transdermal photopolymerization, a minimally invasive method for implantation, was used to subcutaneously place a mixture of polymer and isolated chondrocytes to regenerate cartilage tissue in vivo. Semi-interpenetrating networks of varying proportions of poly(ethylene oxide)-dimethacrylate and poly(ethylene oxide) and primary bovine articular chondrocytes were implanted in athymic mice. Four mice (12 implants) were harvested at 2, 4, and 7 weeks. Chondrocytes survived implantation and photopolymerization and formed neocartilage containing 1.5 to 2.9% wet weight collagen and 4 to 7% glycosaminoglycan. Thirty-five percent of the total collagen was type II collagen. Histologic analysis exhibited tissue structure resembling neocartilage, and safranin O staining demonstrated glycosaminoglycan distribution throughout the hydrogels. This study demonstrates the potential use of transdermal photopolymerization for minimally invasive subcutaneous implantation of hydrogels and chondrocytes for in vivo cartilage regeneration.     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

Injectable tissue-engineered cartilage with different chondrocyte sources

Injectable engineered cartilage that maintains a predictable shape and volume would allow recontouring of craniomaxillofacial irregularities with minimally invasive techniques. This study investigated how chondrocytes from different cartilage sources, encapsulated in fibrin polymer, affected construct mass and volume with time. Swine auricular, costal, and articular chondrocytes were isolated and mixed with fibrin polymer (cell concentration of 40 x 10 cells/ml for all groups). Eight samples (1 cm x 1 cm x 0.3 cm) per group were implanted into nude mice for each time period (4, 8, and 12 weeks). The dimensions and mass of each specimen were recorded before implantation and after explantation. Ratios comparing final measurements and original measurements were calculated. Histological, biochemical, and biomechanical analyses were performed. Histological evaluations (n = 3) indicated that new cartilaginous matrix was synthesized by the transplanted chondrocytes in all experimental groups. At 12 weeks, the ratios of dimension and mass (n = 8) for auricular chondrocyte constructs increased by 20 to 30 percent, the ratios for costal chondrocyte constructs were equal to the initial values, and the ratios for articular chondrocyte constructs decreased by 40 to 50 percent. Constructs made with auricular chondrocytes had the highest modulus (n = 3 to 5) and glycosaminoglycan content (n = 4 or 5) and the lowest permeability value (n = 3 to 5) and water content (n = 4 or 5). Constructs made with articular chondrocytes had the lowest modulus and glycosaminoglycan content and the highest permeability value and water content (p < 0.05). The amounts of hydroxyproline (n = 5) and DNA (n = 5) were not significantly different among the experimental groups (p > 0.05). It was possible to engineer injectable cartilage with chondrocytes from different sources, resulting in neocartilage with different properties. Although cartilage made with articular chondrocytes shrank and cartilage made with auricular chondrocytes overgrew, the injectable tissue-engineered cartilage made with costal chondrocytes was stable during the time periods studied. Furthermore, the biomechanical properties of the engineered cartilage made with auricular or costal chondrocytes were superior to those of cartilage made with articular chondrocytes, in this model.     

Comprehensive Profiling of Cartilage Extracellular Matrix Formation and Maturation Using Sequential Extraction and Label-free Quantitative Proteomics

Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.The cartilage of the mammalian skeletal system has two distinct roles. The epiphyseal cartilage of the growth plate drives endochondral bone growth, and the hyaline cartilage at the weight-bearing surfaces of bones facilitates joint articulation. In both environments, chondrocyte-regulated production, assembly, and turnover of the extracellular matrix (ECM)¹ are essential for the tissue to withstand compressive forces and respond to mechanical loading. The major structural constituents of cartilage ECM are the heterotypic collagen II/IX/XI fibrils and proteoglycan-glycosaminoglycan networks of aggrecan and hyaluronan. Loss of joint function in osteoarthritis (OA) is strongly associated with net loss of aggrecan and collagen breakdown caused by an imbalance of ECM homeostasis (1). In addition, many inherited human chondrodysplasias involve disruption of cartilage matrix assembly or cell-matrix interactions, resulting in abnormal skeletal development and in some cases early onset cartilage degeneration (2, 3).The alterations in chondrocyte metabolism that occur during OA are complex and remain poorly understood (4). An early response to loss or fragmentation of ECM components is attempted tissue repair through secretion of anabolic factors, cell proliferation, and matrix remodeling (5). However, the resulting product is a fibrocartilage that does not recapitulate the composition or precise architecture of the original hyaline articular cartilage. This limited capacity of cartilage for regeneration has driven research into cartilage tissue engineering (6). Production of authentic hyaline cartilage in vitro remains challenging due to the dedifferentiation of primary chondrocytes upon removal from their three-dimensional matrix environment (7). However, improved “neocartilage” culture systems have been developed through evaluation of suitable chondroprogenitor or chondrocyte subpopulations and optimization of exogenous support matrices and growth factors (8, 9). The therapeutic target of neocartilage culture is autologous tissue repair. However, there is fundamental value in using neocartilage systems to elucidate mechanisms of protein integration into the ECM and the role of specific protein interactions during cartilage maturation.Cartilage profiling by 2-DE and mass spectrometry-based proteomics is generating important new insight into mechanisms of cartilage degeneration in vitro and in vivo (10). For example, anabolic factors with potential roles in cartilage repair, including connective tissue growth factor and inhibin βA (activin), were identified in the secretome of human OA cartilage explants (11). Comparison of cartilage protein extracts from normal donors and OA patients revealed significantly increased levels of the serine protease Htra1 in patient cartilage (12) and that Htra1-mediated proteolysis of aggrecan may significantly contribute to OA pathology (13). Targeted analysis of the chondrocyte mitochondrial proteome highlighted OA-related changes in energy production and protection against reactive oxygen species (14). Obtaining sufficient chondrocytes from human donors for proteomics unfortunately requires expansion of the cell population with potential loss of the chondrocyte phenotype during prolonged culture. Other drawbacks encountered with human samples include the clinical heterogeneity of OA, lack of matched controls, and inherent genetic variation of human subjects (15). Alternatively, animal models that recapitulate hallmarks of progressive cartilage degeneration, such as aggrecan loss and articular surface fibrillation, are emerging as a powerful resource, particularly in mice lacking specific proteases or protease target sites (16, 17). The development of techniques for analysis of murine cartilage using proteomics has paved the way for differential analysis of normal and pathological or genetically targeted cartilage (18, 19).Label-free methods for relative peptide quantitation, such as ion intensity measurement and spectral counting, are emerging as reliable and cost-effective alternatives to chemical modification or isotopic peptide labeling (20). Combining orthogonal protein and/or peptide fractionation with high resolution HPLC-MS can achieve proteome-wide coverage (21). Because extensive sample fractionation can introduce redundancy and variation, improved sequence/proteome coverage must be balanced against the cost of additional sample handling and lengthy LC-MS runs (22).Here we describe a novel platform for analysis of mouse cartilage using solubility-based protein fractionation (19) combined with label-free quantitative tandem MS (LTQ-Orbitrap). Sequential extraction of 3-day postnatal (P3) mouse epiphyseal cartilage and 3-week neocartilage cultures revealed a marked transition from a high proportion of readily soluble components in P3 extracts to a greater proportion of poorly soluble proteins in neocartilage. Principal component analysis and hierarchical clustering were used to globally assess the inter-relationships between P3 cartilage and neocartilage NaCl and guanidine hydrochloride (GdnHCl) extracts. At a p value cutoff of 0.05, 403 proteins were classified as extract-specific, whereas 125 proteins were classified as tissue sample-specific. Many of the proteins significantly enriched in neocartilage were annotated by the terms cell adhesion, extracellular matrix, and cytoskeletal remodeling. Further statistical analysis identified a third important protein category in which protein solubility was altered between the P3 and neocartilage. Identification of proteins involved in neocartilage maturation has generated novel insight into the fundamental process of cartilage matrix development with potential for further analysis of engineered cartilaginous tissues with biomedical applications.     

碱性成纤维细胞生长因子与胶原对组织工程软骨体外构建的影响

目的：以三维成团培养为培养系统,探讨bFGF与胶原对组织工程软骨体外构建的影响。方法：成团培养兔生长板软骨细胞,设bFGF、胶原及联合作用组。HE染色观察新生组织形态;免疫组化检测Ⅰ、Ⅱ型胶原表达以观察细胞表型;Hoechst 33258法检测细胞DNA含量;羟脯氨酸法与阿新蓝法测定基质中胶原与蛋白多糖的合成。结果：新生软骨的组织学形态近似自然软骨;各实验组软骨细胞DNA含量明显上升;胶原可以显著促进基质的合成;各实验组Ⅰ型胶原的表达少于对照组,Ⅱ型胶原的表达则高于对照组;联合作用组效果更加明显。结论：三维的成团培养可以促进基质合成,有效维持软骨细胞表型;bFGF与胶原有利于工程化软骨构建,其效果具有协同效应,两者联合应用可进一步促进软骨再生。     

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M^R) and the human hepatic cancer cell line SNU387 (SNU387^R) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387^R cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M^R cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.     

Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit

BACKGROUND: Although accumulating evidence shows that mesenchymal stem cells (MSC) are a promising cell source for articular cartilage repair, the fate of transplanted MSC has not been extensively studied. METHODS: To monitor their persistence and differentiation, we labeled uninduced MSC with a fluorescent dye, PKH26, and transplanted them, in a poly-glycolic-acid scaffold, to full-thickness defects made in the weight-bearing area of rabbit femoral trochleae with hyaluronate sheets. The fate of the labeled cells was monitored for up to 8 weeks. RESULTS: Two weeks after transplantation, immature cartilage containing collagen type II had formed. By 8 weeks, this cartilage had thinned and immunolabeling for collagen type II gradually disappeared from the basal region, which became positive for collagen type I. Most chondrocytes within the regenerated cartilage were PKH26-positive and, therefore, derived from transplanted MSC, whereas osteoblasts within the regenerated bone were a mixture of donor- and host-derived cells. The thickness of the cartilage became thinner up to 8 weeks and then remained stable up to 42 weeks after surgery. DISCUSSION: These results showed that uninduced MSC were able to survive osteochondral defects and differentiated according to the environment, making a major contribution to initial cartilage formation and a partial contribution to bone regeneration.     

Comparison of early passive motion and immobilization after flexor tendon repairs

Thirty-one flexor tendon repairs in 30 patients managed by early passive motion were retrospectively compared with 31 flexor tendon repairs in 30 patients managed by 3 weeks of postoperative immobilization. Repairs were performed by several surgeons, including plastic surgical residents. There were no statistically significant differences between the two groups comparing age, zone of injury, number of tendons repaired, nature of injury, or associated injuries. No statistically significant difference was found between the two groups when total active and total passive range of motion were compared for repairs in zone I, zone II, zones III and IV, and all zones combined. In the early passive motion group in zone II, there were 12 percent excellent results, 15 percent good results, 23 percent fair results, and 50 percent poor results. In the immobilization group, there were 18 percent excellent results, 8 percent good results, 23 percent fair results, and 53 percent poor results. There was no significant difference between the two groups (p less than 0.05).     

A comparison of the effects of immobilization and continuous passive motion on surgical wound healing in mature rabbits

The purpose of this investigation was to compare the effects of continuous passive motion (CPM) and cast immobilization on postoperative wound healing. Medical parapatellar skin incisions and arthrotomies were performed on both knees of 10 mature New Zealand rabbits. After closure of the incisions, one knee was immobilized in a cast while the other was treated by continuous passive motion for 3 weeks. Six standardized skin specimens (2 mm wide) from each wound were tested to failure and one specimen was examined histologically. With respect to the breaking force, tensile strength, strain at failure, stiffness, and toughness, the wounds in the continuous-passive-motion group were significantly stronger, stiffer, and tougher than those in the cast group. Histologically, the structural organization of the collagen fibers was also superior in the scars treated with continuous passive motion. The results of the present investigation indicate that compared to immobilization, continuous passive motion enhances postoperative wound healing in rabbits.     

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.     

CAGE, a novel cancer/testis antigen gene, promotes cell motility by activation ERK and p38 MAPK and downregulating ROS

We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.