Deer antler extract potentially facilitates xiphoid cartilage growth and regeneration and prevents inflammatory susceptibility by regulating multiple functional genes.

BACKGROUND: Deer antler is a zoological exception due to its fantastic characteristics, including amazing growth rate and repeatable regeneration. Deer antler has been used as a key ingredient in traditional Chinese medicine relating to kidney and bone health for centuries. The aim of this study was to dissect the molecular regulation of deer antler extract (DAE) on xiphoid cartilage (XC). METHODS: The DAE used in this experiment was same as the one that was prepared as previously described. The specific pathogen-free (SPF) grade Sprague-Dawley (SD) rats were randomly divided into blank group (n =10) and DAE group (n =10) after 1-week adaptive feeding. The DAE used in this experiment was same as the one that was prepared as previously described. The rats in DAE group were fed with DAE for 3 weeks at a dose of 0.2 g/kg per day according to the body surface area normalization method, and the rats in blank group were fed with drinking water. Total RNA was extracted from XC located in the most distal edge of the sternum. Illumina RNA sequencing (RNA-seq) in combination with quantitative real-time polymerase chain reaction (qRT-PCR) validation assay was carried out to dissect the molecular regulation of DAE on XC. RESULTS: We demonstrated that DAE significantly increased the expression levels of DEGs involved in cartilage growth and regeneration, but decreased the expression levels of DEGs involved in inflammation, and mildly increased the expression levels of DEGs involved in chondrogenesis and chondrocyte proliferation. CONCLUSIONS: Our findings suggest that DAE might serve as a complementary therapeutic regent for cartilage growth and regeneration to treat cartilage degenerative disease, such as osteoarthritis.

Correlation of T2* relaxation times of the retropatellar cartilage with tibial tuberosity-trochlea groove distance in professional soccer players.

The tibial tuberosity-trochlear groove (TT-TG) distance is a radiographic measurement that is used to quantify malalignment of the patellofemoral joint (PFJ) in cross-sectional imaging. There is an ongoing debate about the impact of the TT-TG-distance on lateral patellar instability and the initiating of cartilage degeneration. In this prospective study, the association of T2* relaxation times and TT-TG distances in professional soccer players was analyzed. 36 knees of 18 professional soccer players (age: 21 ± 2.8 years) were evaluated. Participants underwent knee MRI at 3 T. For qualitative image analysis, fat-saturated 2D PD-weighted Fast Spin Echo (FSE) and T1-weighted FSE sequences were used. For quantitative analysis, T2* measurements in 3D data acquisitions were performed. In a qualitative analysis there was no structural cartilage damage and no abnormalities of the patellar and trochlea shape. The highest T2* values (26.7 ± 5.9 ms) were observed in the central compartment of the patella. The mean TT-TG distance was 10 ± 4 mm (range 3-20 mm). There was no significant correlation between TT-TG distance and T2* relaxation times in all three compartments of the retropatellar cartilage. Our study shows that so long as patellar and trochlear morphology is normal, TT-TG distance alone does not affect the tissue structure of the retropatellar cartilage in professional soccer players.

Regenerative effects of hyperbaric oxygen therapy and platelet-rich plasma on the osteochondral defects of rats.

OBJECTIVES: This study aims to investigate the effects of hyperbaric oxygen (HBO) therapy and platelet-rich plasma (PRP) on the regeneration of osteochondral defects of the rats, and the synergistic effect of this combined treatment. MATERIALS AND METHODS: This randomized, controlled, and interventional animal study was conducted between May 2014 and August 2014 Osteochondral regeneration was evaluated in four treatment groups (control, PRP, HBO, and HBO+PRP groups) at the 30th day after iatrogenic injury. Thirty-two female Wistar albino rats (weighing 248-305 g) underwent arthrotomy and osteochondral surgery on left knees. The regenerations of defects were then examined histologically by the modified version of O'Driscoll score. RESULTS: Groups that were treated with either HBO or PRP alone regenerated significantly better than the control group (p=0.01), while no significant difference was found between the HBO- and PRP-treated groups (p>0.05). The defects in group 4 (treated with both HBO and PRP) regenerated significantly better than the control group, the HBO-treated group alone, and the PRP-treated group alone (p=0.01). CONCLUSION: The results of this study showed a synergistic effect of HBO and PRP on knee cartilage regeneration. However, the possible underlying mechanisms should be the subject of future researches. The aggregation and activation of growth factors released from platelets whose activation is increased in the hyperbaric environment may explain this effect. This may result in a better regeneration than the effect of PRP or HBO alone.

In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold.

BACKGROUND: Cartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP). METHODS: Human ADSCs and PRP were characterized. A silk fibroin scaffold with 500 µm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively. RESULTS: Cells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-ß1 (TGF-ß1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21. CONCLUSION: Our study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

Glycosaminoglycans and vitamin C affect broiler bone parameters.

The purpose of this study was to determine if in ovo feeding and rearing with glycosaminoglycans and vitamin C could influence bone and cartilage macroscopy, mineral composition, mineral density and surface area, bone breaking strength, and bone histology in broilers. Fertile eggs from breeders (Cobb) were either uninjected or injected with 4 µg of additive/100 µL water on day 4 of incubation. Every 100 g of in ovo additive contained 30 g of chondroitin sulfate, 30 g of glucosamine, and 5 g of vitamin C. After hatching, the chicks from both incubation treatments were submitted to additional treatments during the growth phase from 1 to 42 D of age (diet without and with 0.74 g of additive/kg of feed). Every 100 kg of feed contained 30 g of glucosamine sulfate, 24 g of chondroitin sulfate, and 20 g of vitamin C. A completely randomized factorial design (2 × 2) was applied. The data were submitted to variance analysis using the general linear model procedure of SAS (SAS Institute). In ovo feeding with 4 µg of additive plus dietary supplementation with 0.74 g of additive/kg of feed resulted in the highest cartilage weight of the femur proximal epiphysis in birds (P = 0.0098). The highest ash, phosphorus and calcium percentage, mineral density and mineral composition were identified for femur and tibia in the following treatments: in ovo feeding plus diet without additive during rearing, or uninjected eggs plus dietary supplementation during rearing. In ovo feeding with 4 µg of the additive reduced (P = 0.0008) the number of chondrocytes in the proximal epiphysis of the tibia cartilage and increased (P < 0.0001) the number of osteocytes in the tibia diaphysis of broilers. We conclude that in ovo feeding or dietary supplementation during broiler rearing with glycosaminoglycans (chondroitin sulfate and glucosamine sulfate) and vitamin C benefits the development of bird bones and cartilage, and may represent a solution to bone problems in broilers.

Biomechanical analysis of the meniscus and cartilage of the knee during a typical Tai Chi movement-brush-knee and twist-step.

This study aimed to analyze the biomechanical response of the knee cartilage and meniscus to a typical Tai Chi (TC) movement, brush-knee and twist-step (BKTS). Kinematic and kinetic data was recorded while an experienced TC practitioner performed normal walking, jogging and BKTS. The kinetic data were then imported into a validated finite element model of the knee joint to examine the biomechanical response of the articular cartilage and meniscus. Compared with walking and jogging, the BKTS movement showed a greater increase in the range of motion (ROM) of the knee. The ROM in the sagittal plane was 56° (walking), 38° (jogging) and 93° (BKTS). In coronal plane, the knee ROM was 8° (walking), 11° (jogging) and 28° (BKTS). And in horizontal plane the ROM was 17° (walking), 15° (jogging) and 29° (BKTS). The finite element simulation demonstrated that the pressure contact stress is much more concentrated during walking and jogging than BKTS, which is consistent with the lower peak contact stresses recorded on the cartilage and meniscus. In conclusion, the TC movement produced a gentler stress state on the meniscus and cartilage, while also requiring a greater knee ROM. Practicing TC may have a lower risk of knee joint injury compared to walking and jogging.

Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen-induced arthritis.

Rheumatoid arthritis (RA) is an inflammatory joint disease that eventually leads to permanent bone and cartilage destruction. Fas has already been established as the regulator of inflammation in RA, but its role in bone formation under arthritic conditions is not completely defined. The aim of this study was to assess the effect of Fas inactivation on the bone damage during murine antigen-induced arthritis. Subchondral bone of wild-type (WT) and Fas-knockout (Fas-/-) mice was evaluated by histomorphometry and microcomputerized tomography. Proportions of synovial bone and cartilage progenitors were assessed by flow cytometry. Synovial bone and cartilage progenitors were purified by fluorescence-activated cell sorting and expression of Fas and Fas-induced apoptosis were analyzed in vitro. Results showed that Fas-/- mice developed attenuated arthritis characterized by preserved epiphyseal bone and cartilage. A proportion of the earliest CD200+ bone and cartilage progenitors was reduced in WT mice with arthritis and was unaltered in Fas-/- mice. During osteoblastic differentiation in vitro, CD200+ cells express the highest levels of Fas and are removed by Fas ligation. These results suggest that Fas-induced apoptosis of early CD200+ osteoprogenitor population represents potential mechanism underlying the impaired bone formation in arthritis, so their preservation may represent the bone-protective mechanism during arthritis.-Lazic Mosler, E., Lukac, N., Flegar, D., Fadljevic, M., Radanovic, I., Cvija, H., Kelava, T., Ivcevic, S., Sucur, A., Markotic, A., Katavic, V., Marusic, A., Grcevic, D., Kovacic, N. Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen-induced arthritis.

Activated platelet-rich plasma improves cartilage regeneration using adipose stem cells encapsulated in a 3D alginate scaffold.

In the current study, the effect of superimposing platelet-rich plasma (PRP) on different culture mediums in a three-dimensional alginate scaffold encapsulated with adipose-derived mesenchymal stem cells for cartilage tissue repair is reported. The three-dimensional alginate scaffolds with co-administration of PRP and/or chondrogenic supplements had a significant effect on the differentiation of adipose mesenchymal stem cells into mature cartilage, as assessed by an evaluation of the expression of cartilage-related markers of Sox9, collagen II, aggrecan and collagen, and glycosaminoglycan assays. For in vivo studies, following induction of osteochondral lesion in a rabbit model, a high degree of tissue regeneration in the alginate plus cell group (treated with PRP plus chondrogenic medium) compared with other groups of cell-free alginate and untreated groups (control) were observed. After 8 weeks, in the alginate plus cell group, functional chondrocytes were observed, which produced immature matrix, and by 16 weeks, the matrix and hyaline-like cartilage became completely homogeneous and integrated with the natural surrounding cartilage in the defect site. Similar effect was also observed in the subchondral bone. The cell-free scaffolds formed fibrocartilage tissue, and the untreated group did not form a continuous cartilage over the defect by 16 weeks.

Design and evaluation of chitosan/poly(l-lactide)/pectin based composite scaffolds for cartilage tissue regeneration.

Poor regenerative potential of cartilage tissue due to the avascular nature and lack of supplementation of reparative cells impose an important challenge in recent medical practice towards development of artificial extracellular matrix with enhanced neo-cartilage tissue regeneration potential. Chitosan (CH), poly (l-lactide) (PLLA), and pectin (PC) compositions were tailored to generate polyelectrolyte complex based porous scaffolds using freeze drying method and crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) solution containing chondroitin sulfate (CS) to mimic the composition as well as architecture of the cartilage extracellular matrix (ECM). The physical, chemical, thermal, and mechanical behaviors of developed scaffolds were done. The scaffolds were porous with homogeneous pore structure with pore size 49-170µm and porosities in the range of 79 to 84%. Fourier transform infrared study confirmed the presence of polymers (CH, PLLA and PC) within the scaffolds. The crystallinity of the scaffold was examined by the X-ray diffraction studies. Furthermore, scaffold shows suitable swelling property, moderate biodegradation and hemocompatibility in nature and possess suitable mechanical strength for cartilage tissue regeneration. MTT assay, GAG content, and attachment of chondrocyte confirmed the regenerative potential of the cell seeded scaffold. The histopathological analysis defines the suitability of scaffold for cartilage tissue regeneration.

Suppression of Cartilage Degradation by Zingerone Involving the p38 and JNK MAPK Signaling Pathway.

Zingerone, an active compound that is present in cooked ginger, has been claimed to be a bioactive ingredient that holds the potential of preventing and/or treating diseases involving inflammation. In this study, zingerone was used to discover its properties against joint inflammation using interleukin-1ß-induced osteoarthritis in cartilage explant and cell culture models. Zingerone was supplemented into the cartilage explant and cell culture media at different concentrations along with the presence of interleukin-1ß, an inducer of osteoarthritis. Markers indicating cartilage degradation, inflammation, and the signaling molecules involved in the inflammatory induction were investigated. Diacerien, an anti-osteoarthritic drug, was used as a positive control. Zingerone at a concentration of 40 µM reduced the level of matrix metalloproteinase-13 to about 31.95 ± 4.33 % compared with the interleukin-1ß-treated group and halted cartilage explant degradation as indicated by reducing the accumulative release of sulfated glycosaminoglycans by falling to the control concomitantly with an elevation of the remaining contents of uronic acid and collagen in the explant tissues when zingerone was added. In the SW1353 cell line model, zingerone efficiently suppressed the expression of TNF-α, interleukin-6, and interleukin-8 mRNA levels and tended to reduce the levels of both p38 and c-Jun N-terminal kinase phosphorylation. From the results of this study, it can be concluded that zingerone potentially reduced cartilage degradation, which is partially involved in p38 and c-Jun N-terminal kinases of the mitogen activator protein kinase signaling pathway leading to the reduction of proinflammatory cytokine amplification effects and cartilage-degrading enzyme syntheses. This finding supports the contention that ginger holds positive pharmaceutical effects against osteoarthritis.

Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis.

Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.

sb203580 preconditioning recharges matrix-expanded human adult stem cells for chondrogenesis in an inflammatory environment - A feasible approach for autologous stem cell based osteoarthritic cartilage repair.

Autologous stem cells are a promising cell source for cartilage regeneration; however, cell replicative senescence and joint posttraumatic inflammation provide challenges in bringing this treatment modality to fruition. In this study, we hypothesized that preconditioning with p38 MAPK inhibitor (sb203580) would recharge decellularized extracellular matrix (dECM) expanded human synovium-derived stem cell (hSDSC) chondrogenesis in an inflammatory environment. We found that preconditioning with sb203580 greatly enhanced dECM expanded hSDSC proliferation and chondrogenic potential while supplementation with sb203580 in an induction medium dramatically retarded hSDSC chondrogenic differentiation, even for dECM expanded cells. We also found that sb203580 preconditioning enhanced matrix-expanded hSDSC chondrogenic capacity even in an interleukin-1 (IL-1) induced inflammatory environment. Non-detectable expression of HLA-DR in the hSDSCs grown on allogeneic dECM indicates the feasibility of commercial preparation of these dECMs from healthy, young donors for patients who need autologous transplantation. Our study indicated that p38 MAPK inhibitor has a distinctive priming effect on dECM mediated stem cell cartilage regeneration. Combined rejuvenation with sb203580 and dECM expansion can precondition hSDSCs' resurfacing capacity for osteoarthritic patients with cartilage defects.

Psoralen activates cartilaginous cellular functions of rat chondrocytes in vitro.

CONTEXT: Psoralen, an active ingredient from Fructus Psoraleae (FP), is used in Traditional Chinese Medicine (TCM) to treat bone diseases. However, the effect of psoralen on cartilage is unknown. OBJECTIVE: To investigate the effects of psoralen on chondrocytes isolated from rats. MATERIALS AND METHODS: Chondrocytes were treated with different concentrations of psoralen (1, 10, and 100 µM) in vitro at 3-d and 9-d intervals. MTS assay, Alcian blue colorimetry, western blotting, and qRT-PCR, respectively, were used to evaluate the effects of psoralen on cell viability, glycosaminoglycan (GAG) synthesis, collagen synthesis, and cartilage-specific gene expression. RESULTS: Psoralen dosages of 1-10 µM exhibited low cytotoxicity toward chondrocytes. However, a dosage of 100 µM suppressed the proliferation of chondrocytes. Different concentrations of psoralen treatments on chondrocytes revealed that GAG and Type II collagen synthesis increased, especially at 100 µM, by 0.39-fold and 0.48-fold, respectively, on day 3, and by 0.51-fold and 0.56-fold, respectively, on day 9. Similarly, gene expression of Type II collagen, aggrecan, and SOX-9 were all up-regulated on days 3 and 9, particularly aggrecan which increased significantly by 9.37-fold and 7.32-fold at 100 µM. Additionally, Type I collagen was inhibited both in gene expression and in protein synthesis. CONCLUSION: The results showed that psoralen promotes cartilaginous extracellular matrix (ECM) synthesis, as well as increased cartilaginous gene expression, and it may be a useful bioactive component for activating the cartilaginous cellular functions of chondrocytes.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in a simulated osteochondral environment is hydrogel dependent.

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.

Duplicated zebrafish co-orthologs of parathyroid hormone-related peptide (PTHrP, Pthlh) play different roles in craniofacial skeletogenesis.

In mammals, parathyroid hormone-related peptide (PTHrP, alias PTH-like hormone (Pthlh)) acts as a paracrine hormone that regulates the patterning of cartilage, bone, teeth, pancreas, and thymus. Beyond mammals, however, little is known about the molecular genetic mechanisms by which Pthlh regulates early development. To evaluate conserved pathways of craniofacial skeletogenesis, we isolated two Pthlh co-orthologs from the zebrafish (Danio rerio) and investigated their structural, phylogenetic, and syntenic relationships, expression, and function. Results showed that pthlh duplicates originated in the teleost genome duplication. Zebrafish pthlha and pthlhb were maternally expressed and showed overlapping and distinct zygotic expression patterns during skeletal development that mirrored mammalian expression domains. To explore the regulation of duplicated pthlh genes, we studied their expression patterns in mutants and found that both sox9a and sox9b are upstream of pthlha in arch and fin bud cartilages, but only sox9b is upstream of pthlha in the pancreas. Morpholino antisense knockdown showed that pthlha regulates both sox9a and sox9b in the pharyngeal arches but not in the brain or otic vesicles and that pthlhb does not regulate either sox9 gene, which is likely related to its highly degraded nuclear localization signal. Knockdown of pthlha but not pthlhb caused runx2b overexpression in craniofacial cartilages and premature bone mineralization. We conclude that in normal cartilage development, sox9 upregulates pthlh, which downregulates runx2, and that the duplicated nature of all three of these genes in zebrafish creates a network of regulation by different co-orthologs in different tissues.

A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.

Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-ß3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation of a more stable chondrogenic phenotype.

Requirement for serum in medium supplemented with insulin-transferrin-selenium for hydrodynamic cultivation of engineered cartilage.

Achievement of viable engineered tissues through in vitro cultivation in bioreactor systems requires a thorough understanding of the complex interplay between hydrodynamic forces and biochemical cues such as serum. To this end, chondrocyte-seeded constructs were cultured under continuous fluid-induced shear forces with reduced serum content (0%-2%, v/v), which was partially or completely replaced by a potential substitute, insulin-transferrin-selenium, to minimize deleterious effects associated with the use of culture media containing high levels of serum (10%-20%). Low-serum cultures yielded constructs with similar biochemical properties to those cultivated with high-serum supplements, whereas the serum-free constructs exhibited poor cell proliferation, insufficient extracellular matrix production, and rapid degradation of and/or shear-induced damage to polyglycolic acid scaffolds. A fibrous outer capsule typically observed in hydrodynamic cultures and characterized by increased cell density and decreased (virtually none) glycosaminoglycan deposition was eliminated when serum concentration was equal to or <0.2% in the presence of hydrodynamic stimuli. Our findings suggest that serum is a requirement in insulin-transferrin-selenium-supplemented cultures in order for constructs to exhibit improved properties in response to hydrodynamic forces, and that mechanical and biochemical stimuli may synergistically modulate tissue properties and morphology through shear-responsive signals.

Cyclic AMP regulates extracellular matrix gene expression and metabolism in cultured primary rat chondrocytes.

In osteo- and rheumatoid arthritis, the synovial fluid surrounding chondrocytes contains increased levels of prostaglandin E(2) (PGE(2)), an agent known to elevate intracellular cyclic AMP (cAMP). However, the effect of PGE(2)/cAMP on mRNA expression in chondrocytes is largely unknown. In this report, we assess the effect of the cell-permeable cAMP analog adenosine 8-(4-chloro-phenylthio)-3',5'-cyclic monophosphate (CPT-cAMP) and PGE(2) on mRNA expression in primary neonatal rat chondrocytes. CPT-cAMP decreased type II collagen, link protein, parathyroid hormone/parathyroid hormone-related peptide receptor and alkaline phosphatase, increased glyceraldehyde-3-phosphate dehydrogenase mRNA and lactate efflux, but did not alter type X collagen or aggrecan mRNA. The effect of CPT-cAMP on type II collagen and link protein mRNAs and chondrocyte metabolism were attenuated by the transcriptional inhibitor actinomycin D, indicating that the ability of CPT-cAMP to suppress mRNA expression was not due to alterations in mRNA stability, but were instead likely due to transcriptional mechanisms. CPT-cAMP-treatment induced GSK3 beta phosphorylation and beta-catenin-mediated transcriptional activity. Pharmacological inhibition of GSK3 beta paralleled the effects of CPT-cAMP on type II collagen, link protein and chondrocyte metabolism, suggesting that the effect of CPT-cAMP on chondrocytes may be GSK3 beta/beta-catenin-dependent. The effects of CPT-cAMP on beta-catenin-mediated transcription, cell metabolism and mRNA expression were mimicked by the cAMP-elevating agent PGE(2), providing a physiologically relevant context for our studies. Collectively, these results suggest that agents that elevate cAMP signaling may impair chondrocyte function in conditions such as arthritis.

Proteomics makes progress in cartilage and arthritis research.

Understanding biology at the systems level is a powerful method for discovery of previously unrecognized molecular pathways and mechanisms in human disease. The application of proteomics to arthritis research has lagged behind many other clinical targets, partly due to the unique biochemical properties of cartilage and associated biological fluids such as synovial fluid. In recent years, however, proteomic-based studies in cartilage and arthritis research have risen sharply and have started to make a significant impact on our understanding of joint disease, including the discovery of new and promising biomarkers of cartilage degeneration, a hallmark of arthritis. In this review we will make the case for the ongoing proteomic analysis of cartilage and other tissues affected by joint disease, overview some of the core proteomic techniques and discuss how the challenge of cartilage proteomics has been met through technical innovation. The major outcomes and information obtained from recent proteomic analysis of synovial fluid, cartilage and chondrocytes will also be described. In addition, we present some novel insights into post-translational regulation of cartilage proteins, through proteomic identification of proteolytic fragments in mouse cartilage extracts and explant culture media. We conclude with our prediction of how emerging proteomic technologies that have yet to be applied in arthritis research are likely to contribute further important information.