Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

Single-cell RNA-seq analysis identifies unique chondrocyte subsets and reveals involvement of ferroptosis in human intervertebral disc degeneration.

OBJECTIVE: Nucleus pulposus (NP) plays a central role in disc degeneration pathogenesis, however, as a heterogeneous tissue, cell subsets in NP and their corresponding biological process in intervertebral disc degeneration (IVDD) are unreported. METHOD: Nucleus pulposus were isolated from normal control and IVDD, and then subjected to single-cell RNA sequencing (scRNA-seq). Unsupervised clustering of the cells based on the gene expression profiles using the Seurat package and passed to tSNE for clustering visualization. Rat model of disc degeneration was built to validate the pathways identified by scRNA-Seq. RESULTS: Seven chondrocyte subsets were revealed in NP based on differential gene expression, among which 4 subsets (C1-C4) were reported for the first time. Furthermore, GO and KEGG analyses discovered that ferroptosis pathways were enriched. Rat model of disc degeneration was built (n = 6/group, control vs. model) to validate the pathways identified by scRNA-Seq. Iron levels of NP were significantly higher in model group than control group (means 0.712 vs. 0.248, respectively, mg/gpro, p = 0.0026), and the levels of Heme Oxygenase 1 (HO-1) were also elevated in model group (means 14.33 vs. 5.16 IOD, respectively, p = 0.0002). However, the levels of ferritin light chain (FTL) were significantly decreased in model group compared to control group (means 26.17 vs. 9.00 FTL+ cell number, respectively, p = 0.0011). CONCLUSIONS: Novel chondrocyte subsets in nucleus pulposus were discovered through scRNA-Seq, which provided novel insight to understand the pathological change during the development of IVDD. Ferroptosis participated in disc degeneration pathogenesis and it might serve as a new target for intervening IVDD.

Post-Treatment Classification for Acetabular Chondral Lesions in Hip Arthroscopy / Classificação pós-tratamento para lesões condrais acetabulares na artroscopia de quadril

Abstract Objective To describe a postarthroscopic treatment classification system for acetabular chondral damage in the hip and to report the intraobserver and interobserver reliability of such classification. Methods This is a retrospective review of ninety-nine digital video recordings made during arthroscopic surgery. Patients who underwent arthroscopic treatment for femoroacetabular impingement and evaluated at the hip arthroscopy outpatient clinic between March 2015 and March 2016 were included in the study. Patients with a history of previous hip surgery, radiologic evidence of advanced osteoarthritis (Tönnis grade > 2), who underwent labral resection, or whose digital recordings were incomplete or of insufficient quality for adequate review were excluded. Two orthopedic surgeons, who did not participate in the surgery, independently reviewed the video recordings and classified the remaining acetabular cartilage using the post-treatment classification system. Intraobserver and interobserver analysis was then conducted using intraclass correlation coefficient (ICC). Results Excellent intraobserver reliability (ICC = 0.790; p < 0.001) and interobserver reliability (ICC = 0.882; p < 0.001) were observed. Both ICC values were statistically significant. Conclusion The posttreatment classification of the remaining acetabular cartilage has excellent intra and interobserver reliability.

Resumo Objetivo Descrever um sistema de classificação de tratamento pós-artroscópico para as lesões condrais acetabulares no quadril e relatar as confiabilidades intra e interobservador deste sistema. Métodos Esta é uma revisão retrospectiva de 99 gravações de vídeo digital realizadas durante artroscopia. Os pacientes submetidos a tratamento artroscópico para impacto femoroacetabular e avaliados no ambulatório de quadril entre março de 2015 e março de 2016 foram incluídos no estudo. Os pacientes com histórico de cirurgia anterior do quadril, evidência radiológica de osteoartrose avançada (Tönnis > 2), pacientes submetidos à ressecção labral ou cujas gravações digitais estavam incompletas ou de qualidade insuficiente para avaliação adequada foram excluídos. Dois ortopedistas, que não participaram da cirurgia, revisaram de forma independente as gravações de vídeo e classificaram a cartilagem acetabular remanescente usando o sistema de classificação pós-tratamento. A análise intra e interobservador foi então realizada utilizando o coeficiente de correlação intraclasse (CCI). Resultados Excelente confiabilidade intraobservador (CCI = 0,790; p < 0,001) e confiabilidade interobservador (CCI = 0,882; p < 0,001). Ambos os valores de CCI foram estatisticamente significativos. Conclusão a classificação pós-tratamento da cartilagem acetabular remanescente possui excelente confiabilidade intra e interobservador.

Bone Marrow Stem Cells and Ear Framework Reconstruction.

BACKGROUND: Repair of total human ear loss or congenital lack of ears is one of the challenging issues in plastic and reconstructive surgery. OBJECTIVE: The aim of the present study was 3D reconstruction of the human ear with cadaveric ear cartilages seeded with human mesenchymal stem cells. METHOD: We used cadaveric ear cartilages with preserved perichondrium. The samples were divided into 2 groups: group A (cartilage alone) and group B (cartilage seeded with a mixture of fibrin powder and mesenchymal stem cell [1,000,000 cells/cm] used and implanted in back of 10 athymic rats). After 12 weeks, the cartilages were removed and shape, size, weight, flexibility, and chondrocyte viability were evaluated. P value <0.05 was considered significant. RESULTS: In group A, size and weight of cartilages clearly reduced (P < 0.05) and then shape and flexibility (torsion of cartilages in clockwise and counterclockwise directions) were evaluated, which were found to be significantly reduced (P > 0.05). After staining with hematoxylin and eosin and performing microscopic examination, very few live chondrocytes were found in group A. In group B, size and weight of samples were not changed (P < 0.05); the shape and flexibility of samples were well maintained (P < 0.05) and on performing microscopic examination of cartilage samples, many live chondrocytes were found in cartilage (15-20 chondrocytes in each microscopic field). CONCLUSION: In samples with human stem cell, all variables (size, shape, weight, and flexibility) were significantly maintained and abundant live chondrocytes were found on performing microscopic examination. This method may be used for reconstruction of full defect of auricles in humans.

Production of serum amyloid A in equine articular chondrocytes and fibroblast-like synoviocytes treated with proinflammatory cytokines and its effects on the two cell types in culture.

OBJECTIVE: To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. SAMPLE: Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). PROCEDURES: Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1ß, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein were assessed by quantitative real-time PCR assay; SAA protein was evaluated by immunoturbidimetry and denaturing isoelectric focusing and western blotting. RESULTS: All cytokine stimulation protocols increased expression of SAA mRNA and resulted in detectable SAA protein production in chondrocytes and FLSs. Isoforms of SAA in lysed chondrocytes and their culture medium corresponded to those previously detected in synovial fluid from horses with joint disease. When exposed to SAA, chondrocytes and FLSs had increased expression of IL-6, SAA, and MMP3, and chondrocytes had increased expression of MMP-1. Chondrocytes had decreased expression of cartilage-derived retinoic acid-sensitive protein. CONCLUSIONS AND CLINICAL RELEVANCE: Upregulation of SAA in chondrocytes and FLSs stimulated with proinflammatory cytokines and the proinflammatory effects of SAA suggested that SAA may be involved in key aspects of pathogenesis of the joint inflammation in horses.

Maximizing phenotype constraint and extracellular matrix production in primary human chondrocytes using arginine-glycine-aspartate concentration gradient hydrogels.

New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine-glycine-aspartic acid peptide (RGD) concentrations from 0mM to 10mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.

Varying regional topology within knee articular chondrocytes under simulated in vivo conditions.

Topographical cartilage variation across the knee joint has been previously reported, but there is only limited information on such gene expression profiles. Articular chondrocytes from eight different topographical regions of bovine knee joints were seeded within three-dimensional scaffolds and further cultured under static conditions (unloaded control group) or subjected to an artificial joint environment within a bioreactor (loaded group). Constructs were analyzed for glycosaminoglycan (GAG), DNA, and expression of Collagen-1,-2,-10, Aggrecan, COMP, Sox9, PRG-4, PTHrp, and MMP-1,-3,-13 mRNA after 2 weeks of in vitro culture. Exclusively among loaded constructs the overall GAG production was significantly different between regions. Patella chondrocytes had overall highest, and cells from the femoral notch had overall lowest GAG/DNA under loaded conditions. Gene expression was significantly different between regions for all targets except for Sox9, PRG-4, and PTHrp among controls and with the exception of Aggrecan, Sox9, and PTHrp among loaded samples. Under mechanical stimulation Collagen-1,-2 and Aggrecan was highest at the patello-femoral joint, whereas it was lowest at typical cartilage biopsy regions. There is a clear topographical variation among distinct regions across the knee joint for gene and matrix expression profiles under static and foremost under dynamic conditions.

SCF, BDNF, and Gas6 are regulators of growth plate chondrocyte proliferation and differentiation.

We previously demonstrated that bovine epiphyseal chondrocytes separated by density gradient centrifugation differ in proliferative response to IGF-I and IGF-I receptor number. To identify novel modifiers of IGF-I action at the growth plate, we used microarray analyses to compare bovine hypertrophic and reserve zones and identified several receptors differentially expressed across the growth plate: NTRK2 [receptor for brain-derived neurotrophic factor (BDNF)], KIT [receptor for stem cell factor (SCF)], and MER and AXL [two receptors for growth arrest-specific 6 (Gas6)]. The corresponding ligands were tested for their ability to stimulate either proliferation of isolated chondrocytes or differentiation in ATDC5 cells. Each factor inhibited IGF-I-mediated proliferation in isolated chondrocytes by attenuating ERK1/2 activation. SCF, BDNF, Gas6, and C-type natriuretic peptide promoted differentiation in ATDC5 cells, each factor producing different expression patterns for collagen X, collagen 2, aggrecan, and lysyl oxidase. Whereas multiple factors stimulated ATDC5 differentiation, only IGF-I and high-dose insulin, out of several factors implicated in chondrocyte maturation, stimulated proliferation of isolated chondrocytes. IGF-I appears to be the primary proliferative signal in growth plate chondrocytes, whereas multiple factors including SCF, BDNF, and Gas6 regulate the pace of differentiation at the growth plate.

[Chondrocytes - one cell type, different subpopulations : characteristics and behavior of different types of chondrocytes and implications for tissue engineering applications]. / Chondrozyten - ein Zelltyp, verschiedene Subpopulationen : Merkmale und Verhalten verschiedener Chondrozyten und Implikationen für deren Einsatz zum "Tissue Engineering"

Chondrocytes represent the most important cell source for engineering of cartilaginous tissues. Depending on the tissue type and the localization within the tissue, these cells may behave differently. Numerous studies have been done to compare articular, nasal, auricular, and costal chondrocytes in order to evaluate differences between knee and ankle joint cartilage and to investigate topographical variations within an articular joint. Moreover, the zonal structure of articular cartilage needs to be considered because it leads to phenotypical differences between chondrocytes of the superficial and the deeper zones. Several studies indicate, however, that even differentiated chondrocytes demonstrate a certain plasticity and strive to adapt their phenotypes to a new mechanical and biochemical environment. The aim of this review is to report on similarities and differences of chondrocytes from different tissues, zones, and topographical locations. In particular, an overview of recent results from comparative studies is presented, and possible consequences for the design of tissue engineering models are discussed.

Cartilage tissue engineering for laryngotracheal reconstruction: comparison of chondrocytes from three anatomic locations in the rabbit.

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 +/- 93 kPa in two samples at 3 weeks and 100 +/- 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity.

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

Retaining zonal chondrocyte phenotype by means of novel growth environments.

The loss of phenotype in articular chondrocytes expanded in monolayer has been established as a possible contributor to the deficiencies associated with in vitro cartilage engineering and autologous cell transplantation procedures. We cultured zonal articular chondrocytes on tissue culture plastic, collagen II-coated polystyrene, and aggrecan-coated polystyrene in an effort to find a surface that can either prevent or slow the loss of phenotype. In addition, we encapsulated passaged cells in agarose to examine the effect of three-dimensional culture on redifferentiating zonal chondrocytes. We used real-time polymerase chain reaction to measure the relative gene expression levels of collagen I and II, aggrecan, and superficial zone protein over relevant passages (P0-P4). Results showed that tissue culture plastic and the collagen II-coated surface induced rapid loss of phenotype in zonal articular chondrocytes. The aggrecan-coated surface had a less detrimental effect on the chondrocytic phenotype of seeded cells, inducing gene expression characteristics comparable to those of agarose-encapsulated cells. Furthermore, when chondrocytes that had been previously passaged on a collagen II surface were placed on an aggrecan surface, the zonal cells showed a dramatic change in gene expression from fibroblastic to chondrocytic. These results indicate that a culture environment using aggrecan as a substratum or agarose as a scaffold is crucial to the development of phenotypically correct articular cartilage.

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

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

Characterization of the cellular origin of a tissue-engineered human phalanx model by in situ hybridization.

Tissue-engineered models of human phalanges have previously been fabricated from a combination of bovine periosteum, cartilage, tendon, and biodegradable polyglycolic acid and poly-L-lactic acid scaffolds. Resulting constructs implanted in athymic mice for more than 40 weeks developed new bone, cartilage, and tendon and became vascularized, but cell types comprising the constructs were unidentified. The origin of cells in middle phalanx models implanted for 20 weeks in nude mice has been studied by in situ hybridization analyzing species-specific gene expression. Oligonucleotide probes homologous to species-specific gene sequences of bovine type II and X collagen, aggrecan, bone sialoprotein, biglycan, and osteopontin, and mouse decorin were labeled with (35)S and hybridized to respective serial sections of bovine tissue, mouse tissue, and phalanx constructs. In situ hybridization showed positive message and tissue-specific localization for all bovine-specific probes examined within cartilaginous and midshaft portions of constructs and negative message for the mouse-specific decorin probe. These data show that osteoblasts and chondrocytes comprising constructs are derived exclusively from their original bovine sources over 20 weeks of implantation. Defining the cellular origin of the models lends insight into their biological, chemical, and physical nature and their growth and development. Maintenance of their initial genotype is crucial for future application of the models in augmenting impaired human phalanges and related tissues.

Development of articular cartilage: what do we know about it and how may it occur?

Articular cartilage has a fundamental role in joint function. While much is known about its structure, organization and biomechanical properties, there is a very poor understanding of how articular chondrocytes develop during embryogenesis and acquire the unique ability to organize and maintain the articular tissue. Given that articular cartilage forms in close juxtaposition with the joint, here we review past studies on limb joint determination and morphogenesis and more recent studies on a number of factors thought to have roles in joint and epiphysis development. These factors include: the homeobox gene Barx-1; the bone morphogenetic protein (BMP) family member GDF-5; the growth factors HGF and PTHrP; and the transcription factor ERG. We summarize current thinking on how these factors participate in joint development and how some of these factors may influence development and behavior of epiphyseal chondrocytes. We also describe pertinent recent studies from our laboratories on ERG and the newly-identified alternatively spliced variant C-1-1, and finally propose a sequela of events that may subtend the process of determination and emergence of articular chondrocytes during limb synovial joint development.