The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

SERPINA9 and SERPINB2: Novel Cartilage Lineage Differentiation Markers of Human Mesenchymal Stem Cells with Kartogenin.

OBJECTIVE: Human mesenchymal stem cells (hMSCs) are a promising source for regenerative medicine, especially mesodermal lineages. Clinical applications require an understanding of the mechanisms for transcriptional control to maintain the desired cell type. The aim of this study was to identify novel markers for differentiation of hMSCs into bone or cartilage with the use of Kartogenin, by RNA analysis using microarray technology, and explore the role of RhoA-Rho associated protein kinase (ROCK) inhibition in these. METHODS: Commercial human bone marrow derived primary mesenchymal stem cells were purchased from ATCC. Cells were differentiated in vitro in 2-dimensional cultures using Kartogenin as the main cartilage inducer and bone morphogenetic protein 2 for bone differentiation; cells were cultured with and without ROCK inhibitor Y-27632. After 21 days of culture, whole RNA was extracted and analyzed via Affimetrix microarrays. The most significant hits were validated by quantitative polymerase chain reaction. RESULTS: We found a total of 1,757 genes that were either up- or downregulated on differentiation, when compared to P1 hMSC (control) at day 0 of differentiation. Two members of the Serpin superfamily, SERPINA9 and SERPINB2, were significantly upregulated in the cartilage groups, whereas they were unchanged in the bone groups with and without ROCK inhibition. CONCLUSIONS: SERPINA9 and SERPINB2 are novel differentiation markers, and molecular regulator candidates for hMSC lineage commitment toward bone and cartilage, providing a new tool for regenerative medicine. Our study highlights the roles of these 2 genes, with significant upregulation of both in cell cultures stimulated with Kartogenin.

Effects of Vitrification Techniques on the Somatic Tissue Preservation of Agouti (Dasyprocta leporina Linnaeus, 1758).

The cryobanks of agouti somatic tissues represent a promising tool for the conservation of this species and of those that are phylogenetically related and endangered. For these purposes, one strategy to guarantee the quality of samples after warming would be to choose the appropriate tissue vitrification technique. Therefore, we evaluated the effects of two different techniques, direct vitrification in cryovials (DVC) and solid-surface vitrification (SSV), on the preservation of ear somatic tissues derived from agoutis kept in a scientific center of creation. Noncryopreserved somatic tissues were used as controls. Although SSV reduced the thickness of the dermis and cartilage (p < 0.05), the epidermal thickness of these samples was observed to be similar to controls (p > 0.05). Notably, the number of fibroblasts was not altered with either technique. However, both vitrification methods led to an increase in the number of perinuclear halos, with a particularly strong increase observed in DVC-derived fragments (p < 0.05). Compared with the DVC group, SSV showed a larger number of normal chondrocytes and smaller number of degenerate chondrocytes. Furthermore, the number of empty lacunae in SSV-derived fragments remained similar to controls (p > 0.05). In summary, SSV was found to be a more efficient method for vitrifying agouti somatic tissues compared with DVC. These results are important for the proper formation of agouti somatic banks, an essential step in the study of biological resources in this species.

Scaffold- and serum-free hypertrophic cartilage tissue engineering as an alternative approach for bone repair.

Human adipose stem/stromal cell (ASC) spheroids were used as a serum-free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced-ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP-13) was upregulated at week 2 in induced-ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real-time PCR. In accordance, secreted levels of IL-6 (P < .0001), IL-8 (P < .0001), IL-10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP-1 was associated with the increase of hypertrophic genes expression at week 2 in induced-ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced-ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum-free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.

Nonwoven membranes for tissue engineering: an overview of cartilage, epithelium, and bone regeneration.

Scaffold-type biomaterials are crucial for application in tissue engineering. Among them, the use of a nonwoven scaffold has grown in recent years and has been widely investigated for the regeneration of different types of tissues. Several polymers, whether they are synthetic, biopolymers or both, have been used to produce a scaffold that can mimic the natural tissue to which it will be applied to. The scaffolds used in tissue engineering must be biocompatible and allow cell adhesion and proliferation to be applied in tissue engineering. In addition, the scaffolds should maintain the mechanical properties and architecture of the desired tissue. Nonwoven fabrics have produced good results and are more extensively applied for the regeneration of cartilage, epithelial and bone tissues. Recent advances in tissue engineering have shown promising results, however, no ideal material or standardization parameters and characteristics of the materials were obtained. The present review provides an overview of the application of nonwoven scaffolds, including the main results obtained regarding the properties of the biomaterials and their applications in vitro and in vivo, focusing on the cartilaginous, the epithelium, and bone tissue regeneration.

Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering.

Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells are responsible for their extracellular matrix synthesis, re-creating structures at the tissue level. Spheroids from adult stem cells can be considered as organoids, since stem cells recapitulate differentiation pathways and also represent a promising approach for identifying new molecular targets (biomarkers) for diagnosis and therapy. Currently, spheroids can be used for scaffold-free (developmental engineering) or scaffold-based approaches. The scaffold promotes better spatial organization of individual spheroids and provides a defined geometry for their 3D assembly in larger and complex tissues. Furthermore, spheroids exhibit potent angiogenic and vasculogenic capacity and serve as efficient vascularization units in porous scaffolds for bone tissue engineering. An automated combinatorial approach that integrates spheroids into scaffolds is starting to be investigated for macro-scale tissue biofabrication.

Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in-vivo animal study.

To compare the quality of the repair tissue in three-dimensional co-culture of human chondrocytes implanted in an in vivo model. Six cadaveric and five live human donors were included. Osteochondral biopsies from the donor knees were harvested for chondrocyte isolation. Fifty percent of cadaveric chondrocytes were expanded until passage-2 (P2) while the remaining cells were cryopreserved in passage-0 (P0). Fresh primary chondrocytes (P0f) obtained from live human donors were co-cultured. Three-dimensional constructs were prepared with a monolayer of passage-2 chondrocytes, collagen membrane (Geistlich Bio-Gide®), and pellet of non-co-cultured (P2) or co-cultured chondrocytes (P2 + P0c, P2 + P0f). Constructs were implanted in the subcutaneous tissue of athymic mice and left for 3 months growth. Safranin-O and Alcian blue staining were used to glycosaminoglycan content assessment. Aggrecan and type-II collagen were evaluated by immunohistochemistry. New-formed tissue quality was evaluated with an adaptation of the modified O'Driscoll score. Histological quality of non-co-cultured group was 4.37 (SD ±4.71), while co-cultured groups had a mean score of 8.71 (SD ±3.98) for the fresh primary chondrocytes and 9.57 (SD ±1.27) in the cryopreserved chondrocytes. In immunohistochemistry, Co-culture groups were strongly stained for type-II and aggrecan not seen in the non-co-cultured group. It is possible to isolate viable chondrocytes from cadaveric human donors in samples processed in the first 48-h of dead. There is non-significant difference between the numbers of chondrocytes isolated from live or cadaveric donors. Cryopreservation of cadaveric primary chondrocytes does not alter the capability to form cartilage like tissue. Co-culture of primary and passaged chondrocytes enhances the histological quality of new-formed tissue compared to non-co-cultured cells.

* Thermosensitive Poly(N-vinylcaprolactam) Injectable Hydrogels for Cartilage Tissue Engineering.

Injectable hydrogels have gained prominence in the field of tissue engineering for minimally invasive delivery of cells for tissue repair and in the filling of irregular defects. However, many injectable hydrogels exhibit long gelation times or are not stable for long periods after injection. To address these concerns, we used thermosensitive poly(N-vinylcaprolactam) (PNVCL) hydrogels due to their cytocompatibility and fast response to temperature stimuli. Changes in the PNVCL molecular weight and concentration enabled the development of hydrogels with tunable mechanical properties and fast gelation times (<60 s when the temperature was raised from room temperature to physiologic temperature). Chondrocytes (CHs) and mesenchymal stem cells were encapsulated in PNVCL hydrogels and exhibited high viability (∼90%), as monitored by Live/Dead staining and Alamar Blue assays. Three-dimensional constructs of CH-laden PNVCL hydrogels supported cartilage-specific extracellular matrix production both in vitro and after subcutaneous injection in nude rats for up to 8 weeks. Moreover, biochemical analyses of constructs demonstrated a time-dependent increase in glycosaminoglycans (GAGs) and collagen, which were significantly augmented in the implants cultured in vivo. Histological analyses also demonstrated regular distribution of synthesized cartilage components, including abundant GAGs and type II collagen. The findings from this study demonstrate thermosensitive PNVCL as a candidate injectable biomaterial to deliver cells for cartilage tissue engineering.

Isolation of Primary Fibroblast Culture from Wildlife: the Panthera onca Case to Preserve a South American Endangered Species.

Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult-to-find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar. The fibroblasts obtained are a part of the Genetic Bank of Buenos Aires Zoo with the 6700 samples, including tissues such as muscle, ovarian, testicular, blood, fibroblast cultures, sperm, hair, and fluids and cells from 450 individuals of 87 different species. © 2016 by John Wiley & Sons, Inc.

In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide.

BACKGROUND: Platelet-rich plasma (PRP) preparations are a common treatment in equine osteoarthritis (OA). However, there are controversies regarding the ideal concentration of platelets and leukocytes in these biological substances necessary to induce an adequate anti-inflammatory and anabolic response in articular cartilage. The aims were to study the influence of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the histological changes of cartilage, the degree of chondrocyte apoptosis, the production of hyaluronan (HA) and the gene expression of nuclear factor kappa beta (NFkß), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1) and cartilage oligomeric matrix protein (COMP) in normal cartilage explants (CEs) challenged with lipopolysaccharide (LPS). RESULTS: Overall, 25 % L-PRG supernatant (followed in order of importance by, 50 % P-PRG, 25 % P-PRG and 50 % L-PRG) represented the substance with the most important anti-inflammatory and anabolic effect. 25 % P-PRG supernatant presented important anabolic effects, but it induced a more severe chondrocyte apoptosis than the other evaluated substances. CONCLUSIONS: 25 % L-PRG supernatant presented the best therapeutic profile. Our results demonstrate that the biological variability of PRP preparations makes their application rather challenging. Additional in vivo research is necessary to know the effect of PRP preparations at different concentrations.

Effects of cartilage-derived morphogenetic protein 1 (CDMP1) transgenic mesenchymal stem cell sheets in repairing rabbit cartilage defects.

The aim of this study was to investigate the abilities of cartilage-derived morphogenetic protein 1 (CDMP1) transgenic cell sheets in repairing rabbit cartilage defects. Rabbit CDMP1 transgenic bone marrow mesenchymal stem cell (BMSC) sheets (CDMP1-BMSCs) were cultured on temperature-sensitive culture dishes, and CDMP1 expression and type II collagen protein in the cell sheets were detected. Tissue-engineered cell sheets were constructed and transplanted into defect rabbit thyroid cartilage, to investigate the expression of engineered cartilage collagen protein and proteoglycan (GAG). The experiment was divided into three groups; A) BMSC sheet, B) Ad-CMV-eGFP-transfected cell sheet, and C) Ad-CMV-hCDMP1-IRES-eGFP-transfected cell sheet. The expression of CDMP1 was detected in the transgenic cell sheets. The engineered cartilage exhibited positive immunohistochemical and Alcian blue staining. The expression levels of type II collagen protein and GAG in group A were positive, whereas those in group B and group C were negative (P < 0.05). The CDMP1-BMSC sheets had a good cartilage differentiation activity, and could effectively repair rabbit laryngeal cartilage defects.

Nestin-positive stem cells participate in chondrocyte renewal in healthy adult lung cartilage / Células troncales nestin-positivas participan en la renovación de condrocitos en el cartílago pulmonar del adulto sano

It is considered that healthy adult cartilage has little or no capacity for renewal, and that chondrocytes maintain a stable resting phenotype and resist proliferation and differentiation throughout life. Recently we found that cell turnover in lung cartilage is possible and that nestin-positive cells may have a role in it. In this paper, we report additional findings about chondrocyte renewal in lung cartilage. Lung specimens from CD1 mice at the age of 2, 6, 12, 18 or 24 months were fixed in 10% neutral-buffered formalin and paraffin-embedded. Nestin expression was examined by an immunohistochemical peroxidase-based method. We found nestin-positive cells inside of cartilage islets and cells in division very close from them. Our findings indicate that there exist nestin-positive mesenchymal stem cells in the adult that are able to differentiate into lung chondrocytes, perhaps to maintain homeostasis or repair damaged tissue. These findings may improve our knowledge about the cartilage biology and could provide new cell candidates for cartilage tissue engineering.

Se considera que el cartílago adulto sano tiene poca o ninguna capacidad para renovarse, y que sus condrocitos permanecen en un estado de reposo estable, careciendo de las propiedades de proliferación y diferenciación. Recientemente encontramos que el recambio celular en el cartílago pulmonar es posible y que células troncales positivas para nestin pudieran tener algún papel en el mismo. En este artículo, reportamos nuevos hallazgos acerca de la renovación de condrocitos en el cartílago pulmonar. Pulmones de ratones CD1 de 2, 6, 12, 18 o 24 meses de edad se fijaron en formalina amortiguada al 10% y se incluyeron en parafina. Se analizó la expresión de nestin utilizando un método inmunohistoquímico basado en un sistema de detección con peroxidasa. Encontramos células positivas para nestin en el interior de los islotes de cartílago y células en división muy cercanas a ellas. Estos hallazgos indican que existen células madre mesenquimales positivas para nestin en el adulto con capacidad para diferenciarse en condrocitos pulmonares, probablemente para mantener la homeostasis tisular o reparar daños en el tejido. Asimismo, estos hallazgos pueden aumentar nuestra comprensión acerca de las propiedades biológicas del cartílago y podrían proporcionar nuevos candidatos para la ingeniería celular en la terapia regenerativa en enfermedades de las articulaciones.

Bioengineered cartilage in a scaffold-free method by human cartilage-derived progenitor cells: a comparison with human adipose-derived mesenchymal stromal cells.

The objective of our study was to investigate chondrogenesis potential of human adipose-derived mesenchymal stromal cells (MSCs), using as a positive control a human source of cartilage-derived progenitor cells (PCs). This source of PCs was recently described by our group and dwells on the surface of nasoseptal cartilage. Histological analysis using Safranin O staining and immunofluorescence for actin filaments and collagen type II was performed on three-dimensional (3D) pellet cultures. Cartilage PCs and adipose MSCs showed similarities in monolayer culture related to cell morphology and proliferation. Our 3D pellet cultures substantially reduced the actin stress and after 21 days under chondrogenic medium, we observed an increase in the pellet diameter for cartilage PCs (7.4%) and adipose MSCs (21.2%). Adipose-derived MSCs responded to chondrogenic stimulus, as seen by positive areas for collagen type II, but they were not able to recreate a mature extracellular matrix. Using semi-quantitative analysis, we observed a majority of Safranin O areas rising from blue (no stain) to orange (moderate staining) and no changes in fibroblastic morphology (P < 0.0001). For cartilage PCs, chondrogenic induction is responsible for morphological changes and a high percentage of matrix area/number of cells (P ≤ 0.0001), evaluated by computerized histomorphometry. Morphological analyses reveal that adipose-derived MSCs were not able to recreate a bioengineered cartilage. The cost of culture was reduced, as the cartilage PCs under growth-factor free medium exhibit a high score for cartilage formation compared with the induced adipose mesenchymal stromal cells (P = 0.0021). Using a pellet 3D culture, our cartilage PCs were able to produce a cartilage tissue in vitro, leading to the future development of bioengineered products.

Evidence of chondrocyte turnover in lung cartilage, with the probable participation of nestin-positive cells.

Healthy adult cartilage is thought to have little or no capacity to renewal, and cell turnover has not been reported in lung cartilage. We report that chondrocyte turnover occurs in lung cartilage, found in an unrelated study. Lung specimens from CD1 mice of 2, 6, 12, 18 or 24 months were fixed in 10% neutral-buffered formalin and paraffin-embedded. Apoptosis was analysed by in situ end-labelling of fragmented DNA. Proliferating cell nuclear antigen (PCNA) and nestin were examined by immunohistochemistry. Apoptosis and PCNA were detected in lung chondrocytes. Serial section analysis showed that cells in apoptosis were different from PCNA-positive cells, indicating that turnover was occurring. Chondrocytes were negative for nestin. Nestin-positive cells were present in connective tissue associated with cartilage, in some specimens in close proximity of it and in perivascular cells. Thus cell turnover in lung cartilage is possible, which may be mediated by nestin-positive cells.

Matrigel supports neural, melanocytic and chondrogenic differentiation of trunk neural crest cells.

The neural crest (NC) is composed of highly multipotent precursor cells able to differentiate into both neural and mesenchymal phenotypes. Until now, most studies focusing on NC cell differentiation have been performed with traditional two-dimensional (2D) cell culture systems. However, such culture systems do not reflect the complex three-dimensional (3D) microenvironments of in vivo NC cells. To address this limitation, we have developed a method of Matrigel™ coating to create 2D and 3D microenvironments in the same culture well. When we performed cultures of trunk neural crest cells (TNCCs) on three different lots of basement membrane matrix (Matrigel™), we observed that all analyzed Matrigel™ lots were equally efficient in allowing the appearance of glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes. We further observed that chondrocytes were found predominantly in the 3D microenvironment, whereas smooth muscle cells were almost exclusively located in the 2D microenvironment. Glial cells were present in both environments, but with broader quantities on the 2D surface. Melanocytes and neurons were equally distributed in both 2D and 3D microenvironments, but with distinct morphologies. It is worth noting the higher frequency of chondrocytes detected in this study using the 3D Matrigel™ microenvironment compared to previous reports of chondrogenesis obtained from TNCCs on traditional 2D cultures. In conclusion, Matrigel™ represents an attractive scaffold to study NC multipotentiality and differentiation, since it permits the appearance of the major NC phenotypes.

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats.

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.

Evaluation of the potential of novel PCL-PPDX biodegradable scaffolds as support materials for cartilage tissue engineering.

Cartilage is a specialized tissue represented by a group of particular cells (the chondrocytes) and an abundant extracellular matrix. Because of the reduced regenerative capacity of this tissue, cartilage injuries are often difficult to handle. Nowadays tissue engineering has emerged as a very promising discipline, and biodegradable polymeric scaffolds are widely used as tissue supports. In cartilage injuries, the use of autologous chondrocyte implantation from non-affected cartilage zones has emerged as a very interesting technique, where chondrocytes are expanded in order to obtain a greater number of cells. Nevertheless, it has been reported that chondrocytes in bidimensional cultures suffer a dedifferentiation process. The present study sought, in the first place, to standardize a novel protocol in order to obtain primary cultures of chondrocytes from newborn rabbit hyaline cartilage from the xiphoid process. Second, the potential of porous three-dimensional (3D) biodegradable polymeric matrices as support materials for chondrocytes was evaluated: a novel poly(ε-caprolactone)-poly(p-dioxanone) (PCL-PPDX) blend in a 90:10 w:w ratio and poly(ε-caprolactone) (PCL). After achieving the standardization, a typical round-shaped chondrocyte morphology and the expression of collagen type II and aggrecan, evaluated by RT-PCR, were observed. Second-passage chondrocytes adhered effectively to these scaffolds, although cell growth at 7 days in culture was significantly less in the PCL-PPDX blend. After 3 weeks of culture on PCL-PPDX or PCL, the cells expressed collagen type II. The present study demonstrates the potential, unknown until now, of PCL-PPDX blend scaffolds in the field of cartilage tissue engineering.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components.

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads.

Preservation of the chondrocytic phenotype in vitro requires a 3D (three-dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [35S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [35S]Na2SO4 was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra-bead) and EB (extra-bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.