Chondrogenic Medium Research Articles

Articular Cartilage Repair with Mesenchymal Stem Cells After Chondrogenic Priming: A Pilot Study.

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess a protocol that involved autologous transplantation of BMSCs into full-thickness cartilage defects in sheep following isolation, expansion, and a short period (4 days) of chondrogenic priming. The impact of oxygen tension during preimplantation culture was investigated. It was hypothesized that chondrogenically primed BMSCs would produce superior cartilaginous repair tissue relative to control defects, and that culture under hypoxia would yield improved repair tissue in comparison to normoxia. Ovine BMSCs were isolated, expanded to passage 2, seeded within a hyaluronic acid (HYAFF) scaffold, and primed ex vivo in chondrogenic medium for 4 days under normoxia (21% oxygen) or hypoxia (3% oxygen). Full-thickness, 7-mm-diameter articular cartilage defects were created in the femoral condyles of five sheep. Twenty defects were treated with normoxia-cultured, autologous BMSC-seeded scaffolds (eight); hypoxia-cultured, autologous BMSC-seeded scaffolds (eight); cell-free scaffolds (two); or no implants (two). Preimplantation priming was evaluated through gene expression analysis using reverse transcription quantitative polymerase chain reaction. After 6 months, histological assessment was performed on repair tissues with a modified O'Driscoll scoring system and tissue dimension analysis. Priming of preimplantation BMSC-seeded scaffolds in chondrogenic medium for 4 days resulted in significantly increased gene expression of hyaline cartilage-related collagen II and aggrecan relative to unprimed BMSCs (p < 0.05). Defects implanted with chondrogenically primed BMSC-seeded scaffolds developed cartilaginous repair tissues that contained safranin O-positive proteoglycans, and had significantly larger repair tissue areas, higher percentages of defect fill, and improved histological scores than cell-free controls (p < 0.05). Although hypoxic culture improved the preimplantation gene expression profile, a consistent difference in histological scores was not found between normoxia- and hypoxia-seeded BMSC-seeded scaffolds after 6 months (p = 0.90). This study demonstrates in a sheep model that (1) chondrogenic priming ex vivo improves the gene expression profile of BMSCs; (2) chondrogenically primed BMSCs are associated with the development of superior cartilaginous tissue to cell-free controls within cartilage defects; and (3) oxygen tension during preimplantation ex vivo culture does not consistently modulate cartilaginous repair tissue formation following BMSC transplantation into cartilage defects.

Formation of Tissue-Engineered Construct of Human Cartilage Tissue in a Flow-Through Bioreactor.

We performed culturing of a cell-engineered construct of human cartilage tissue consisting of biopolymer microstructured collagen-containing hydrogel, human adipose tissue mesenchymal stromal cells, and induction chondrogenic culture medium in a specially designed flow-through bioreactor. On day 16 of the experiment, human adipose tissue mesenchymal stromal cells acquired flattened shape typical for chondroblasts, demonstrated high proliferative activity, and formed extracellular matrix. The observed histological changes in the cultured system attested to the beginning of the formation of a tissue-engineered construct of human cartilage tissue.

Study on the effect of adiponectin on the differentiation of cartilage derived stem cells into chondrocytes

Objective To observe the effect of adiponectin on the differentiation of cartilage derived stem cells into chondrocytes from the articular cartilage of patients with advanced osteoarthritis. Methods The remaining cartilage tissue was collected from 5 patients with advanced osteoarthritis underwent total knee arthroplasty, then cultured and selected chondroprogenitors, to observe and compare the expression of type Ⅰ, type Ⅱ collagen, Aggrecan and sex determining region Y box-9 (SOX-9) by immunohistochemistry and the related genes expression between simple cartilage induced group and the group with adiponectin. Results The chondroprogenitors selected from articular cartilage of patients with advanced osteoarthritis can differentiate into chondrocytes under appropriate conditions; when in chondrogenic medium added adiponectin, compared with the group without adiponectin, the gene and protein expression of type Ⅱ collagen and Aggrecan decreased (type Ⅱ collagen: 1.004±0.125 vs. 0.588±0.101, P=0.001; Aggrecan: 1.002±0.050 vs. 0.584±0.138, P=0.000), the SOX-9 downregulation was not obvious (0.998±0.080 vs. 0.946±0.149, P=0.513), but the expression of type I collagen increased significantly (2.290±0.349 vs. 1.010±0.059, P=0.000). Conclusion Adiponectin can inhibit chondrogenic potential of chondroprogenitors. Key words: Adiponectin; Chondrogprogenitors; Chondrogenic differentiation; Sex determining region Y box-9

Dose-dependent effect of triiodothyronine on the chondrogenic differentiation of mesenchymal stem cells from the bone marrow of female rats.

Verify the in-vitro effect of triiodothyronine (T3) on the chondrogenic differentiation of female rat bone marrow mesenchymal stem cells (BMMSCs) over several time periods and at several doses. CD54+/CD73+/CD90+ BMMSCs from Wistar female rats were cultured in chondrogenic medium with or without T3 (0.01; 1; 100; 1000nm). At seven, 14 and 21 days, the cell morphology, chondrogenic matrix formation and expression of Sox9 and collagen II were evaluated. The dose of 100nm did not alter the parameters evaluated in any of the periods studied. However, the 0.01nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and expression of Sox9 and collagen II in at least one of the evaluated periods; the 1nm T3 dose also improved the chondrogenic potential by increasing the chondrogenic matrix formation and the expression of collagen II in at least one of the evaluated periods. The 1000nm T3 dose improved the chondrogenic potential by increasing the chondrogenic matrix formation and Sox9 expression in at least one of the evaluated periods. T3 has a dose-dependent effect on the differentiation of BMMSCs from female rats.

Platelet-rich concentrate in serum-free medium enhances cartilage-specific extracellular matrix synthesis and reduces chondrocyte hypertrophy of human mesenchymal stromal cells encapsulated in alginate

Platelet-rich concentrate (PRC), used in conjunction with other chondroinductive growth factors, have been shown to induce chondrogenesis of human mesenchymal stromal cells (hMSC) in pellet culture. However, pellet culture systems promote cell hypertrophy and the presence of other chondroinductive growth factors in the culture media used in previous studies obscures accurate determination of the effect of platelet itself in inducing chondrogenic differentiation. Hence, this study aimed to investigate the effect of PRC alone in enhancing the chondrogenic differentiation potential of human mesenchymal stromal cells (hMSC) encapsulated in three-dimensional alginate constructs. Cells encapsulated in alginate were cultured in serum-free medium supplemented with only 15% PRC. Scanning electron microscopy was used to determine the cell morphology. Chondrogenic molecular signature of hMSCs was determined by quantitative real-time PCR and verified at protein levels via immunohistochemistry and enzyme-linked immunosorbent assay. Results showed that the cells cultured in the presence of PRC for 24 days maintained a chondrocytic phenotype and demonstrated minimal upregulation of cartilaginous extracellular matrix (ECM) marker genes (SOX9, TNC, COL2, ACAN, COMP) and reduced expression of chondrocyte hypertrophy genes (Col X, Runx2) compared to the standard chondrogenic medium (p < 0.05). PRC group had correspondingly higher levels of glycosaminoglycan and increased concentration of chondrogenic specific proteins (COL2, ACAN, COMP) in the ECM. In conclusion, PRC alone appears to be very potent in inducing chondrogenic differentiation of hMSCs and offers additional benefit of suppressing chondrocyte hypertrophy, rendering it a promising approach for providing abundant pool of chondrogenic MSCs for application in cartilage tissue engineering.

Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation

BackgroundMesenchymal stem cells (MSCs) hold promising translational potential in cartilage regeneration. However, the efficacy of MSC-based tissue engineering is not satisfactory in the treatment of cartilage defect because of the inevitable cellular functional changes during ex vivo cell expansion. How to maintain the chondrogenic capacity of MSCs to improve their therapeutic outcomes remains an outstanding question.MethodsBone marrow-derived MSCs were firstly primed in chondrogenic induction medium which was then replaced with normal growth medium to attain the manipulated cells (M-MSCs). Methacrylated hyaluronic acid (MeHA) was synthesized as a scaffold to encapsulate the cells. The MSC- or M-MSC-laden constructs were treated with dynamic compressive loading (DL) in a bioreactor or with free loading (FL) for 14 days. Afterwards, the constructs were implanted in nude mice or rat models of osteochondral defects to test their efficiency in cartilage regeneration or repair.ResultsData showed that the resulting M-MSCs exhibited superior chondrogenic differentiation potential and survivability compared with untreated MSCs. More importantly, we found that DL significantly promoted neocartilage formation in the MeHA hydrogel encapsulated with M-MSCs after 30 days of implantation in nude mice. Furthermore, the constructs laden with M-MSCs after DL for 14 days significantly enhanced cartilage healing in a rat model of osteochondral defect.ConclusionsFindings from this study highlight the importance of maintaining chondrogenic potential of MSCs by in-vitro chondrogenic preconditioning and a synergistic effect of mechanical stimulation in cartilage engineering, which may shed light on the stem cell-based tissue engineering for cartilage repair.

RGD-functionalized polyethylene glycol hydrogels support proliferation and in vitro chondrogenesis of human periosteum-derived cells.

The combination of progenitor cells with appropriate scaffolds and in vitro culture regimes is a promising area of research in bone and cartilage tissue engineering. Mesenchymal stem cells (MSCs), when encapsulated within hydrogels composed of the necessary cues and/or preconditioned using suitable culture conditions, have been shown to differentiate into bone or cartilage. Here, we utilized human periosteum-derived cells (hPDCs), a progenitor cell population with MSC characteristics, paired with protease-degradable, functionalized polyethylene glycol (PEG) hydrogels to create tissue-engineered constructs. The objective of this study was to investigate the effects of scaffold composition, exploring the addition of the cell-binding motif Arginine-Glycine-Aspartic Acid (RGD), in combination with various in vitro culture conditions on the proliferation, chondrogenic gene expression, and matrix production of encapsulated hPDCs. In growth medium, the hPDCs in the RGD-functionalized hydrogels maintained high levels of viability and demonstrated an enhanced proliferation when compared with hPDCs in non-functionalized hydrogels. Additionally, the RGD-containing hydrogels promoted higher glycosaminoglycan (GAG) synthesis and chondrogenic gene expression of the encapsulated hPDCs, as opposed to the non-functionalized constructs, when cultured in two different chondrogenic media. These results demonstrate the potential of hPDCs in combination with enzymatically degradable PEG hydrogels functionalized with adhesion ligands for cartilage regenerative applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 33-42, 2018.

Application of tissue engineering technology for formation of human articular cartilage in perfusion bioreactor

Formation of tissue-engineered construct was performed in a specially developed bioreactor. At first, a cellengineered construct of human cartilage tissue consisting of biopolymer microstructured collagen-containing hydrogel, mesenchymal stromal cells of human adipose tissue (hADMSCs) and induction chondrogenic culture medium was prepared and placed in a perfusion bioreactor. As a result, on the 16th day of the study hADMSCs obtain a flattened shape typical for chondroblasts and demonstrate high proliferative activity with the formation of their own extracellular matrix. Histological analysis of the cultured system indicates the beginning of the formation of a tissue-engineered construct of human cartilage tissue.

ID: 1076 Establishing an artificial cartilage from rabbit bone marrow derived mesenchymal stem cell and polycaprolactone scaffold

Nowaday, the regenerative medicine based on the use of biomaterial scaffold combine to stem cells was considered as a necessary treatment strategy for injury or diseases involving cartilage. In this study, we investigated the adherence, proliferation and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (rBM-MSCs) on biodegradable polycaprolactone scaffold (PCL). MSCs were isolated from bone marrow of rabbit. rBM-MSC (2x105cell/scaffold) were seeded on PCL and collagen-coated PCL (PCL/col) scaffolds. The cell adhesion and proliferation abilities on the PCL and PCL/col scaffolds were compared to each other to find the better using MTT assay. Then 3D cultured-cells were induced in chondrogenic differentiation medium. The change of scaffold surface structure was evaluated by scanning electron microscope (SEM). The accumulation of aggrecan protein was detected by Alcian Blue staining. The chondrogenic-related gene expression such as sox9, collagen type 1, collagen type 2, collagen type 10, aggrecan, runx2 was assessed by RT-PCR. The existence of artificial cartilage after transplantation of chondrocyte-seeded scaffold under rabbit skin was evaluated by staining histological sections with Alcian Blue. The results showed that rBM-MSCs attached and grown on PCL/col and PCL scaffolds. However, the adhesion efficient of rBM-MSC on PCL/col scaffold was better than PCL scaffolds. Therefore, PCL/col scaffold was used for next experiments. The morphology of rBM-MSCs on PLC/col scaffold were changed and the aggrecan accumulation increased after cultured in chondrogenic differentiation medium. The scaffold surface modification was detected with the presentation of collagen-like fiber structures. The expression of chondrogenesis-associated genes increased after 3, 7, 21, 28 days of chondrogenic induction, especially for collagen type II, collagen type I, aggrecan and collagen type X. Transplanted artificial cartilage was existed in rabbit for six weeks. The mature chondrocyte-like cells were detected and cartilage specific-proteoglycan accumulated more in differentiated cell-scaffold than scaffold seeded undifferentiated-cells. Thus artificial cartilage could be created when differentiating rBM-MSC into chondrocyte on collagen-coated PCL scaffold.

Initiating human articular chondrocyte re-differentiation in a 3D system after 2D expansion

Cartilage damage affects a large population via acute and chronic injury and disease. Since native cartilage does not self-renew, cartilage tissue engineering has gained traction as a potential treatment. However, a limiting factor is that the primary cell type in cartilage, the articular chondrocyte, tends to de-differentiate when grown on 2D surfaces for in vitro expansion. Thus, 3D systems are being developed and used to counter this loss of chondrogenic capabilities. We hypothesize that a 3D matrix that can be remodeled may be more supportive of the chondrogenic phenotype of encapsulated articular chondrocytes than a 2D surface and may allow for the re-differentiation of chondrocytes after 2D expansion. Hence, in this study, enzymatically degradable polyethylene glycol (PEG) hydrogels containing two different protease degradable peptide segments, with different degradation rates, were tested in combination with chondrogenic medium as a 3D in vitro culture system to better recapitulate the native environment of human articular chondrocytes (hACs). In addition, the effect of incorporation of the integrin binding ligand Arg-Gly-Asp (RGD) in the hydrogels was explored. Hydrogels crosslinked with a slower degrading crosslinker and not functionalized with RGD maintained hAC viability and led to increased GAG production and chondrogenic gene expression over time, suggesting that this system can initiate hAC re-differentiation after 2D expansion.Graphical abstract

Effects of Micronized Cartilage Matrix on Cartilage Repair in Osteochondral Lesions of the Talus

Category: Ankle, Arthroscopy, Sports Introduction/Purpose: A promising new technique in the treatment of osteochondral lesions of the talus (OLT) involves the use of an acellular micronized cartilage matrix (MCM), BioCartilage, to fill the lesions. The micronized cartilage matrix is thought to improve the production of hyaline-like cartilage by resident cells in a cartilage defect, but its effect on bone marrow cells remains untested. Here we hypothesized that adding bone-marrow derived stem cells to the BioCartilage would result in the chondrogenic differentiation of the stem cells. We designed an in-vitro model to mimic the clinical situation to determine if the combination of MCM and human bone marrow derived mesenchymal stem cells (MSCs) would produce a hyaline-like cartilage in- vitro to ultimately provide a reliable, one-step treatment for osteochondral lesions in the talus. Methods: Human bone marrow-derived stem cells were obtained from consented patients and expanded in monolayer culture using standard protocols, to a maximum passage of 4. Viability was measured using Live/Dead cell viability assays (Thermofisher), and imaged on a Nikon TE2000 inverted fluorescent microscope. A custom-manufactured polysulfone device was created with four 6 mm diameter 3 mm deep indentations in agarose within each well of standard 6-well culture plates (Figure 1A-C). In each well, we placed chrondrogenic media with cells+micronized matrix to a depth of 2 mm and covered with a 1 mm layer of TISSEEL fibrin glue as is done clinically. Control groups had either no cells, or no MCM. At the end of 3 weeks, cartilage constructs were extracted and divided to perform viability, histology, and gene expression analysis (Figure 1D). Experiments were performed with 4 technical replicates, and repeated at least 3 times. Statistical analysis was performed using ANOVA with Dunnett’s test. Results: We found that stem cells were almost immediately killed when added directly to the dry micronized cartilage powder. Rehydrating the micronized cartilage prior to addition of cells was required to maintain the viability of the added stem cells, with no statistically significant difference between rehydration with serum or saline. After 3 weeks of culture in chondrogenic media, we observed that the combination of stem cells and micronized cartilage produced a cohesive structures that were easily handled, suggesting chondrogenic differentiation of the stem cells. Without the micronized matrix, the stem cells did not form viable constructs. In constructs that contained both cells and micronized cartilage, the 3-week cell viability was over 98%, with no dead cells visible in many constructs. Conclusion: Our study demonstrates that the micronized cartilage matrix is a suitable scaffold for the chondrogenic differentiation of bone marrow-derived stem cells, given that the matrix is first rehydrated before adding cells. Technical observations include that the MCM itself generated a “dead cell” signal initially, therefore the normalized total number of live cells in each condition was used for statistical comparisons. After 3 weeks of culturing under chondrogenic media conditions, we observed robust cell survival with nearly 100% viability. Preliminary results suggest cartilage matrix deposition occurred surrounding the cells after 3 weeks of chondrogenic culture.

A Perfusion Bioreactor for Making Tissue-Engineered Constructs

Experiments were performed to develop a design for a flow bioreactor. A system was constructed consisting of a small-scale bioreactor placed in a CO2 incubator, with four chambers for making tissue-engineered constructs, providing for long-term experiments in flow conditions maintaining sterility with different humidity and temperature factors in the culture and gaseous media. The adverse influences of flow on cell viability were minimized by optimizing the culture medium flow rate and the excess pressure in the flow chambers. The functional effectiveness of the bioreactor was demonstrated by chondrogenic differentiation of cells in a cell-engineered construct consisting of a microheterogeneous collagen-containing hydrogel matrix biopolymer, mesenchymal stromal cells from human fatty tissue, and chondrogenic differentiation medium as an example.

Efficacy of thermoresponsive, photocrosslinkable hydrogels derived from decellularized tendon and cartilage extracellular matrix for cartilage tissue engineering.

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin-soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow MSCs and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell-mediated contraction, as compared against non-methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition and histology. In particular, methacrylated cartilage hydrogels supported minimal due to chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell-matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non-methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan and collagen type II) but increased collagen type I expression. Non-methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non-methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogeneous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin-solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal- and light-induced polymerization but compromises chondrogenic differentiation of seeded MSCs.

Effect of Various Ratios of Co-Cultured ATDC5 Cells and Chondrocytes on the Expression of Cartilaginous Phenotype in Microcavitary Alginate Hydrogel.

The present study introduced a direct co-culture of mouse ATDC5 cells and primary porcine chondrocytes into a microcavitary hydrogel, which possessed advantages in promoting the growth of chondrocytes and retaining the phenotype. These two types of cells were encapsulated with gelatin microspheres in alginate hydrogels in either of the three ratios (3:1, 1:1, or 1:3 of ATDC5 cells to chondrocytes) and cultured in chondrogenic medium for 28 days. Simultaneously, the single encapsulation of ATDC5 cells or chondrocytes was set as a control. Cell Counting Kit-8 (CCK-8), real-time PCR, and immunohistochemistry staining were used to evaluate the effect of various ratios of co-cultured ATDC5 cells and chondrocytes on the expression of the cartilaginous phenotype. The CCK-8 data indicated that the ratio of 3:1 group had an outstanding ability of cell growth. The other results demonstrated that higher the ATDC5 ratios and longer the culture duration, greater the expression of cartilage-specific genes (including type II collagen and aggrecan) and more the synthesized cartilaginous extracellular matrix. Also, the Western blot analysis suggested that p44/42 MAP Kinase was involved in cell proliferation. However, due to the direct co-culture of the two cell types, the underlying mechanism necessitates further investigation. Overall, the co-culture system in microcavitary hydrogel improved the effect of chondrogenesis and exhibited promising strategy for cartilage tissue engineering therapies. J. Cell. Biochem. 118: 3607-3615, 2017. © 2017 Wiley Periodicals, Inc.

Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses.

Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is considered potentially effective for cartilage repair and in-vitro chondrogenesis. We thus aimed to harness chondro-promotive effects of PRP and hypertrophic-suppressive effects of AC:MSC co-culture to achieve a more functional cartilage neo-tissue. We used PRP or conventional-differentiation chondrogenic media (ConvDiff) in MSC mono-cultures and AC:MSC co-cultures. We assessed gene expression of chondrogenic and hypertrophic markers using real-time RT-PCR and immunostaining. Alkaline-phosphatase activity (ALP) and calcium content of the pellets were quantified. We also measured VEGF and TNF-α secretion via ELISA. We showed PRP had higher chondrogenic potential (in mRNA and protein level) and hypertrophic-suppressive effects than Conv-Diff (mRNA level). Co-culturing reduced ALP while PRP increased calcium deposition. In all four groups, TNF-α was down-regulated compared to MSC controls, with co-cultures receiving ConvDiff media secreting the least. Meanwhile, the only group with increased VEGF secretion was PRP-mono-cultures. We observed synergistic effects for PRP and AC:MSC co-culture in enhancing chondrogenesis. Inclusion of AC reduced hypertrophic markers and angiogenic potential in PRP groups. We thus propose that combination of PRP and co-culture would favor chondrogenesis while alleviate but not totally eradicate undesired hypertrophic and pathologic responses.

Combination of bone marrow mesenchymal stem cells and cartilage fragments contribute to enhanced repair of osteochondral defects.

Cartilage tissue engineering using stem cells and biomaterials is considered a promising approach despite poor outcomes. We hypothesise that articular cartilage fragments provides native environmental cues to enhance stem cell differentiation. As such we evaluated the chondrogenic differentiation and repair of critical size defect in a human explant osteochondral model (OD) using bone marrow derived mesenchymal stem cells (BM-MSCs) and homogenised cartilage. BM-MSCs were established from the bone-marrow plugs of patients undergoing total knee arthroplasty and characterized. Osteochondral tissue was trimmed and a central drill defect (∼2mm) was made. Chondrogenic repair was evaluated by filling the OD defect area with either BM-MSCs (Group II), homogenized cartilage (Group III) or a combination of both BM-MSCs and homogenized cartilage (Group IV). OD with no added cell or tissue served as control (Group I). Samples were maintained in chondrogenic differentiation medium for 28 days. Microscopic images showed maximal OD closure in Group IV. Partial OD closure was observed in Group II and to a lesser extent in Group III. Haematoxylin-eosin staining revealed immature cartilaginous matrix in Group II and more mature matrix in Group IV. Sircol™ Assay showed increased collagen deposition in both Group II and Group IV. Immunostaining for both groups revealed positive staining for type II collagen. Combining BM-MSCs and homogenised cartilage demonstrated enhanced cartilage formation and defect filling in a human ex-vivo osteochondral model.

Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture.

Chondrogenesis of mesenchymal stem cells (MSCs) is induced in culture conditions that have been associated with oxidative stress, although the extent to which the oxidative environment affects differentiation and extracellular matrix (ECM) accumulation is not known. The objectives of this study were to evaluate the oxidative environment during MSCs chondrogenesis in conventional serum-free medium, and the effect of serum-supplementation on intracellular reactive oxygen species (ROS) and chondrogenesis. Young adult equine MSCs were seeded into agarose and cultured in chondrogenic medium, with or without 5% fetal bovine serum (FBS), for up to 15 days. Samples were evaluated for intracellular ROS, the antioxidant glutathione, ECM and gene expression measures of chondrogenesis, and carbonylation as an indicator of oxidative damage. Intracellular ROS increased with time in culture, and was lower in medium supplemented with FBS. Glutathione decreased ∼12-fold during early chondrogenesis (p < 0.0001), and was not affected by FBS (p = 0.25). After 15 days of culture, FBS supplementation increased hydroxyproline accumulation ∼80% (p = 0.0002); otherwise, measures of chondrogenesis were largely unaffected. Protein carbonylation in chondrogenic MSCs cultures was not significantly different between serum-free and FBS cultures (p = 0.72). Supplementation with adult equine serum increased hydroxyproline accumulation by 45% over serum-free culture (p = 0.0006). In conclusion, this study characterized changes in the oxidative environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:506-514, 2018.

Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors

BackgroundEvaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions.MethodsFresh human bone marrow aspirates were transduced with rAAV-FLAG-hsox9 (40 μl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of rAAV vector; reporter rAAV-lacZ, rAAV-RFP, and rAAV-luc gene transfer).ResultsProlonged, significant sox9 overexpression via rAAV was achieved in the aspirates for at least 28 days when applying the rAAV-FLAG-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments.ConclusionsThe current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by rAAV sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces.

Modified methods for efficiently differentiating human embryonic stem cells into chondrocyte-like cells.

Human articular cartilage has a poor regenerative capacity. This often results in the serious joint disease- osteoarthritis (OA) that is characterized by cartilage degradation. An inability to self-repair provided extensive studies on AC regeneration. The cell-based cartilage tissue engineering is a promising approach for cartilage regeneration. So far, numerous cell types have been reported to show chondrogenic potential, among others human embryonic stem cells (hESCs). However, the currently used methods for directed differentiation of human ESCs into chondrocyte-like cells via embryoid body (EB) formation, micromass culture (MC) and pellet culture (PC) are not highly efficient and require further improvement. In the present study, these three methods for hESCs differentiation into chondrocyte-like cells in the presence of chondrogenic medium supplemented with diverse combination of growth factors (GFs) were evaluated and modified. The protocols established here allow highly efficient, simple and inexpensive production of a large number of chondrocyte-like cells suitable for transplantation into the sites of cartilage injury. The most crucial issue is the selection of appropriate GFs in defined concentration. The obtained stem-derived cells reveal the presence of chondrogenic markers such as type II collagen, Sox6 and Sox9 as well as the lack or significantly lower level of pluripotency markers including Nanog and Oct3/4. The most efficient method is the differentiation throughout embryoid bodies. In turn, chondrogenic differentiation via pellet culture is the most promising method for implementation on clinical scale. The most useful GFs are TGF-β1, -3 and BMP-2 that possess the most chondrogenic potential. These methods can also be used to obtain chondrocyte-like cells from differentiating induced pluripotent stem cells (iPSCs).

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets.

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.