Cartilage progenitor cells combined with PHBV in cartilage tissue engineering.
ABSTRACT: BACKGROUND:Bone marrow-derived stem cells (BMSCs) and chondrocytes have been reported to present "dedifferentiation" and "phenotypic loss" during the chondrogenic differentiation process in cartilage tissue engineering, and cartilage progenitor cells (CPCs) are novel seeding cells for cartilage tissue engineering. In our previous study, cartilage progenitor cells from different subtypes of cartilage tissue were isolated and identified in vitro, but the study on in vivo chondrogenic characteristics of cartilage progenitor cells remained rarely. In the current study, we explored the feasibility of combining cartilage progenitor cells with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) to produce tissue-engineered cartilage and compared the proliferation ability and chondrogenic characteristics of cartilage progenitor cells with those of bone marrow-derived stem cells and chondrocytes. METHODS:These three cells combined with PHBV were cultured in vitro for 1 week without chondrogenic induction and then transplanted subcutaneously into nude mice for 6 weeks. The cell-PHBV constructs were evaluated by gross observation, histological staining, glycosaminoglycan content measurement, biomechanical analysis and RT-PCR. RESULTS:The chondrocyte-PHBV constructs and CPC-PHBV constructs became an ivory-whitish cartilage-like tissue, while the BMSC-PHBV constructs became vascularized 6 weeks after the subcutaneous implantation. Histological examination showed that many typical cartilage structures were present in the chondrocyte group, some typical cartilage structures were observed in the CPC group, while no typical cartilage structures were observed in the BMSC group. CONCLUSIONS:Cartilage progenitor cells may undergo chondrogenesis without chondrogenic induction and are better at chondrogenesis than BMSCs but worse than chondrocytes in the application of cartilage tissue engineering.
Project description:The regeneration of diseased hyaline cartilage remains a great challenge, mainly because degeneration activities after major injury or due to age-related processes overwhelm the self-renewal capacity of the tissue. We show that repair tissue from human articular cartilage of late stages of osteoarthritis harbor a unique progenitor cell population, termed chondrogenic progenitor cells exhibiting stem cell characteristics, such as multipotency, lack of immune system activation and, in particular, migratory activity. The isolated CPC exhibit a high chondrogenic potential and were able to populate diseased tissue in vivo. Moreover, down-regulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9 and consequently the matrix synthesis potential of chondrogenic progenitor cells. Our results, while offering new insight into the biology of progenitor cells from diseased cartilage tissue, might assist future strategies to treat late stages of osteoarthritis. Experiment Overall Design: Characterization chondrogenic progenitor cells in P1 of 3 male and 3 female patients with late-stage OA in comparison to healthy chondrocytes in P1
Project description:The regeneration of diseased hyaline cartilage remains a great challenge, mainly because degeneration activities after major injury or due to age-related processes overwhelm the self-renewal capacity of the tissue. We show that repair tissue from human articular cartilage of late stages of osteoarthritis harbor a unique progenitor cell population, termed chondrogenic progenitor cells exhibiting stem cell characteristics, such as multipotency, lack of immune system activation and, in particular, migratory activity. The isolated CPC exhibit a high chondrogenic potential and were able to populate diseased tissue in vivo. Moreover, down-regulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9 and consequently the matrix synthesis potential of chondrogenic progenitor cells. Our results, while offering new insight into the biology of progenitor cells from diseased cartilage tissue, might assist future strategies to treat late stages of osteoarthritis. Keywords: cell type comparison Overall design: Characterization chondrogenic progenitor cells in P1 of 3 male and 3 female patients with late-stage OA in comparison to healthy chondrocytes in P1
Project description:For the generation of multi-layered full thickness osteochondral tissue substitutes with an individual geometry based on clinical imaging data, combined extrusion-based 3D printing (3D plotting) of a bioink laden with primary chondrocytes and a mineralized biomaterial phase was introduced. A pasty calcium phosphate cement (CPC) and a bioink based on alginate-methylcellulose (algMC) - both are biocompatible and allow 3D plotting with high shape fidelity - were applied in monophasic and combinatory design to recreate osteochondral tissue layers. The capability of cells reacting to chondrogenic biochemical stimuli inside the algMC-based 3D hydrogel matrix was assessed. Towards combined osteochondral constructs, the chondrogenic fate in the presence of CPC in co-fabricated and biphasic mineralized pattern was evaluated. Majority of expanded and algMC-encapsulated cells survived the plotting process and the cultivation period, and were able to undergo redifferentiation in the provided environment to produce their respective extracellular matrix (ECM) components (i.e. sulphated glycosaminoglycans, collagen type II), examined after 3 weeks. The presence of a mineralized zone as located in the physiological calcified cartilage region suspected to interfere with chondrogenesis, was found to support chondrogenic ECM production by altering the ionic concentrations of calcium and phosphorus in in vitro culture conditions.
Project description:: The regeneration of tissue-engineered cartilage in an immunocompetent environment usually fails due to severe inflammation induced by the scaffold and their degradation products. In the present study, we compared the tissue remodeling and the inflammatory responses of engineered cartilage constructed with bone marrow mesenchymal stem cells (BMSCs), chondrocytes, or both and scaffold group in pigs. The cartilage-forming capacity of the constructs in vitro and in vivo was evaluated by histological, biochemical, and biomechanical analyses, and the inflammatory response was investigated by quantitative analysis of foreign body giant cells and macrophages. Our data revealed that BMSC-based engineered cartilage suppressed in vivo inflammation through the alteration of macrophage phenotype, resulting in better tissue survival compared with those regenerated with chondrocytes alone or in combination with BMSCs. To further confirm the macrophage phenotype, an in vitro coculture system established by engineered cartilage and macrophages was studied using immunofluorescence, enzyme-linked immunosorbent assay, and gene expression analysis. The results demonstrated that BMSC-based engineered cartilage promoted M2 polarization of macrophages with anti-inflammatory phenotypes including the upregulation of CD206, increased IL-10 synthesis, decreased IL-1? secretion, and alterations in gene expression indicative of M1 to M2 transition. It was suggested that BMSC-seeded constructs have the potential to ameliorate scaffold-induced inflammation and improve cartilaginous tissue regeneration through M2 polarization of macrophages.Finding a strategy that can prevent scaffold-induced inflammation is of utmost importance for the regeneration of tissue-engineered cartilage in an immunocompetent environment. This study demonstrated that bone marrow mesenchymal stem cell (BMSC)-based engineered cartilage could suppress inflammation by increasing M2 polarization of macrophages, resulting in better tissue survival in a pig model. Additionally, the effect of BMSC-based cartilage on the phenotype conversion of macrophages was further studied through an in vitro coculture system. This study could provide further support for the regeneration of cartilage engineering in immunocompetent animal models and provide new insight into the interaction of tissue-engineered cartilage and macrophages.
Project description:BACKGROUND: Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage. METHODS AND FINDINGS: Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect. CONCLUSIONS: In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.
Project description:Osteoarthritis (OA) is characterized by a slowly progressing, irreversible loss of articular cartilage. Tissue engineering approaches for cartilage regeneration include stem cell-based strategies but not much is known about their repair capacity in an OA microenvironment. The aim of the present study was to identify factors regulating collagen expression during chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSC) in an OA microenvironment. Coculture with OA cartilage induced miR-29b expression in BMSC which inhibited collagen I and III expression. Elevated miR-29b expression resulted in higher caspase 3/7 activity and promoted apoptosis of BMSC in part by directly inhibiting the anti-apoptotic proteins Bcl-2 and Mcl-1. Stimulation with IFN-? induced miR-29b expression in BMSC. Our results suggest that miR-29b affects BMSC-based OA cartilage regeneration because expression of collagen III, mainly produced by undifferentiated BMSC, and collagen I, a marker for dedifferentiated chondrocytes, are inhibited by miR-29b thus influencing composition of the newly formed ECM. This might be critical to avoid formation of inferior fibrocartilage instead of hyaline cartilage. Furthermore, higher miR-29b expression promotes apoptosis either preventing excessive cell growth or reducing the number of BMSC undergoing chondrogenesis. Thus, miR-29b has both supportive but possibly also unfavourable effects on BMSC-based OA cartilage regeneration.
Project description:Adult articular cartilage has a limited capacity for growth and regeneration and, with injury, new cellular or biomaterial-based therapeutic platforms are required to promote repair. Tissue engineering aims to produce cartilage-like tissues that recreate the complex mechanical and biological properties found in vivo. In this study, a unique composite scaffold was developed by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-assembling nanofibers to obtain multi-scale functional and biomimetic tissue-engineered constructs. The scaffold was seeded with expanded dedifferentiated human articular chondrocytes and cultured for four weeks in control and chondrogenic growth conditions. The composite constructs were compared to control constructs obtained by culturing cells with 3D woven PCL scaffolds or RAD16-I independently. High viability and homogeneous cell distribution were observed in all three scaffolds used during the term of the culture. Moreover, gene and protein expression profiles revealed that chondrogenic markers were favored in the presence of RAD16-I peptide (PCL/RAD composite or alone) under chondrogenic induction conditions. Further, constructs displayed positive staining for toluidine blue, indicating the presence of synthesized proteoglycans. Finally, mechanical testing showed that constructs containing the PCL scaffold maintained the initial shape and viscoelastic behavior throughout the culture period, while constructs with RAD16-I scaffold alone contracted during culture time into a stiffer and compacted structure. Altogether, these results suggest that this new composite scaffold provides important mechanical requirements for a cartilage replacement, while providing a biomimetic microenvironment to re-establish the chondrogenic phenotype of human expanded articular chondrocytes.
Project description:The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1?) as well as tumor necrosis factor alpha (TNF-?) were tested with a modified Boyden chamber assay. The influence of IL-1? and TNF-? was additionally examined by scratch assays and outgrowth experiments.A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1? and TNF-? significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1? and TNF-? inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.
Project description:Articular cartilage was thought to be one of the first tissues to be successfully engineered. Despite the avascular and non-innervated nature of the tissue, the cells within articular cartilage - chondrocytes - account for a complex phenotype that is difficult to be maintained in vitro. The use of bone marrow-derived stromal cells (BMSCs) has emerged as a potential solution to this issue. Differentiation of BMSCs toward stable and non-hypertrophic chondrogenic phenotypes has also proved to be challenging. Moreover, hyaline cartilage presents a set of mechanical properties - relatively high Young's modulus, elasticity, and resilience - that are difficult to reproduce. Here, we report on the use of additive manufactured biodegradable poly(ester)urethane (PEU) scaffolds of two different structures (500 ?m pore size and 90° or 60° deposition angle) that can support the loads applied onto the knee while being highly resilient, with a permanent deformation lower than 1% after 10 compression-relaxation cycles. Moreover, these scaffolds appear to promote BMSC differentiation, as shown by the deposition of glycosaminoglycans and collagens (in particular collagen II). At gene level, BMSCs showed an upregulation of chondrogenic markers, such as collagen II and the Sox trio, to higher or similar levels than that of traditional pellet cultures, with a collagen II/collagen I relative expression of 2-3, depending on the structure of the scaffold. Moreover, scaffolds with different pore architectures influenced the differentiation process and the final BMSC phenotype. These data suggest that additive manufactured PEU scaffolds could be good candidates for cartilage tissue regeneration in combination with microfracture interventions.
Project description:Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21?days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14?days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo. STATEMENT OF SIGNIFICANCE:Current cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.