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B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

ABSTRACT: This study investigated whether the synthetic peptide B2A (B2A2-K-NS) could induce in vitro chondrogenic differentiation and enhance the in vivo repair of damaged cartilage in an osteoarthritis model. In vitro, micromass cultures of murine and human stem cells with and without B2A were used as models of chondrogenic differentiation. Micromasses were evaluated for gene expression using microarray analysis and quantitative PCR; and for extracellular matrix production by Alcian blue staining for sulfated glycosaminoglycan and immunochemical detection of collagen type II. In vivo, osteoarthritis was chemically induced in knees of adult rats by an injection of mono-iodoacetate (MIA) into the synovial space. Treatment was administered at 7- and 14 days after the MIA by injection into the synovial space of B2A or saline and terminated at 21 days, after which knee cartilage damage was determined and scored by histological analysis. In murine C3H10T1/2 micromass culture, B2A induced the expression of more than 11 genes associated with growth factors/receptors, transcription, and the extracellular matrix, including PDGF-AA. B2A also significantly increased the sulfated glycosaminoglycan and collagen of murine- and human micromass cultures. In the knee osteoarthritis model, B2A treatment enhanced cartilage repair compared to untreated knees as determined histologically by a decrease in damage indicators. These findings suggest that B2A induces stem cells chondrogenic differentiation in vitro and enhances cartilage repair in vivo. The results suggest that B2A might be useful to promote cartilage repair.

SUBMITTER: Lin X

PROVIDER: S-EPMC3349005 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

Lin Xinhua X Shanmugasundaram Shobana S Liu Yi Y Derrien Alexandrine A Nurminskaya Maria M Zamora Paul O PO

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20120123 8

This study investigated whether the synthetic peptide B2A (B2A2-K-NS) could induce in vitro chondrogenic differentiation and enhance the in vivo repair of damaged cartilage in an osteoarthritis model. In vitro, micromass cultures of murine and human stem cells with and without B2A were used as models of chondrogenic differentiation. Micromasses were evaluated for gene expression using microarray analysis and quantitative PCR; and for extracellular matrix production by Alcian blue staining for su ...[more]

PMID: 22271086

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Project description:Surgical strategies to treat articular cartilage injury such as microfracture, expose human bone marrow stem cells (hMSCs) to synovial fluid and its components. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules of healthy synovial fluid (hSF) and it plays an important role in the protection of opposing articular cartilage surfaces within the synovial joint. Although hMwt HA has been extensively used to attempt the engineering of the cartilage tissue, its effect as media supplement has not been established. Indeed, current media are often simple in their composition and doesn't recapitulate the rheological and biological features of hSF. In addition, critical in vivo molecules that can potentially change the chondrogenic behavior of hBMSCs to make the in vitro results more predictive of the real in vivo outcome, are lacking. In order to be one step closer to the in vivo physiology of hSF, a new culture media supplemented with physiological level of hMwt HA was developed and the effect of the hMwt HA on the chondrogenesis of hMSCs that would be present in a traumatic defect after marrow stimulation techniques, was investigated. hBMSC-seeded fibrin-polyurethane constructs were cultured in a serum free chondropermissive control medium (HA- TGF?-). This medium was further supplemented with 10 ng/mL TGF?1 (HA- TGF?+) or 2 mg/ml hMwt HA 1.8 MDa (HA+ TGF?-) or both (HA+ TGF?+). Alternatively, 1 MDa HA was mixed with the fibrin at 0.2 mg/ml (HASc TGF?+). The effect of hMwt HA on hMSC differentiation was investigated at the gene expression level by RT-qPCR and total DNA, sulfated glycosaminoglycans and Safranin O staining were evaluated. Addition of hMwt HA to the culture media, significantly increased the synthesis of sulfated glycosaminoglycans, especially in the early days of chondrogenesis, and reduced the upregulation of the hypertrophic cartilage marker collagen X. hMwt HA added inside the fibrin gel(HASc TGF+) led to the best matrix deposition. hMwt HA can be one key medium component in a more reliable in vitro/ex vivo system to reduce in vitro artifacts, enable more accurate pre-screening of potential cartilage repair therapies and reduce the need for animal studies.

Project description:Objectives: Repairing articular cartilage damage is challenging. Clinically, tissue engineering technology is used to induce stem cell differentiation and proliferation on biological scaffolds to repair defective joints. However, no ideal biological scaffolds have been identified. This study investigated the effects of amniotic membrane/collagen scaffolds on the differentiation of adipose-derived stem cells (ADSCs) and articular cartilage repair. Methods: Adipose tissue of New Zealand rabbits was excised, and ADSCs were isolated and induced for differentiation. An articular cartilage defect model was constructed to identify the effect of amniotic membrane/collagen scaffolds on cartilage repair. Cartilage formation was analyzed by imaging and toluene blue staining. Knee joint recovery in rabbits was examined using hematoxylin and eosin, toluidine, safranine, and immunohistochemistry at 12 weeks post-operation. Gene expression was examined using ELISA, RT-PCR, Western blotting, and immunofluorescence. Results: The adipose tissue was effectively differentiated into ADSCs, which further differentiated into chondrogenic, osteogenic, and lipogenic lineages after 3 weeks' culture in vitro. Compared with platelet-rich plasmon (PRP) scaffolds, the amniotic membrane scaffolds better promoted the growth and differentiation of ADSCs. Additionally, scaffolds containing the PRP and amniotic membrane efficiently enhanced the osteogenic differentiation of ADSCs. The levels of COL1A1, COL2A1, COL10A1, SOX9, and ACAN in ADSCs + amniotic membrane + PRP group were significantly higher than the other groups both in vitro and in vivo. The Wakitani scores of the ADSC + amniotic membrane + PRP group were lower than that in ADSC + PRP (4.4 ± 0.44**), ADSC + amniotic membrane (2.63 ± 0.38**), and control groups (6.733 ± 0.21) at week 12 post-operation. Osteogenesis in rabbits of the ADSC + amniotic membrane + PRP group was significantly upregulated when compared with other groups. Amniotic membranes significantly promoted the expression of cartilage regeneration-related factors (SOX6, SOX9, RUNX2, NKX3-2, MEF2C, and GATA4). The ADSC + PRP + amniotic membrane group exhibited the highest levels of TGF-β, PDGF, and FGF while exhibiting the lowest level of IL-1β, IL6, and TNF-α in articular cavity. Conclusion: Amniotic membrane/collagen combination-based scaffolds promoted the proliferation and cartilage differentiation of ADSCs, and may provide a new treatment paradigm for patients with cartilage injury.

Project description:Even small cartilage defects could finally degenerate to osteoarthritis if left untreated, owing to the poor self-healing ability of articular cartilage. Stem cell transplantation has been well implemented as a common approach in cartilage tissue engineering but has technical complexity and safety concerns. The stem cell homing-based technique emerged as an alternative promising therapy for cartilage repair to overcome traditional limitations. In this study, we constructed a composite hydrogel scaffold by combining an oriented acellular cartilage matrix (ACM) with a bone marrow homing peptide (BMHP)-functionalized self-assembling peptide (SAP). We hypothesized that increased recruitment of endogenous stem cells by the composite scaffold could enhance cartilage regeneration. Methods: To test our hypothesis, in vitro proliferation, attachment and chondrogenic differentiation of rabbit mesenchymal stem cells (MSCs) were tested to confirm the bioactivities of the functionalized peptide hydrogel. The composite scaffold was then implanted into full-thickness cartilage defects on rabbit knee joints for cartilage repair, in comparison with microfracture or other sample groups. Stem cell recruitment was monitored by dual labeling with CD29 and CD90 under confocal microcopy at 1 week after implantation, followed by chondrogenic differentiation examined by qRT-PCR. Repaired tissue of the cartilage defects was evaluated by histological and immunohistochemistry staining, microcomputed tomography (micro-CT) and magnetic resonance imaging (MRI) at 3 and 6 months post-surgery. Macroscopic and histological scoring was done to evaluate the optimal in vivo repair outcomes of this composite scaffold. Results: The functionalized SAP hydrogels could stimulate rabbit MSC proliferation, attachment and chondrogenic differentiation during in vitro culture. At 7 days after implantation, increased recruitment of MSCs based on CD29+ /CD90+ double-positive cells was found in vivo in the composite hydrogel scaffold, as well as upregulation of cartilage-associated genes (aggrecan, Sox9 and type II collagen). After 3 and 6 months post-surgery, the articular cartilage defect in the composite scaffold-treated group was fully covered with cartilage-like tissue with a smooth surface, which was similar to the surrounding native cartilage, according to the results of histological and immunohistochemistry staining, micro-CT and MRI analysis. Macroscopic and histological scoring confirmed that the quality of cartilage repair was significantly improved with implantation of the composite scaffold at each timepoint, in comparison with microfracture or other sample groups. Conclusion: Our findings demonstrated that the composite scaffold could enhance endogenous stem cell homing and chondrogenic differentiation and significantly improve the therapeutic outcome of chondral defects. The present study provides a promising approach for in vivo cartilage repair without cell transplantation. Optimization of this strategy may offer great potential and benefits for clinical application in the future.

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 significanceCurrent 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.

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B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

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B2A peptide induces chondrogenic differentiation in vitro and enhances cartilage repair in rats.

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