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Extracellular Matrix Components and Culture Regimen Selectively Regulate Cartilage Formation by Self-Assembling Human Mesenchyma

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Extracellular Matrix Components and Culture Regimen Selectively Regulate Cartilage Formation by Self-Assembling Human Mesenchyma Ng et al. Stem Cell Research & Therapy (2016) 7:183 DOI 10.1186/s13287-016-0447-4 RESEARCH Open Access Extracellular matrix components and culture regimen selectively regulate cartilage formation by self-assembling human mesenchymal stem cells in vitro and in vivo Johnathan Ng1, Yiyong Wei1, Bin Zhou1,3, Aonnicha Burapachaisri1, Edward Guo1,3 and Gordana Vunjak-Novakovic1,2* Abstract Background: Cartilage formation from self-assembling mesenchymal stem cells (MSCs) in vitro recapitulate important cellular events during mesenchymal condensation that precedes native cartilage development. The goal of this study was to investigate the effects of cartilaginous extracellular matrix (ECM) components and culture regimen on cartilage formation by self-assembling human MSCs in vitro and in vivo. Methods: Human bone marrow-derived MSCs (hMSCs) were seeded and compacted in 6.5-mm-diameter transwell inserts with coated (type I, type II collagen) or uncoated (vehicle) membranes, at different densities (0.5 × 106,1.0×106, 1.5 × 106 per insert). Pellets were formed by aggregating hMSCs (0.25 × 106) in round-bottomed wells. All tissues were cultured for up to 6 weeks for in vitro analyses. Discs (cultured for 6, 8 or 10 weeks) and pellets (cultured for 10 weeks) were implanted subcutaneously in immunocompromised mice to evaluate the cartilage stability in vivo. Results: Type I and type II collagen coatings enabled cartilage disc formation from self-assembling hMSCs. Without ECM coating, hMSCs formed dome-shaped tissues resembling the pellets. Type I collagen, expressed in the prechondrogenic mesenchyme, improved early chondrogenesis versus type II collagen. High seeding density improved cartilage tissue properties but resulted in a lower yield of disc formation. Discs and pellets exhibited compositional and organizational differences in vitro and in vivo. Prolonged chondrogenic induction of the discs in vitro expedited endochondral ossification in vivo. Conclusions: The outcomes of cartilage tissues formed from self-assembling MSCs in vitro and in vivo can be modulated by the control of culture parameters. These insights could motivate new directions for engineering cartilage and bone via a cartilage template from self-assembling MSCs. Background complications. Thus, there is an ongoing effort toward The articular cartilage has a limited ability to regenerate developing stem cell-based therapies [3]. upon injury. Current approaches to treat focal cartilage In cartilage tissue engineering (TE), scaffolding lesions include autograft, mosaicplasty, and autologous methods used successfully with chondrocytes have re- chondrocyte implantation [1, 2]. However, these sulted in subnormal cartilage formation by mesenchymal methods are limited by donor site morbidity and other stem cells (MSCs) – the most attractive cell source for clinical application [4]. In contrast, self-assembly * Correspondence: [email protected] methods recapitulated mesenchymal condensation 1Department of Biomedical Engineering, Columbia University, 622 West 168th Street, VC12-234, New York, NY 10032, USA during joint development, and enhanced the in vitro 2Department of Medicine, Columbia University, New York, NY, USA chondrogenesis of MSCs [5–8]. Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ng et al. Stem Cell Research & Therapy (2016) 7:183 Page 2 of 12 In pellet culture, cells form spherical aggregates that of the discs and pellets in vitro and in vivo. Finally, we deposit matrix and grow over time [5, 6]. In disc culture, investigated the in vivo fate of discs cultured under dif- multiple layers of cells deposit matrix and form a disc ferent durations in vitro. Overall, the study was designed that grows in thickness over time [8, 9]. Cartilage to clarify the effects of in vitro culture parameters on formed in pellet culture of self-assembling MSCs lacks the in vitro properties and in vivo fate of cartilage physiological stratification and is not well suited for formed by self-assembling hMSCs. cartilage repair due to its tendency to undergo endo- chondral ossification in vivo [3, 10, 11]. While the pellet Methods culture has been well established, the exact conditions Cell source and preparation for cartilage disc culture are not known [8, 9, 11–23]. In Fresh bone marrow aspirates were obtained from some cases, MSCs could not form discs due to the Cambrex (East Rutherford, NJ, USA) and processed as forces associated with self-assembly (Table 1). Further- in our previous studies. To compare multiple treatment more, it remains to be determined how the conditions of groups in vitro and in vivo, we used an aspirate that has MSC self-assembly in vitro affect cartilage fate in vivo. been well characterized and shown to undergo robust We hypothesized that (i) the membrane extracellular differentiation [7, 24]. Bone marrow-derived hMSCs matrix (ECM) coating and (ii) the initial cell seeding were isolated and expanded in high-glucose Dulbecco’s density determine functional disc formation in vitro. To modified Eagle’s medium (DMEM) containing 10% fetal this end, we investigated if the control of these two bovine serum, 1% Pen-Strep and 1 ng/mL of fibroblast parameters would enable reliable formation of cartilage growth factor-2 (FGF-2). At the end of the fourth discs by self-assembling MSCs. Further, we asked passage, hMSCs were seeded for experiments. Several whether the in vivo fate of cartilage can be modulated groups have reported the use of hMSCs at the end of by the self-assembly regimen (disc versus pellet) and the the fourth passage for chondrogenic differentiation pre- length of in vitro culture. viously [4, 25]. All reagents were from Life Technologies To test the hypotheses, we analyzed gene expression, (Carlsbad, CA, USA), unless specified otherwise. biochemical, mechanical, and morphological changes of self-assembling human MSCs (hMSCs) in membrane Cell seeding and differentiation inserts with or without ECM coating, following chon- For chondrogenic differentiation, serum-free chondro- drogenic induction at different cell seeding densities. genic media consisting of high-glucose DMEM, ITS+ Further, we compared the composition and organization supplement (BD Biosciences, San Jose, CA, USA), Table 1 Summary of studies on disc culture of self-assembling chondrocytes or MSCs for in vitro cartilage formation Cell type Source Density (× 106/mm2) Insert type Substrate Disc formation Thickness (mm) Reference (Yes/No) Chondrocytes Bovine MCC joint 0.050 Millicell PTFE Type II collagen Yes - [9] Bovine MCC joint 0.016 Millicell PTFE Type II collagen Yes 0.3 [12] Bovine MCC joint 0.160 - CPP Yes 0.94 [13] Sheep MCC joint 0.160 - CPP Yes 0.5 [14] Bovine hock joint 0.067 Millicell PTFE Type II collagen Yes 0.45 [15] Bovine MCC joint 0.033 Millicell PTFE Type II collagen Yes - [16] Bovine distal femur 0.172 - Agarose Yes 0.15 [17] Bovine distal femur 0.289 - Agarose Yes 0.8 [18] Human femoral cartilage 0.006 Millicell PTFE Type I/Type II collagen Yes 0.15 [19] Human articular cartilage 0.102 - Agarose Yes 0.6 [20] MSCs Human BM 0.015 Corning PC None Yes 0.8 [8] Porcine BM 0.392 Millicell PET None Yes 1.3 [21] Human BM 0.015 Corning PC None No - [11] Sheep BM 0.033 Millicell PTFE Type IV collagen/CPP Yes 0.5 [22] Equine cord blood 0.033 Millicell Fibronectin Yes 0.1 [23] Human BM 0.102 - Agarose No - [20] In some cases, MSCs failed to form discs MSC mesenchymal stem cell, MCC convex condyle of the metacarpus, CPP calcium polyphosphate, BM bone marrow Ng et al. Stem Cell Research & Therapy (2016) 7:183 Page 3 of 12 5 mM L-proline, HEPES, sodium pyruvate, dexametha- we determined that 4 weeks was the shortest duration of sone, 50 μM ascorbic acid (Sigma-Aldrich, St. Louis, implantation for evaluating endochondral ossification. MO, USA) and 10 ng/mL transforming growth factor Samples were explanted at 4 weeks and analyzed. beta-3 (TGF-β3) (PeproTech, Rocky Hill, NJ, USA) was used. All reagents were from Life Technologies, unless Gene expression analysis otherwise specified. Samples (n = 4 per group) were crushed with pestles and For disc formation, 6.5-mm-diameter transwell inserts homogenized by mixing in the TRIzol reagent. RNA was with polycarbonate membranes were used (Corning, extracted according to the manufacturer’s instructions Corning, NY, USA). To each transwell insert, 50 μLof using the TRIzol method. The quantity of RNA was vehicle (1 mM acetic acid with ethanol), 1.5 mg/mL type measured on the Nanodrop ND1000 (Thermo Fisher I collagen (BD Biosciences, San Jose, CA, USA) or type Scientific). Following treatment with DNase I removal II collagen (Elastin Products, Owensville, MI, USA) was kit, cDNA synthesis was performed using the Applied added and the transwell inserts were air dried overnight. Biosystems (Waltham, MA, USA) High Capacity kit ac- The coated transwell inserts were rinsed briefly with cording to the manufacturer’s instructions. Quantitative phosphate-buffered saline (PBS) and hMSCs were seeded RT-PCR analysis was done using 20 ng cDNA per reac- at various densities (0.5 × 106,1×106, or 1.5 × 106 cells tion and the Applied Biosystems SYBR® Green PCR per well). After which, the 24-well plates loaded with Master Mix. The expression of target genes was normal- transwells were centrifuged at 200 g for 5 minutes. The ized to GAPDH, unless otherwise stated, and the mean next day, chondrogenic media was added so that every cycle threshold (CT) values of technical duplicates were transwell was fully submerged.
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