Genetic Engineering of Juvenile Human Chondrocytes Improves Scaffold-free Mosaic Neocartilage Grafts

Ng, Vincent Y.; Jump, Seth S.; Santangelo, Kelly S.; Russell, Duncan S.; Bertone, Alicia L.

doi:10.1007/s11999-012-2615-x

Cited by 8 publications

(4 citation statements)

References 50 publications

(75 reference statements)

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“…Alternatively, initial high BMP2 expression and release from fMSCs could have caused a transient increase in number of osteoclasts derived from hematopoietic stem cells [24] but there was no evidence of bone remodeling in our histology to suggest this mechanism was at work. Based on our in vitro findings with fMSC, and recent work from our laboratory demonstrating superior outcomes of neocartilage growth with a mosaic of 90% naïve and 10% Ad-BMP2-transduced juvenile chondrocytes [25] , it was anticipated that the dilution of BMP2 expressing cells in the rapidly proliferating fMSC population may be an advantage in vivo. In addition, Brady and colleagues recently demonstrated that recombinant BMP2 can induce chondrogenesis in pellet culture of fetal but not adult MSCs [23] .…”

Section: Discussionmentioning

confidence: 92%

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

Santiago-Torres

Lovasz

Bertone

2015

WJSC

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AIM:To investigate adenoviral transduction in mesenchymal stem cells (MSCs) and effects on stemness in vitro and function as a cell therapy in vivo . METHODS:Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine (PEI)-mediated transfection of pcDNA3-eGFP or adenoviral transduction of green fluorescent protein (GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness (i.e. , cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2 (BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology.RESULTS: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs (81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs (78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs (7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression (0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs (1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitro Clinical Trials Studywere not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult (P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells (P < 0.05). Ad-BMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs (83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone (1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05). CONCLUSION:Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo .

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Section: Discussionmentioning

confidence: 92%

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

Santiago-Torres

Lovasz

Bertone

2015

WJSC

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“…As a next step, to distinguish between spontaneous and focused regeneration, load‐bearing regions of the knee such as the medial and femoral condyle will be explored in rodents and other animal models. The trochlear groove in a lower weight‐bearing region is considered an appropriate model for proof of concept studies . Earlier studies that employed biomaterials on rat trochlear groove regeneration for critical sized defects have shown similar rates of regeneration .…”

Section: Discussionmentioning

confidence: 99%

In vivo evaluation of stem cell aggregates on osteochondral regeneration

et al. 2016

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To date, many osteochondral regenerative approaches have utilized varied combinations of biocompatible materials and cells to engineer cartilage. Even in cell-based approaches, to date, no study has utilized stem cell aggregates alone for regenerating articular cartilage. Thus, the purpose of this study was to evaluate the performance of a novel stem cell-based aggregate approach in a fibrin carrier to regenerate osteochondral defects in the Sprague-Dawley rat trochlear groove model. Two different densities of rat bone marrow mesenchymal stem cell (rBMSC) aggregates were fabricated by the hanging drop technique. At 8 weeks, the cell aggregates supported the defects and served as a catalyst for neo-cartilage synthesis, and the experimental groups may have been beneficial for bone and cartilage regeneration compared to the fibrin-only control and sham groups, as evidenced by histological assessment. The cell density of rBMSC aggregates may thus directly impact chondrogenesis. The usage of cell aggregates with fibrin as a cell-based technology is a promising and translational new treatment strategy for repair of cartilage defects. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1606-1616, 2017.

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“…Using our experience with the nude rodent model to study knee cartilage healing [ 25 , 26 ] we determined the healing potential of the sConstructs. The time point of healing, 5 weeks, was selected such that we could detect either a delay or acceleration of repair among our comparison groups.…”

Section: Introductionmentioning

confidence: 99%

Synovium extra cellular matrices seeded with transduced mesenchymal stem cells stimulate chondrocyte maturation in vitro and cartilage healing in clinically-induced rat-knee lesions in vivo

et al. 2019

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Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is “proof of concept” that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA.

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Genetic Engineering of Juvenile Human Chondrocytes Improves Scaffold-free Mosaic Neocartilage Grafts

Abstract: Genetic augmentation of jCh and creation of mosaic neocartilage may improve graft viability and articular healing compared with naïve neocartilage.

Cited by 8 publications

References 50 publications

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

In vivo evaluation of stem cell aggregates on osteochondral regeneration

Synovium extra cellular matrices seeded with transduced mesenchymal stem cells stimulate chondrocyte maturation in vitro and cartilage healing in clinically-induced rat-knee lesions in vivo

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