RUNX2-EXPRESSING PRIMARY DERMAL FIBROBLASTS … · 2017. 1. 30. · into subcutaneous pockets made...
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RUNX2-EXPRESSING PRIMARY DERMAL FIBROBLASTS MINERALIZE COLLAGEN SCAFFOLDS IN VIVO *Phillips, J E ; **Guldberg, R E; **García, A J *Georgia Tech/Emory Department of Biomedical Engineering, **Woodruff School of Mechanical Engineering, Georgia Institute of T echnology, Atlanta, GA 30322 INTRODUCTION: Bone tissue engineering strategies are limited by inadequate availability of a robust mineralizing cell source. Non- osteogenic cells, such as skin fibroblasts, are an attractive cell alternative because they are easy to harvest from autologous donors and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer [1]. However, sustained expression of Runx2 was not sufficient to promote functional osteogenesis and co- treatment with the steroid hormone dexamethasone was required for biologically -equivalent matrix mineralization in this model system. Furthermore, transient adenoviral Runx2 expression was also reported insufficient to produce significant levels of mineralization in primary murine fibroblasts and the C3H10T1/2 pluripotent fibroblastic cell-line both in vitro and in vivo [2, 3]. The objective of this study was to investigate Runx2-genetic engineering as a mineralization induction strategy in fibroblasts seeded on polymeric scaffolds in vitro and in vivo . Constructs were implanted in a heterotopic, subcutaneous site in order to assess the mineralization capacity of these cells in the absence of osteoinductive cues typically present in an orthotopic defect . MATERIALS AND METHODS: Primary fibroblasts were harvested from 16-week-old male Wistar rats by enzymatic digestion of the dermis. Passage four cells were seeded in monolayer on 1 mg/ml collagen coated dishes and transduced with Runx2 retrovirus or left as unmodified controls. Runx2-expressing or unmodified cells (1x10 6 ) were seeded 2 days post infection onto fibrous collagen scaffolds (8 mm x 1.5 mm, average pore size 61.7 μm, 93.7% pore volume) coated with 20 μg/ml fibronectin. In vivo constructs were implanted 1 day post -seeding into subcutaneous pockets made by blunt dissection in the backs of 7- week-old syngeneic rats in accordance to an IACUC-approved protocol. Each animal received two implants (one Runx2 and one unmodified cell-seeded scaffold) on opposite sides of a midline incision. In vitro constructs were cultured in DMEM with 10% FBS, 1% pen-strep, 3 mM sodium ß-glycerophosphate, 50 ug/ml ascorbic acid, and 10nM dexamethasone. Retroviral transduction efficiency was found to be 65% by eGFP expression levels using flow cytometry. Matrix mineralization was quantified by micro-CT image analysis at 28 and 42 days post- seeding for in vitro cultures and 28 days post-implantation for in vivo cultures. Data was analyzed by ANOVA and Tukey’s test for pairwise comparisons, where * represents differences from control cells (p< 0.05). RESULTS: Runx2-expressing and unmodified cells were evenly distributed throughout collagen scaffolds after 1 day in culture (Figure 1). These cells remained viable and exhibited a marked increase in proliferation throughout the 42 day in vitro culture period. DNA quantification verified equivalent cell seeding efficiency (75 ± 9%) between Runx2-modified and control constructs (data not shown). Figure 1: Scaffold colonization and cellular viability were assessed at 1 , 21, and 42 days post- seeding by confocal microscopy and Live/Dead fluorescence staining. Runx2-expressing fibroblasts deposited significantly higher amounts of mineralized matrix on collagen scaffolds cultured in vitro for 28 and 42 days compared to unmodified cells (Figure2). Surprisingly , unmodified cells showed a low level of radiopaque material, which may be due to accumulation of calcium phosphate precipitates. Future analysis of the mineral phase with FTIR spectroscopy will offer insights into the chemical composition and origin of these deposits. von Kossa and hematoxylin -eosin histological staining at 42 days post-seeding revealed that mineral deposits and a dense cell layer were preferentially localized to the construct periphery, likely due to mass transport limitations (Figure 3). Figure 2: (A) Representative micro-CT images of Runx2-expressing and unmodified fibroblasts seeded on collagen scaffolds and cultured in osteogenic media for 28 and 42 days (B) Quantification of bone volume by micro-CT image analysis of constructs after 28 and 42 days in vitro culture (Mean + SEM, n=3 ; ANOVA, p<0.05). Figure 3 : Histological sections were stained with hematoxylin-eosin or von Kossa-nuclear fast red to observe cellular distribution and mineralization on Runx2-fibroblast -seeded scaffolds. Importantly, scaffolds seeded with Runx2-expressing cells displayed a significant increase in mineral volume after 28 days in vivo, while controls (unmodified cells, empty vector cells, empty scaffold) showed minimal radiodense regions (Figure 4). Figure 4: (A) Quantification of bone volume by micro-CT image analysis of constructs after 28 days subcutaneous implantation (Mean + SEM, n=6, ANOVA, p<0.05). (B) Representative micro-CT images of explanted constructs after 28 days subcutaneous implantation DISCUSSION: We have demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days implantation in a subcutaneous, heterotopic site. This finding is in contrast to previous reports that transient adenoviral Runx2 expression is insufficient to produce significant levels of mineralization in vivo [2, 3] and suggests that sustained retroviral expression of the transcription factor is necessary for mineral formation. These results establish Runx2-genetic engineering as a strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizin g cell source for bone tissue engineering applications. ACKNOWLEDGMENTS: This research is supported by NIH R01-EB003364, the NSF GTEC Engineering Research Center (EEC-9731643), and an NSF graduate fellowship (JEP). Collagen scaffolds were generously donated by Kensey Nash Corporation. REFERENCES: [1] Phillips et al., Trans ORS, 30:0290, 2005. [2] Hirata, et al., Bone, 32:502-512, 2003. [3] Yang, et al., JBMR, 18:705- 715, 2003. Day 1 Day 21 df control Runx2 Day 42 Day 1 Day 21 df control Runx2 Day 42 0 5 10 15 20 Day28 in vitro Day42 in vitro Bone Volume (mm 3 ) df control Runx2 Day 28 in vitro Day 42 in vitro Control Runx2 A B * 0 5 10 15 20 0 5 10 15 20 Day28 in vitro Day42 in vitro Bone Volume (mm 3 ) df control Runx2 df control Runx2 Day 28 in vitro Day 42 in vitro Control Runx2 Day 28 in vitro Day 42 in vitro Control Runx2 A B * 4x 10x 4x 10x 0 0.2 0.4 0.6 0.8 1.0 1.2 empty scaffold empty vector df control Runx2 Bone Volume (mm 3 ) empty scaffold empty vector df control Runx2 * A B MicroCT Day 28 in vivo 0 0.2 0.4 0.6 0.8 1.0 1.2 empty scaffold empty vector df control Runx2 Bone Volume (mm 3 ) empty scaffold empty vector df control Runx2 * A B 0 0.2 0.4 0.6 0.8 1.0 1.2 empty scaffold empty vector df control Runx2 Bone Volume (mm 3 ) empty scaffold empty vector df control Runx2 0 0.2 0.4 0.6 0.8 1.0 1.2 0 0.2 0.4 0.6 0.8 1.0 1.2 empty scaffold empty vector df control Runx2 Bone Volume (mm 3 ) empty scaffold empty vector df control Runx2 * A B MicroCT Day 28 in vivo Paper No: 0104 52nd Annual Meeting of the Orthopaedic Research Society
Transcript of RUNX2-EXPRESSING PRIMARY DERMAL FIBROBLASTS … · 2017. 1. 30. · into subcutaneous pockets made...
RUNX2-EXPRESSING PRIMARY DERMAL FIBROBLASTS MINERALIZE COLLAGEN SCAFFOLDS IN VIVO *Phillips, J E; **Guldberg, R E; **García, A J
*Georgia Tech/Emory Department of Biomedical Engineering, **Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30322
INTRODUCTION: Bone tissue engineering strategies are limited by inadequate availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell alternative because they are easy to harvest from autologous donors and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer [1]. However, sustained expression of Runx2 was not sufficient to promote functional osteogenesis and co-treatment with the steroid hormone dexamethasone was required for biologically -equivalent matrix mineralization in this model system. Furthermore, transient adenoviral Runx2 expression was also reported insufficient to produce significant levels of mineralization in primary murine fibroblasts and the C3H10T1/2 pluripotent fibroblastic cell-line both in vitro and in vivo [2, 3]. The objective of this study was to investigate Runx2-genetic engineering as a mineralization induction strategy in fibroblasts seeded on polymeric scaffolds in vitro and in vivo . Constructs were implanted in a heterotopic, subcutaneous site in order to assess the mineralization capacity of these cells in the absence of osteoinductive cues typically present in an orthotopic defect . MATERIALS AND METHODS: Primary fibroblasts were harvested from 16-week-old male Wistar rats by enzymatic digestion of the dermis. Passage four cells were seeded in monolayer on 1 mg/ml collagen coated dishes and transduced with Runx2 retrovirus or left as unmodified controls. Runx2-expressing or unmodified cells (1x106) were seeded 2 days post infection onto fibrous collagen scaffolds (8 mm x 1.5 mm, average pore size 61.7 µm, 93.7% pore volume) coated with 20 µg/ml fibronectin. In vivo constructs were implanted 1 day post -seeding into subcutaneous pockets made by blunt dissection in the backs of 7-week-old syngeneic rats in accordance to an IACUC-approved protocol. Each animal received two implants (one Runx2 and one unmodified cell-seeded scaffold) on opposite sides of a midline incision. In vitro constructs were cultured in DMEM with 10% FBS, 1% pen-strep, 3 mM sodium ß-glycerophosphate, 50 ug/ml ascorbic acid, and 10nM dexamethasone. Retroviral transduction efficiency was found to be 65% by eGFP expression levels using flow cytometry. Matrix mineralization was quantified by micro-CT image analysis at 28 and 42 days post-seeding for in vitro cultures and 28 days post-implantation for in vivo cultures. Data was analyzed by ANOVA and Tukey’s test for pairwise comparisons, where * represents differences from control cells (p< 0.05). RESULTS: Runx2-expressing and unmodified cells were evenly distributed throughout collagen scaffolds after 1 day in culture (Figure 1). These cells remained viable and exhibited a marked increase in proliferation throughout the 42 day in vitro culture period. DNA quantification verified equivalent cell seeding efficiency (75 ± 9%) between Runx2-modified and control constructs (data not shown).
Figure 1: Scaffold colonization and cellular viability were assessed at 1, 21, and 42 days post-seeding by confocal microscopy and Live/Dead fluorescence staining.
Runx2-expressing fibroblasts deposited significantly higher amounts of mineralized matrix on collagen scaffolds cultured in vitro for 28 and 42 days compared to unmodified cells (Figure2). Surprisingly , unmodified cells showed a low level of radiopaque material, which may be due to accumulation of calcium phosphate precipitates. Future analysis of the mineral phase with FTIR spectroscopy will offer insights into the chemical composition and origin of these deposits. von Kossa and hematoxylin -eosin histological staining at 42 days post-seeding revealed that mineral deposits and a dense cell layer were preferentially localized
to the construct periphery, likely due to mass transport limitations (Figure 3). Figure 2: (A) Representative micro-CT images of Runx2-expressing and unmodified fibroblasts seeded on collagen scaffolds and cultured in osteogenic media for 28 and 42 days (B) Quantification of bone volume by micro-CT image analysis of constructs after 28 and 42 days in vitro culture (Mean + SEM, n=3 ; ANOVA, p<0.05).
Figure 3: Histological sections were stained with hematoxylin-eosin or von Kossa-nuclear fast red to observe cellular distribution and mineralization on Runx2-fibroblast -seeded scaffolds.
Importantly, scaffolds seeded with Runx2-expressing cells displayed a significant increase in mineral volume after 28 days in vivo, while controls (unmodified cells, empty vector cells, empty scaffold) showed minimal radiodense regions (Figure 4). Figure 4: (A) Quantification of bone volume by micro-CT image analysis of constructs after 28 days subcutaneous implantation (Mean + SEM, n=6, ANOVA, p<0.05). (B) Representative micro-CT images of explanted constructs after 28 days subcutaneous implantation DISCUSSION: We have demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days implantation in a subcutaneous, heterotopic site. This finding is in contrast to previous reports that transient adenoviral Runx2 expression is insufficient to produce significant levels of mineralization in vivo [2, 3] and suggests that sustained retroviral expression of the transcription factor is necessary for mineral formation. These results establish Runx2-genetic engineering as a strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizin g cell source for bone tissue engineering applications. ACKNOWLEDGMENTS: This research is supported by NIH R01-EB003364, the NSF GTEC Engineering Research Center (EEC-9731643), and an NSF graduate fellowship (JEP). Collagen scaffolds were generously d onated by Kensey Nash Corporation. REFERENCES: [1] Phillips et al., Trans ORS, 30:0290, 2005. [2] Hirata, et al., Bone, 32:502-512, 2003. [3] Yang, et al., JBMR, 18:705-715, 2003.
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Paper No: 010452nd Annual Meeting of the Orthopaedic Research Society
