Biomaterials xxx (2014) 1e12
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration Falguni Pati a, Tae-Ha Song a, Girdhari Rijal a, Jinah Jang b, Sung Won Kim c, d, Dong-Woo Cho a, * a
Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, Korea Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, Korea c Department of Otolaryngology-Head and Neck Surgery, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea d Department of Biomedical Science, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2014 Accepted 2 October 2014 Available online xxx
3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and b-tricalcium phosphate (b-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-theshelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals. © 2014 Elsevier Ltd. All rights reserved.
Keywords: 3D printed scaffolds Cell-laid extracellular matrix Osteoinduction Osteoconduction Bone regeneration
1. Introduction The extracellular matrix (ECM) strongly influences the biological responses of cells and is crucial in regulating cellular adhesion, proliferation, migration, and differentiation [1e3]. Hence, creation of a microenvironment that mimics natural ECM may greatly improve the biological responses of tissue-engineering scaffolds [4]. This recognition has inspired the design of biomimetic substrates for bone regeneration aiming to provide both structural
* Corresponding author. E-mail address: [email protected] (D.-W. Cho).
support and bioactive signals that mimic key attributes of the native tissue microenvironment. Decellularized ECM extracted from animal tissues has been used directly as a bioscaffold for tissue engineering [5e7]. However, ECM scaffolds suffer from several drawbacks like mismatch in dimensions and form with those of original tissue, lack of micro-tailored geometry, and potential risk of introducing pathogens. ECM has also been extracted from cultured cells and used as biological scaffolds for tissue engineering, but the generated scaffolds lack form and structural support [8]. Several studies have attempted to mimic the ECM microenvironment by integrating one or more ECM proteins on supportive synthetic biomaterials such as natural or synthetic polymers,
http://dx.doi.org/10.1016/j.biomaterials.2014.10.012 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pati F, et al., Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.012
2
F. Pati et al. / Biomaterials xxx (2014) 1e12
hydrogels, and ceramics [9e11]. However, use of naturallyoccurring molecules has the drawback that they are not easily processed within compliant biomaterials. Most of these investigations concentrated on coating the scaffolds with one or several ECM protein solutions or on grafting proteins or short peptide sequences by silanization or etching on the surface of a scaffold [12,13]. These methods provide only fragmental components that to present specific functional receptors for cell attachment or proliferation [14,15] and cannot replicate the complexity of natural ECM [3]. Moreover, these approaches consider only the organic components of the bony microenvironment, whereas bone ECM is composed of an inorganic mineralized phase along with collagenous and non-collagenous proteins [16,17]. Use of cell-laid ECM has been evaluated as a physiologically source of naturally-occurring bioactive signals. Rat marrow stromal cells form bone matrix in vitro when cultured on titanium fiber mesh in the presence of osteogenic supplements [2,18,19]. Furthermore, synthetic scaffolds that contain cell-laid bone-like ECM significantly improve the deposition of mineralized matrix by marrow stromal cells compared to those on bare titanium or polymeric scaffolds [18e20]. Scaffolds constructed of b-tricalcium phosphate (b-TCP) and cell-derived ECM enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) in vitro, possibly due to activation of MAPK/ERK signaling pathway [21,22]. However, whether a similar result could be obtained in vivo remains uncertain. Moreover, control over the architecture of the scaffolds was limited because they were produced by template-casting [21]. Polymeric scaffolds with controlled architecture can be prepared by 3D printing technology using a multi-head deposition system (MHDS) [23,24]. This process has the advantages of high throughput and high resolution, but MHDS can use only thermostable materials. Although the 3D printed scaffolds have demonstrated a wide acceptability for bone tissue regeneration [25], the performance of synthetic polymer is generally limited by a suboptimal biological interaction with cells. Efforts have been made to improve polymers' bioactivity by combining them with specific bone ECM components, such as calcium phosphate-based particles [23]. Nonetheless, use of this strategy is complicated by the difficulty of identifying optimal amounts and combinations of defined factors. We thus hypothesized that ornamenting the 3D-printed tailored synthetic substrates with cell-laid ECM may replicate the natural bony ECM microenvironment including the inorganic phase, in addition to providing structural support. In addition, this process could generate off-the-self bone substitute materials with enhanced biological functionality and possibly with osteoinductivity. We used human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs) to ornament 3D printed scaffolds with cell-laid ECM, because these cells have a high intrinsic capacity to proliferate, thereby providing large cell numbers and high osteogenic differentiation potential [26,27]. These cells were isolated from the inferior turbinate tissue that was discarded after surgery [27]. Use of hTMSCs is feasible because passage number and donor age do not affect differentiation characteristics of hTMSCs significantly [28]. The goal of the present work was to increase the biological functionality of the 3D-printed synthetic scaffolds by ornamenting them with cell-laid bone like ECM. We evaluated whether the ECM laid by hTMSCs could induce osteogenic differentiation of newlyseeded stem cells in vitro and drive osteogenesis in vivo. The ECM-ornamented scaffolds were decellularized and their capacity to enhance in vitro differentiation of newly-seeded stem cells was evaluated. In vivo bone regeneration capability of the decellularized cell-laid ECM-ornamented scaffolds was assessed after both ectopic
and orthotopic implantation in subcutaneous and calvarial defects, respectively, in rats. 2. Materials and methods 2.1. Printing and characterization of 3D synthetic scaffolds Synthetic scaffolds were prepared using an in-house Multi-Head Deposition System (MHDS) [29], using (1) a blend of polycaprolactone (PCL; 19561-500G, MW 43,000e50,000; Polysciences Inc., Warrington, PA, USA) and poly(lactic-co-glycolic acid) (PLGA; 430471-5G, MW 50,000e75,000; SigmaeAldrich, St. Louis, MO, USA) blend (PCL/PLGA); and (2) a PCL/PLGA blend with b-tricalcium phosphate (b-TCP; Ca3O8P2, 310.08 g/mol, 49963, SigmaeAldrich, MO, USA) (PCL/PLGA/TCP). The phase composition (by XRD analysis) of the TCP was b-TCP: >98%; hydroxyapatite:
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration Falguni Pati a, Tae-Ha Song a, Girdhari Rijal a, Jinah Jang b, Sung Won Kim c, d, Dong-Woo Cho a, * a
Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, Korea Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheongam ro, Nam-gu, Pohang, Kyungbuk 790-784, Korea c Department of Otolaryngology-Head and Neck Surgery, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea d Department of Biomedical Science, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2014 Accepted 2 October 2014 Available online xxx
3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and b-tricalcium phosphate (b-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-theshelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals. © 2014 Elsevier Ltd. All rights reserved.
Keywords: 3D printed scaffolds Cell-laid extracellular matrix Osteoinduction Osteoconduction Bone regeneration
1. Introduction The extracellular matrix (ECM) strongly influences the biological responses of cells and is crucial in regulating cellular adhesion, proliferation, migration, and differentiation [1e3]. Hence, creation of a microenvironment that mimics natural ECM may greatly improve the biological responses of tissue-engineering scaffolds [4]. This recognition has inspired the design of biomimetic substrates for bone regeneration aiming to provide both structural
* Corresponding author. E-mail address: [email protected] (D.-W. Cho).
support and bioactive signals that mimic key attributes of the native tissue microenvironment. Decellularized ECM extracted from animal tissues has been used directly as a bioscaffold for tissue engineering [5e7]. However, ECM scaffolds suffer from several drawbacks like mismatch in dimensions and form with those of original tissue, lack of micro-tailored geometry, and potential risk of introducing pathogens. ECM has also been extracted from cultured cells and used as biological scaffolds for tissue engineering, but the generated scaffolds lack form and structural support [8]. Several studies have attempted to mimic the ECM microenvironment by integrating one or more ECM proteins on supportive synthetic biomaterials such as natural or synthetic polymers,
http://dx.doi.org/10.1016/j.biomaterials.2014.10.012 0142-9612/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pati F, et al., Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.10.012
2
F. Pati et al. / Biomaterials xxx (2014) 1e12
hydrogels, and ceramics [9e11]. However, use of naturallyoccurring molecules has the drawback that they are not easily processed within compliant biomaterials. Most of these investigations concentrated on coating the scaffolds with one or several ECM protein solutions or on grafting proteins or short peptide sequences by silanization or etching on the surface of a scaffold [12,13]. These methods provide only fragmental components that to present specific functional receptors for cell attachment or proliferation [14,15] and cannot replicate the complexity of natural ECM [3]. Moreover, these approaches consider only the organic components of the bony microenvironment, whereas bone ECM is composed of an inorganic mineralized phase along with collagenous and non-collagenous proteins [16,17]. Use of cell-laid ECM has been evaluated as a physiologically source of naturally-occurring bioactive signals. Rat marrow stromal cells form bone matrix in vitro when cultured on titanium fiber mesh in the presence of osteogenic supplements [2,18,19]. Furthermore, synthetic scaffolds that contain cell-laid bone-like ECM significantly improve the deposition of mineralized matrix by marrow stromal cells compared to those on bare titanium or polymeric scaffolds [18e20]. Scaffolds constructed of b-tricalcium phosphate (b-TCP) and cell-derived ECM enhance the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) in vitro, possibly due to activation of MAPK/ERK signaling pathway [21,22]. However, whether a similar result could be obtained in vivo remains uncertain. Moreover, control over the architecture of the scaffolds was limited because they were produced by template-casting [21]. Polymeric scaffolds with controlled architecture can be prepared by 3D printing technology using a multi-head deposition system (MHDS) [23,24]. This process has the advantages of high throughput and high resolution, but MHDS can use only thermostable materials. Although the 3D printed scaffolds have demonstrated a wide acceptability for bone tissue regeneration [25], the performance of synthetic polymer is generally limited by a suboptimal biological interaction with cells. Efforts have been made to improve polymers' bioactivity by combining them with specific bone ECM components, such as calcium phosphate-based particles [23]. Nonetheless, use of this strategy is complicated by the difficulty of identifying optimal amounts and combinations of defined factors. We thus hypothesized that ornamenting the 3D-printed tailored synthetic substrates with cell-laid ECM may replicate the natural bony ECM microenvironment including the inorganic phase, in addition to providing structural support. In addition, this process could generate off-the-self bone substitute materials with enhanced biological functionality and possibly with osteoinductivity. We used human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs) to ornament 3D printed scaffolds with cell-laid ECM, because these cells have a high intrinsic capacity to proliferate, thereby providing large cell numbers and high osteogenic differentiation potential [26,27]. These cells were isolated from the inferior turbinate tissue that was discarded after surgery [27]. Use of hTMSCs is feasible because passage number and donor age do not affect differentiation characteristics of hTMSCs significantly [28]. The goal of the present work was to increase the biological functionality of the 3D-printed synthetic scaffolds by ornamenting them with cell-laid bone like ECM. We evaluated whether the ECM laid by hTMSCs could induce osteogenic differentiation of newlyseeded stem cells in vitro and drive osteogenesis in vivo. The ECM-ornamented scaffolds were decellularized and their capacity to enhance in vitro differentiation of newly-seeded stem cells was evaluated. In vivo bone regeneration capability of the decellularized cell-laid ECM-ornamented scaffolds was assessed after both ectopic
and orthotopic implantation in subcutaneous and calvarial defects, respectively, in rats. 2. Materials and methods 2.1. Printing and characterization of 3D synthetic scaffolds Synthetic scaffolds were prepared using an in-house Multi-Head Deposition System (MHDS) [29], using (1) a blend of polycaprolactone (PCL; 19561-500G, MW 43,000e50,000; Polysciences Inc., Warrington, PA, USA) and poly(lactic-co-glycolic acid) (PLGA; 430471-5G, MW 50,000e75,000; SigmaeAldrich, St. Louis, MO, USA) blend (PCL/PLGA); and (2) a PCL/PLGA blend with b-tricalcium phosphate (b-TCP; Ca3O8P2, 310.08 g/mol, 49963, SigmaeAldrich, MO, USA) (PCL/PLGA/TCP). The phase composition (by XRD analysis) of the TCP was b-TCP: >98%; hydroxyapatite:
