Acta Biomaterialia 31 (2016) 301–311

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Acta Biomaterialia

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Full length article Recapitulating cranial osteogenesis with neural crest cells in 3-D microenvironments ⇑ Bumjin Namkoong a,b,1, Sinan Güven c,d,1, Shwathy Ramesan e, Volha Liaudanskaya c, Arhat Abzhanov a,f,g, ,2, ⇑ Utkan Demirci c,e, ,2 a Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA b Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA c Demirci BAMM Labs, Canary Center at Stanford for Early Cancer Detection, Department of Radiology, Department of Electrical Engineering (By courtesy), Stanford School of Medicine, Palo Alto, CA 94304, USA d Izmir Biomedicine and Genome Center, Dokuz Eylul University Health Campus, Balcova, 35350 Izmir, Turkey e Demirci BAMM Labs, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA f Current address: Department of Life Sciences, Imperial College London, Silwood Park Campus Buckhurst Road, Ascot, Berkshire SL5 7PY, United Kingdom g Current address: Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom article info abstract

Article history: The experimental systems that recapitulate the complexity of native tissues and enable precise control Received 9 June 2015 over the microenvironment are becoming essential for the pre-clinical tests of therapeutics and tissue Received in revised form 11 November 2015 engineering. Here, we described a strategy to develop an in vitro platform to study the developmental Accepted 2 December 2015 biology of craniofacial osteogenesis. In this study, we directly osteo-differentiated cranial neural crest Available online 7 December 2015 cells (CNCCs) in a 3-D in vitro bioengineered microenvironment. Cells were encapsulated in the gelatin-based photo-crosslinkable hydrogel and cultured up to three weeks. We demonstrated that this Keywords: platform allows efficient differentiation of p75 positive CNCCs to cells expressing osteogenic markers cor- 3-D culture responding to the sequential developmental phases of intramembranous ossification. During the course Controlling 3-D tissue microenvironment Ectodermal to mesodermal differentiation of culture, we observed a decrease in the expression of early osteogenic marker Runx2, while the other Bone mature osteoblast and osteocyte markers such as Osterix, Osteocalcin, Osteopontin and Bone sialoprotein increased. We analyzed the ossification of the secreted matrix with alkaline phosphatase and quantified the newly secreted hydroxyapatite. The Field Emission Scanning Electron Microscope (FESEM) images of the bioengineered hydrogel constructs revealed the native-like osteocytes, mature osteoblasts, and cra- nial bone tissue morphologies with canaliculus-like intercellular connections. This platform provides a broadly applicable model system to potentially study diseases involving primarily embryonic craniofacial bone disorders, where direct diagnosis and adequate animal disease models are limited. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction are regulated by the multitude of chemical and physical stimuli [1–4]. Tissue engineering and regenerative medicine hold a great Tissues and organs comprise cells and intercellular components promise to create models that mimic the natural biological tissue that are highly organized at the organismic level, which have a microenvironments and recapitulate tissue and organ formation dynamic, three-dimensional (3-D) nature especially highlighted and maturation. Cell-encapsulating 3-D tissue models are emerging during embryonic development. Tissue functionality arises from tools that bring inspiring in vitro venues to study the development the finely orchestrated interactions of all such elements, which of complex physiological systems. These models overcome the lim- itations of the two-dimensional (2-D) monolayer cultures in study- ⇑ Corresponding authors at: Department of Life Sciences, Imperial College ing the significantly complex biological mechanisms involved in London, Silwood Park Campus Buckhurst Road, Ascot, Berkshire SL5 7PY, United multicellular behavior [5,6]. Microscale tissue models can effi- Kingdom (A. Abzhanov). Demirci BAMM Labs, Canary Center at Stanford for Early ciently utilize rare cells that are challenging for monolayer expan- Cancer Detection, Department of Radiology, Department of Electrical Engineering sion [7]. Such microscale models are capable of recapitulating (By courtesy), Stanford School of Medicine, Palo Alto, CA 94304, USA (U. Demirci). regulatory dynamics of the germ layers and cellular condensations E-mail addresses: [email protected] (A. Abzhanov), utkan@stanford. edu (U. Demirci). forming at the early developmental stages. The design and fabrica- 1 These authors contributed equally to this work. tion of 3-D microenvironments with well-defined architecture and 2 Corresponding authors contributed equally. http://dx.doi.org/10.1016/j.actbio.2015.12.004 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 302 B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311

Fig. 1. Recapitulation of the osteogenic differentiation of CNCCs with direct differentiation in the 3-D hydrogels in vitro. Neural crest cells were isolated from E10.5 mouse embryo and initially expanded in monolayer with neural crest-specific media and later on encapsulated in hydrogels forming 3-D in vitro culture. Osteogenic differentiation phases with specific gene expressions in 3-D microenvironments were monitored for three weeks. material compositions specific for progenitor cell types are differentiation of neural crest cells, forming cranial skeletal tissues essential for mimicking intricate events taking place in vivo. and mimicking the embryonic phases of organogenesis. Cell-encapsulating hydrogel microenvironments are fabricated by gelation of extracellular matrix mimicry polymer solutions using 2. Materials and methods various techniques including thermal, enzymatic, chemical and UV photo-crosslinking methods. In particular, the flexibility to 2.1. Mouse cranial neural crest cell isolation and culture fine-tune the microscale tissue size and shape, together with the high throughput and reproducibility aspect of the UV photo- Mouse cranial neural crest cells were isolated according to crosslinking platform, underscores its broad utility to create Etchevers [24] under Harvard IACUC, Animal Experimentation Pro- successful tissue models [8,9]. tocol No 26-04. Briefly, frontonasal, maxillary and mandibular Often referred to as the ‘‘fourth embryonic germ layer” [10,11], prominences of E10.5 mouse embryos were dissected into small neural crest is a transient, multipotent embryonic population of pieces in a petri dish containing DMEM/F12 media (Life Technolo- cells, which migrate from their site of origin at the dorsal neural gies) with 1% penicilin–streptomycin (Life Technologies). These tis- tube and differentiate into a diverse array of specialized cell types sue blocks were digested for 5 min in a mixture of warm 0.1% during vertebrate development [12]. Cranial neural crest cells collagenase (Sigma) and 0.025% trypsin (Thermo Scientific), while (CNCCs) can differentiate into chondrogenic and osteogenic cells being pipetted gently up and down to generate single cell suspen- that, in turn, generate most of the cranial skeletal tissues (both sion of mouse CNCCs. These cells were washed with fresh medium bones and cartilages) in the vertebrate head [12,13]. Cranial der- to stop the digestion, counted and seeded in laminin-coated plates. mal bones are unique in the way they are formed – dermal bones Cells were maintained and expanded in a monolayer culture in form directly from the neural crest of the cranial dermis without DMEM/F12 media with N2 supplement (Life Technologies), forming a cartilage precursor, through a process known as 10 ng/ml EGF and 10 ng/ml bFGF recombinant proteins (R&D intramembranous ossification [12,14–20]. Despite these unique Systems). developmental features of the cranial bones, most of our current understanding of bone development, in general, is from studies on the limb and trunk bones of mesodermal origin. The skeletoge- 2.2. Synthesis of methacrylated gelatin hydrogel nesis using CNCCs, and the morphogenesis of cranial bone forma- ° tion, are not very well understood, particularly at the tissue level, Gelatin (Sigma) was dissolved in 50 C phosphate buffered sal- at least in part due to the lack of 3-D microscale tools to model ine (PBS) with 10% (w/v) of final concentration. Methacrylic anhy- and manipulate cranial skeletal tissues in vitro. dride (Sigma) was added (0.8 ml/g) drop wise to the dissolved Recent studies investigating cranial neural crest cell biology gelatin and stirred for two and a half hours. The unreacted utilize 2-D monolayer culture models, which do not replicate the methacrylic anhydride was dialyzed, and the final product was lyo- native, three-dimensionally defined microenvironments [21–23]. philized. Methacrylation was monitored and controlled with NMR In this study, we created a broadly applicable platform that allows analysis (Oxford Instruments, NMR AS600) in deuterium oxide the direct differentiation of multipotent progenitor cells, including (Supplementary Fig. 1). those of neural crest origin. We produced in vitro 3-D tissue model constructs with relatively high throughput efficiency to mimic the 2.3. Cell encapsulation osteogenic differentiation conditions of the mammalian post- migratory CNCCs (Fig. 1). The objective was to recapitulate the Cell-encapsulated 3-D hydrogels were generated using in vivo development of cranial dermal bone tissue from CNCCs in methacrylated gelatin (GelMA) as a prepolymer solution. CNCCs well-defined 3-D bio-mimicking hydrogel units. Such a platform between passage 3 and 5 were resuspended in 5% (w/v) provides in vitro study models for embryonic craniofacial disorders, GelMA prepolymer solution containing 0.3% (w/v) 2-hydroxy-1- where our understanding of etiology is limited, and few adequate (4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone photoinitiator animal disease models exist. To this purpose, we have developed (Irgacure 2959; CIBA Chemicals) with the cell densities of an array of microenvironments allowing successful 3-D direct 1 106,5 106,1 107 or 4 107 cells/ml. The mixture of the B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311 303 cell-prepolymer solution was placed between 150 lm-thick crest cells were used as control samples to calculate the relative spacers and covered with a glass coverslip (Fig. 1). The geometry gene expression levels in fold change (2DDCT value). of the hydrogel units was shaped by a photomask with circular motifs of 500 lm in diameter, placed on top of the glass coverslip. 2.6. Immunohistochemistry The prepolymer solution with cells was crosslinked with UV light (1 mW/cm2) for 20 s, and the excess uncrosslinked polymer For each time point, samples were fixed in 4% paraformalde- solution was washed out with PBS. The resulting cell- hyde solution for 20 min, permeabilized with 0.1% TritonX-100 encapsulated 3-D hydrogels were cultured in osteogenic media (Sigma) and blocked with 1% bovine serum albumin for 2 h at room (OM) composed of alpha-MEM (Life Technologies), 10% fetal temperature. Primary antibodies for Osterix (OSX) (Abcam), bovine serum (Life Technologies), 100 U/mL penicillin (Life RUNX2 (Novus-Biologicals), Osteopontin (OPN) (GenWay Biotech), Technologies), 100 mg/mL streptomycin (Life Technologies), Osteocalcin (OCN) (EMD Millipore), bone sialoprotein (BSP) (Bioss) 100 nM dexamethasone (Sigma), 10 mM b-glycerophosphate and Ki67 (Abcam) were used at the concentrations recommended (Sigma), 200 lM ascorbic acid (Sigma) and 50 ng/ml BMP2 (R&D by the manufacturers, and the hydrogel samples were incubated Systems) for direct differentiation toward osteogenic lineage. with the antibodies overnight at 4 °C. AlexaFluor 488 and Alexa- These cell-encapsulated 3-D hydrogels were harvested weekly for Fluor 568 (Life Technologies) were used as secondary antibodies. three weeks for further analysis. 3-D hydrogel samples cultured Alexa Fluor 647 Phalloidin (Life Technologies) was used to visual- in the neural crest growth media (control media, CM) were used ize the actin cytoskeleton. The images were taken with Zeiss LSM as controls. 780 confocal microscope. 2.4. Characterization of cranial neural crest cells 2.7. Mineralization assay Prior to 3-D hydrogel encapsulation, the isolated and monolayer-expanded post-migratory CNCCs were characterized The mineralization matrixes produced by the osteo- with ectoderm- and mesenchyme-specific cell surface markers. differentiated CNCCs were detected and quantified with alkaline Cells were stained with p75 antibody conjugated with fluorescein phosphatase staining kit (Sigma) and hydroxyapatite quantifying isothiocyanate (FITC) (Abcam) and CD73 antibody conjugated with OsteoimageTM (Lonza) assay, according to manufacturers’ instruc- phycoerythrin (PE) (BD Biosciences) in 1% BSA solution for 40 min tions. The mineralization levels in the cell-encapsulated hydrogels at 4 °C and analyzed using flow cytometry (BD FACS Calibur) and were assessed weekly for three weeks. The secreted hydroxyap- FlowJo software (Fig. 2A). Cells stained with IgG FITC and IgG PE atite was quantified by the relative fluorescence intensities of the were used as negative controls. The viability and proliferation of cell-encapsulated hydrogels using ImageJ software (NIH, Bethesda, the encapsulated CNCCs in 3-D in vitro culture were assessed with Maryland, USA). Live–Dead staining (Life Technologies).

2.5. Quantitative real-time PCR 2.8. Image quantification

Total RNA (3 replicates for each group) was extracted by TRIzolÒ The unprocessed raw confocal z-stack files were analyzed with (Life Technologies) and RNeasy MINI kit with QIAshedder columns Fiji software (ImageJ, NIH, Bethesda, Maryland, USA) with 3-D (Qiagen) according to the manufacturer’s instructions. After objects counter plugins. The total number of cells were counted extraction, total RNA was resuspended in the final volume of using DAPI staining. For OSX, cells with a positive signal in the 30 ll with RNAse-free water and quantified using spectropho- nucleus were counted while cells with the positive signal in the tometer (Nanodrop ND-1000, Delaware, USA). For the best results cytoplasm were counted for OCN and OPN. Signals above a in the PCR array, all RNA samples were selected for purity using an threshold set by the software were counted as positive and were A260/A280 UV spectrophotometry ratio greater the 1.99. 1 lgof confirmed manually. total RNA was used for the reverse transcription reaction using High-Capacity cDNA Reverse Transcription Kit (Applied Biosys- 2.9. Freeze-fracture cryo-scanning electron microscopy tems). Amplification and detection were carried out using SYBR Green (Kapa Biosystems) with Eppendorf Mastercycler using 2- Freeze-fracture cryo-SEM imaging was performed with a Zeiss step cycle for 40 cycles. Primer sequences: (i) GAPDH Forward Supra55VP FESEM. A fixed single hydrogel was placed on a sample (50-CTA CAC TGA GGA CC AGG TTG TCT-30) Reverse (50-TTG TCA stub and submerged into a slurry bath of liquid nitrogen. The TAC CAG GAA ATG AGC TT-30), (ii) p75 Forward (50-GCA TTG TGG frozen sample was transferred to a cryo-transfer chamber and TAG GCC AGA CC-30) Reverse (50-CCT GAA AGT CAC TCC ATC was fractured in a vacuum with a sharp blade to expose the cross CC-30) (iii) Sox9 Forward (50-AGC TCA CCA GAC CCT GAG AA-30) sections of the sample. The surfaces of the fractured samples were Reverse (50-TCC CAG CAA TCG TTA CCT TC-30), (iv) Osterix Forward sputter coated with platinum and imaged using the SE2 detector at (50-CTG CTT GAG GAA GAA GCT C-30) Reverse (50-TTC TTT GTG CCT a voltage of 4 kV while maintained at 160 °C. CCT TTC C-30), (v) Osteocalcin Forward (50-CTG ACA AAG CCT TCA TGT CCA A-30) Reverse (50-GCG CCG GAG TCT GTT CAC TA-30), (vi) Bone Sialoprotein Forward (50-CAG GGA GGC AGT GAC TCT 2.10. Statistical analysis TC-30) Reverse (50-AGT GTG GAA AGT GTG GCG TT-30), (vii) Alkaline phosphatase Forward (50-AGT TAC TGG CGA CAG CAA GC-30) Rev- Statistical analyses were performed with GraphPad Prism, ver- erse (50-GAG TGG TGT TGC ATC GCG-30), (viii) Aggrecan Forward sion 5 (GraphPad Software, Inc., San Diego). Results were analyzed (50-TGG CTT CTG GAG ACA GGA CT-30) Reverse (50-TTC TGC TGT using analysis of variance with Tukey’s post hoc test for multiple CTG GGT CTC CT-30), (ix) Collagen Type 2 Forward (50-CAA CAC comparisons and Student’s two-tailed t test for single comparisons, AAT CCA TTG CGA AC-30) Reverse (50-TCT GCC CAG TTC AGG TCT with statistical significance set at p < 0.05. Unless otherwise stated, CT-30). mRNA expressions were assayed in triplicate for each the mean values represent three experiments with two or three sample and normalized to GAPDH housekeeping gene expressions channels per experiment, and the error bars represent the standard to calculate DCT. The undifferentiated and unencapsulated neural error of mean. 304 B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311

Fig. 2. The characterizations of the CNCCs prior and after hydrogel encapsulation. (A) FACS analysis of the CNCCs prior to the encapsulation in 3-D hydrogels. 63.21% ± 20.28 of cells were p75 positive indicating the neural crest cells phenotype (left), and 66.22% ± 11.64 were CD73 positive showing mesenchymal lineage characteristic (right). Corresponding IgG staining was used as a negative control (blue contour). (B) The Live–Dead staining of neural crest cells encapsulated in the 3-D hydrogel. Live cells were stained for green and dead cell are shown in red. (C) The proliferation capacity of neural crest cells was evaluated with Ki67 staining after 1 week of encapsulation in the 3-D hydrogels. Proliferating cells express Ki67 (red) in the nuclei, co-localizing with DAPI (blue). The cell cytoskeletons were shown with phalloidin staining (green). (D–I) Effects of cell encapsulation density on osteogenic differentiation of primary CNCCs. (D–F) Bright field images of the cell-encapsulated hydrogels generated using different cell densities. (G–I) The immunocytochemistry results for OSX (green) and cytoskeleton (phalloidin, magenta) in 3-week osteo-differentiated hydrogels showed that high cell density is more favorable for osteogenic differentiation. Scale bars 100 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311 305

3. Results differentiation phase, and RUNX2 expression later gradually decreased as the bone tissue started to form (Fig. 4G and Supple- 3.1. Characterization of hydrogel and neural crest cells mentary Fig. 2). On the other hand, more than 30% of encapsulated cells cultured in the osteogenic media showed OSX expression in 3-D cellular microenvironments were designed from highly the nucleus after three weeks of incubation (Fig. 4D). The OPN biocompatible gelatin-based hydrogels, supporting cell viability and OCN protein depositions could be detected in the cytoplasm and growth [25]. Gelatin was methacrylated and lyophilized for and the extracellular matrix of osteogenic cells cultured for 3 weeks the generation of UV photo-crosslinkable hydrogels (Supplemen- in osteogenic media (Fig. 4E and F), as in the late development tary Fig. 1). Mouse CNCCs were isolated from the frontonasal, max- phase of the in vivo bone tissue, confirming the successful osteo- illary and mandibular prominences of E10.5 mouse embryos and genic differentiation of the CNCCs using the 3-D hydrogel platform. expanded in the monolayer culture prior to 3-D differentiation We detected BSP depositions in the peripheral parts of the 3-D (Fig. 1). The CNCCs before encapsulation showed 63.21% ± 20.28 hydrogels starting from week 1, which gradually expanded its positivity for ectodermal marker p75 and 66.22% ± 11.64 positivity expression toward the core parts of the hydrogel during the 3- for mesodermal marker CD73 confirming the mesenchymal-neural week osteogenic differentiation period (Supplementary Fig. 2). By phenotype as previously shown (Fig. 2A) [26,27]. The effect of the week 3, BSP accumulation could be observed around the lacunae encapsulation process on cell viability were assessed with of the osteogenic cells. Live–Dead staining (Fig. 2B). More than 90% of encapsulated cells were alive after the formation of 3-D constructs. The encapsulation 3.3. Osteogenic mineralization and the morphological analysis of the process or the hydrogel itself do not restrict or alter the prolifera- neo-tissues tion capacity of neural crest cells as shown with Ki67 immunos- taining (Fig. 2C). The encapsulated cells were then differentiated ALP expression starts from the early osteogenic differentiation toward the osteogenic lineage in the 3-D microenvironment to steps and continues through the maturation of the newly formed evaluate their potential for ectomesenchymal transformation and bone tissues. The engineered 3-D cell-encapsulated hydrogels were the natural state osteogenesis. We investigated the effect of the cell analyzed for mineralization by examining the ALP activity in the encapsulation density on cranial osteogenesis under four different cells differentiated over the 3-week culture period (Fig. 5A). The 6 7 cell concentrations (1 10 to 4 10 cells/ml). Cell densities cells cultured in the osteogenic condition inside the hydrogels 7 above 10 cells/ml showed significant expressions of the osteo- exhibited high level of ALP activity after two weeks, which genic markers (Fig. 2D–I), whereas the lower cell concentrations increased 2.56 folds by week 3. The undifferentiated cells in the resulted with poor osteogenic morphology. All following control hydrogels did not show any ALP activity (Fig. 5A). We eval- 7 experiments were performed at a cell density of 4 10 cell/ml. uated the functionality of osteo-differentiated CNCCs also with mineralization of hydrogels and the secretion of hydroxyapatite. The newly secreted hydroxyapatite in the 3-D cell-encapsulated 3.2. Monitoring the osteogenic differentiation of cranial neural crest Ò hydrogels was quantified with Osteoimage Mineralization assay cells in 3-D platform (Fig. 5B). The secretion of hydroxyapatite crystals was detected in the osteogenic hydrogels starting from week 1, which gradually 3-D differentiated CNCCs were analyzed for chondrogenic and increased with further osteogenic differentiation and maturation osteogenic gene expressions with qRT-PRC (Fig. 3). The neural crest by week 3. The undifferentiated control hydrogels did not show marker p75 only maintained a high level of expression in the cells any fluorescent signals indicative of hydroxyapatite secretion. cultured in the neural crest cell-specific media and its expression Importantly, osteo-differentiated CNCC encapsulated hydrogels decreased by more than 70% after the first week of culture in the showed formation of in vivo-grade osteo lacuna structures, charac- osteogenic media (Fig. 3A). Since CNCCs have the potential to dif- teristic of the living bone tissue with embedded mature osteocytes ferentiate into chondrocytes, we also checked the expression level (Fig. 6, Supplementary Fig. 3). Within the 3-D constructs osteo- of Sox9, collagen type II, and aggrecan. Sox9 is required for chondro- differentiated cells also build intercellular connections providing genesis, as it induces the expressions of cartilage-specific extracel- cell–cell interactions (Fig. 6C, Supplementary Video 1 and Video lular matrix molecules, such as collagen type II, IX and XI and 2). Morphological analysis of the osteo-differentiated hydrogels aggrecan [28–31]. The Sox9 expression was slightly increased up with FESEM revealed round, osteocyte-shaped cells located in the to the second week but eventually decreased below the original lacunae, and also mature osteoblasts throughout the hydrogel level, confirming the cells were not in the course of chondrogenic (Fig. 7A–C). Moreover, we observed differentiated bone cells with differentiation (Fig. 3B) [32,33]. The expression of collagen type II a large number of long projections called filopodia extending from and aggrecan did not show differences in expression between the the cell body, which is another key morphological characteristic of control and the osteogenic samples (Fig. 3C–D). To evaluate the the mature osteocytes (Fig. 7A, red colored) [34]. osteogenic differentiation, expression levels of four major bone specific genes, Osterix (Osx, also known as Sp7), Alkaline phos- phatase (Alp), Osteocalcin (Ocn) and Bone sialoprotein (Bsp) were 4. Discussion analyzed. All four genes showed dramatic increase in their expres- sion when cultured in the osteogenic differentiation media, espe- Along with many other important roles in the embryonic devel- cially by the third week of incubation, where markers indicated opment, neural crest cells are essential for the proper development the presence of mixed population of osteoblasts and osteocytes of the vertebrate head. The human skull is composed of more than (Fig. 3E–H). twenty intricately shaped and interconnected bones, which the The expressions of bone-specific markers of 3-D encapsulated majority is originated from the neural crest cells. Thus, deeper CNCC were also evaluated at the protein level for three weeks with knowledge of the cranial neural crest cell behavior, and eventually immunocytochemistry (Fig. 4). The differentiation of CNCCs to bone how they properly differentiate to bone, is essential for under- cells was demonstrated by the accumulation of OSX (Fig. 4A and D), standing both the normal head development and the myriad of OPN (Fig. 4B and E) and OCN proteins (Fig. 4C and F). The expression craniofacial abnormal conditions present in up to one-third of all of the early osteogenic marker RUNX2 was higher in cells cultured congenital birth defects in humans [35]. The vertebrate cranium in the osteogenic media compared to the control cells at the initial is also an important and unique structure from the evolutionary 306 B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311

Fig. 3. qRT-PCR analysis of the neural crest-specific gene p75 and bone and cartilage-specific genes in cells cultured in control or osteogenic media for three weeks (n = 3 for each sample. Error bar indicates standard error). (A) p75 was highly expressed in encapsulated cells cultured in control media. (B) The expression of cartilage-specific marker Sox9 did not change in both control and osteogenic media. (C) Col II expression slightly decreased after week 2. (D) Expression of chondrogenic marker aggrecan did not alter significantly during the differentiation. (E–H) Bone specific marker Osx, Alp, Ocn, and Bsp were highly expressed in the cells cultured in osteogenic media especially at the third week of incubation. (One asterisks denote significance at P < 0.05, two asterisks denote significance at P < 0.01, Student t-test). perspective as it represents one of the innovations that defines the in vitro differentiation platform that recapitulates the in vivo vertebrate lineage, and vertebrate heads display a variety of adap- osteogenic differentiation of CNCCs, we monitored and analyzed tive and species-specific characteristics [36]. 3-D differentiation the developmental phases of cranial bone in our system. In the culture systems support efficient tissue formation and allow the course of embryogenesis, the neural crest cells have a unique manipulation of the cells in their native-like microenvironments status as these cells of neuroectodermal origin can give rise to [23]. Notably, our work demonstrates the generation of 3-D cellu- the specialized cell types that are typically derived from the meso- lar niches for the efficient differentiation of CNCCs while maintain- dermal germ layer, such as connective tissue, cartilage and bones ing their viability and proliferation capacity. To achieve a 3-D [26]. In this study, after monolayer amplification we encapsulated B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311 307

Fig. 4. The cell-encapsulated hydrogels cultured either in control media (CM) (A–C) or osteogenic media (OM) (D–F) were stained for OSX, OPN and OCN after three weeks. (A, D) The nuclear localization of osteoblastic marker OSX (green) could be seen in the differentiated cells cultured in the osteogenic media. (B, C, E, F) Late osteogenic markers OPN (red) and OCN (green) were expressed in the cytoplasm and the extracellular matrix of osteo-differentiated cells (E and F). (G) The expression of early osteogenic marker RUNX2 decreased during the course of osteogenic differentiation of CNCCs, whereas the expression of OPN and OSX increased (n = 3 for each sample. Error bar indicates standard error). All scale bars 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) post-migratory CNCCs expressing both neural and mesenchymal hydrogels cultured in osteogenic media lost the expression of p75 markers, p75 and CD73 (Fig. 2A). Clonal study performed on neural and even in early phase (week 1) are committed to osteogenic lin- crest cells demonstrated that those cells are mostly formed as eage. On the other hand, the ectodermal phenotype of CNCCs in heterogeneous populations consisting of multiple progenitor cell control condition resumed up to week 2 with elevaded expression types (e.g., melanocytes, glia, neuron, and myofibroblast) [37].To of p75. Intramembranous ossification differs from the endochon- reach the high CNCC differentiation yield, we investigated the dral ossification, as it lacks the formation of hypertrophic cartilage effects of hydrogel properties and differential cell density in template prior to generating mineralized calcified bone [39]. Sox9 micro-tissue formation. Substrate stiffness and focal polarization is a well-known marker for chondrogenic cells in the process of of progenitor cells in conventional 2-D culture conditions dramat- endochondral ossification, but it also plays an important role in ically affect the fate and physiology of cells [22,38]. The 3-D plat- the differentiation of CNCCs to early bone progenitor cells during form that we generated allows tuning of both hydrogel stiffness intramembranous ossification [19]. Accordingly, we detected the and cell encapsulation density. According to our findings, high elevated levels of Sox9 expression during the early phase of the cell-to-cell contact dramatically assists the osteogenic differentia- osteogenic differentiation, which then decreased below the initial tion in the microscale hydrogels. The highest level of cell-to-cell level at the later stages of the culture process (Fig. 3B). We also contacts and the native-like osteogenic differentiation in both mor- evaluated the chondrogenesis for potential endrochondral ossifica- phological and molecular aspects were achieved in our 3-D culture tion with regulation of Col II and aggrecan expression. After week 2, system with the cell density of 4 107 cell/ml. Col II expression slightly decreased in osteogenic conditions and We systematically investigated the intramembranous aggrecan expression did not alter significantly for both study osteogenic differentiation progress of CNCCs in our 3-D platform groups. These findings indicate there is no chondrogenic tissue for- by analyzing the expression patterns of multiple neural crest and mation and the osteogenic differentiation is through the skeletogenic markers, including p75, Sox9, Col II, aggrecan, Runx2, intramembranous course. Early phases of the osteogenesis process Osx, Alp, Ocn, Opn and Bsp (Fig. 3 and 4). Cells encapsulated in are directly regulated by Runx2, an early marker and regulator of 308 B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311

Fig. 5. The osteogenic mineralization in the control and osteo-differentiated cell-encapsulated 3-D hydrogels were determined and quantified at weeks 1, 2 and 3 with alkaline phosphatase (A), and hydroxyapatite (B) staining. The osteogenic differentiation of CNCCs showed hydroxyapatite secretion and mineralized the hydrogels gradually after week 1 (C). Scale bars 100 lm. many other osteoblast markers, such as Bsp, Ocn and Alp [19].In process was successfully recapitulated and led to the formation our 3-D differentiation system, RUNX2 was expressed during the of mature osteoblasts and osteocytes. During the second and first week of osteogenesis and its expression decreased signifi- third week of 3-D osteo-differentiatiation CNCCs demonstrate the cantly at the later stages of bone tissue culture (Fig. 4G). These expression of (i) OSX, a bone-specific transcription factor that is later stage osteogenically committed cells no longer require specifically expressed in osteoblasts of all skeletal elements [41], RUNX2 for further ossification [40]. All of the later stage osteogenic (ii) OCN, an osteoblast-derived hormone expressed only in fully dif- markers (e.g., Osx, Ocn, Opn, BSP) suggest that skeletal development ferentiated osteoblasts and osteocytes, (iii) OPN, a late osteogenic B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311 309

Fig. 6. The hydrogel-encapsulated CNCCs change their morphology in the 3-D osteogenic differentiation platform. (A) Hydrogel-encapsulated CNCCs incubated in control media exhibited elongated morphology typical for mesenchymal cells. (B) Spherical morphology of osteo-differentiated cells in lacunae-like niches. Cytoskeletons were stained with phalloidin (magenta) and cell nuclei with DAPI (blue). (C) The confocal image of the hydrogel with encapsulated cells differentiated for two weeks in osteogenic media. The immunostaining results showed early osteogenic cells positive for RUNX2 (red) and osteocytes positive for OSX (green). The cytoskeletons stained with phalloidin (white) revealed filopodia projections and cell-to-cell connections (red arrowheads). Scale bars: 100 lm in A and B, 10 lm in C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) protein that regulates the biomineralization and boneremodelling osteoblasts and osteocytes is to analyze their morphologies com- [42,43] and (iv) BSP, a non-collagenous protein that is a key pared to the native mature bone tissues with osteoblasts and component of the bone extracellular matrix [44,45] (Fig. 3E, F, osteocytes embedded. As early as one week after the initiation of Fig. 4B, C, E, F, G and Supplementary Fig. 2). The formation of the 3-D osteogenic differentiation process, the cell morphology of mature bone tissue is further characterized by the mineralization the neural crest cells began to change to reflect their differentiation in terms of hydroxyapatite deposition in the newly formed extra- into osteoblasts (Figs. 6, 7, Supplementary Video 1 and Video 2). cellular matrix [46]. We demonstrate the ability of newly differen- The appearance of the lacunae cavities containing mature tiated osteogenic cells to mineralize the hydrogels with osteoblasts and osteocytes within the hydrogel is strikingly similar hydroxyapatite from second week. Similarly, high expression of to those seen in the natural bone tissue [48]. Such cell morphology alkaline phosphatase (ALP), which is highly involved in the miner- changes during the course of osteogenesis can be observed through alization of neo-tissues [47] supports the osteogenesis and in vitro the freeze-fractured FESEM imaging. While the newly formed, lar- tissue maturation (Fig. 5). Another decisive method to evaluate the ger osteoblast cells were tightly embedded in the hydrogel matrix formation of bone tissue and the emergence of the differentiated (Fig. 7B), their morphology changed to the small and round 310 B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311

Fig. 7. The representative freeze-fractured hydrogel FESEM images of the bioengineered neo-tissues, after three weeks of osteogenic differentiation. Cell-encapsulated hydrogels revealed cells in different phases of osteogenic differentiations, recapitulating native-like microenvironment. (A) An osteocyte (red) in a lacuna with extended filopodia that could also be observed in the bone canaliculus-like structure (white arrowheads), connecting to the neighboring cells. (B) A focused image of a single osteoblast starting to remodel the surrounding hydrogel into a lacuna-like structure. (C) Representative image of the osteogenic niche in 3-D hydrogel microenvironment. Scale bars; 3 lm in A and B, 10 lm in C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) osteocyte-looking cells that reside in the spacious lacunae at later research. The adaptive and flexible nature of the platform will also stages, and send multiple filopodia extensions through the sur- allow it to be used to investigate the evolution of the diverse cran- rounding matrix and the neighboring bone cells (Fig. 7A). Filopodia iofacial bone shapes by including morphogenic factors in the speci- penetrate the bone tissue through canal-like structures called bone fic regions of the assembled multi-hydrogels to recapitulate and canaliculi and are believed to sense biomechanical stress and com- manipulate the morphogenic processes in vitro. municate with other bone cells within the same tissue [34]. These cellular projections also observed in our mature 3-D bone tissues extended beyond the immediate lacunae boundaries and contin- Acknowledgements ued through the surrounding matrix toward other neighboring bone cells (Fig. 7A, white arrowheads). These cell–cell interactions UD acknowledges that this material is based in part upon work were clearly detected in the cell-encapsulated 3-D hydrogels, start- supported by the NSF CAREER Award Number 1150733, NIH ing from the second week of osteogenic differentiation. R01EB015776, NIH R15HL115556 and NIH R21HL112114. AA In summary, we demonstrate a 3-D differentiation platform acknowledges NSF 1257122 and Templeton Foundation RFP-12- that enables to recapitulate an important developmental process 01. Dr. U Demirci is a founder of, and has an equity interest in: in vitro. The platform we generated successfully supports the (i) DxNow Inc., a company that is developing microfluidic and intramembranous ossification of cranial bone tissue using primary imaging technologies for point-of-care diagnostic solutions, and CNCCs. Thus, it is an essential tool added for studying the normal (ii) Koek Biotech, a company that is developing microfluidic IVF and abnormal osteogenic development in the model and non- technologies for clinical solutions. U.D.’s interests were viewed model vertebrates. This platform has the potential to be used in and managed in accordance with the conflict of interest policies. a broader context, especially when combined with other bioengi- The other authors indicated no potential conflicts of interest. neering technologies [6,8,49,50]. This platform can be easily scaled Authors thank to Yesim Bagatur (M.S.) and Zeynep Esencan (M.S.) up by assembly technologies, and be utilized in bottom-up tissue for their technical assistance. engineering, enabling to generate larger, multi-hydrogel constructs needed to understand the morphogenesis of specific skeletal struc- tures. Neural crest cells differentiated from induced pluripotent Appendix A. Supplementary data stem cells (iPSCs) that are derived from human patients with cran- iofacial diseases could be used to monitor disease-specific pheno- Supplementary data associated with this article can be found, in types during osteogenic differentiation and eventually bone and the online version, at http://dx.doi.org/10.1016/j.actbio.2015.12. other skeletal tissue formations for medical and pharmaceutical 004. B. Namkoong et al. / Acta Biomaterialia 31 (2016) 301–311 311

References [27] C. von Levetzow et al., Modeling initiation of Ewing sarcoma in human neural crest cells, PLoS ONE 6 (4) (2011) e19305. [28] V. Lefebvre et al., SOX9 is a potent activator of the chondrocyte-specific [1] R.J. Davenport, What controls organ regeneration, Science 309 (5731) (2005) 84. enhancer of the pro alpha1(II) collagen gene, Mol. Cell Biol. 17 (4) (1997) [2] R. Perez-Castillejos, Replication of the 3D architecture of tissues, Mater. Today 2336–2346. 13 (1–2) (2010) 32–41. [29] W. Bi et al., Sox9 is required for cartilage formation, Nat. Genet. 22 (1) (1999) [3] Y.S. Song et al., Engineered 3D tissue models for cell-laden microfluidic 85–89. channels, Anal. Bioanal. Chem. 395 (1) (2009) 185–193. [30] C. Healy, D. Uwanogho, P.T. Sharpe, Regulation and role of Sox9 in cartilage [4] F. Xu et al., A three-dimensional in vitro ovarian cancer coculture model using a formation, Dev. Dyn. 215 (1) (1999) 69–78. high-throughput cell patterning platform, Biotechnol. J. 6 (2) (2011) 204–212. [31] Y. Mori-Akiyama et al., Sox9 is required for determination of the chondrogenic [5] S. Guven et al., Engineering of large osteogenic grafts with rapid engraftment cell lineage in the cranial neural crest, Proc. Natl. Acad. Sci. USA 100 (16) capacity using mesenchymal and endothelial progenitors from human adipose (2003) 9360–9365. tissue, Biomaterials 32 (25) (2011) 5801–5809. [32] L.J. Ng et al., SOX9 binds DNA, activates transcription, and coexpresses with [6] S. Guven et al., Multiscale assembly for tissue engineering and regenerative type II collagen during chondrogenesis in the mouse, Dev. Biol. 183 (1) (1997) medicine, Trends Biotechnol. 33 (5) (2015) 269–279. 108–121. [7] C.A. Bichsel et al., Diagnostic microchip to assay 3D colony-growth potential of [33] Q. Zhao et al., Parallel expression of Sox9 and Col2a1 in cells undergoing captured circulating tumor cells, Lab Chip 12 (13) (2012) 2313–2316. chondrogenesis, Dev. Dyn. 209 (4) (1997) 377–386. [8] S. Tasoglu et al., Untethered micro-robotic coding of three-dimensional [34] E.M. Aarden, E.H. Burger, P.J. Nijweide, Function of osteocytes in bone, J. Cell. material composition, Nat. Commun. 5 (2014) 3124. Biochem. 55 (3) (1994) 287–299. [9] S. Tasoglu et al., Guided and magnetic self-assembly of tunable [35] N.C. Jones, P.A. Trainor, The therapeutic potential of stem cells in the treatment magnetoceptive gels, Nat. Commun. 5 (2014) 4702. of craniofacial abnormalities, Expert Opin. Biol. Ther. 4 (5) (2004) 645–657. [10] B.K. Hall, The neural crest as a fourth germ layer and vertebrates as [36] C. Gans, R.G. Northcutt, Neural crest and the origin of vertebrates: a new head, quadroblastic not triploblastic, Evol. Dev. 2 (1) (2000) 3–5. Science 220 (4594) (1983) 268–273. [11] B.K. Hall, The neural crest and neural crest cells: discovery and significance for [37] G.W. Calloni, N.M. Le Douarin, E. Dupin, High frequency of cephalic neural theories of embryonic organization, J. Biosci. 33 (5) (2008) 781–793. crest cells shows coexistence of neurogenic, melanogenic, and osteogenic [12] N. Le Douarin, C. Kalcheim, The Neural Crest, second ed., Developmental and differentiation capacities, Proc. Natl. Acad. Sci. USA 106 (22) (2009) 8947– Cell Biology Series, vol. 36, Cambridge University Press, Cambridge, UK, New 8952. York, NY, USA, 1999. Xxiii, 445 p. [38] M.A. Bucaro et al., Fine-tuning the degree of stem cell polarization and [13] G.F. Couly, P.M. Coltey, N.M. Le Douarin, The triple origin of skull in higher alignment on ordered arrays of high-aspect-ratio nanopillars, ACS Nano 6 (7) vertebrates: a study in quail-chick chimeras, Development 117 (2) (1993) (2012) 6222–6230. 409–429. [39] C. Scotti et al., Recapitulation of endochondral bone formation using human [14] D.M. Noden, The role of the neural crest in patterning of avian cranial skeletal, adult mesenchymal stem cells as a paradigm for developmental engineering, connective, and muscle tissues, Dev. Biol. 96 (1) (1983) 144–165. Proc. Natl. Acad. Sci. USA 107 (16) (2010) 7251–7256. [15] D.M. Noden, Cell movements and control of patterned tissue assembly during [40] T. Takarada et al., An analysis of skeletal development in osteoblast-specific craniofacial development, J. Craniofac. Genet. Dev. Biol. 11 (4) (1991) 192–213. and chondrocyte-specific runt-related transcription factor-2 (Runx2) knockout [16] B.K. Hall, T. Miyake, The membranous skeleton: the role of cell condensations mice, J. Bone Miner. Res. 28 (10) (2013) 2064–2069. in vertebrate skeletogenesis, Anat. Embryol. (Berl) 186 (2) (1992) 107–124. [41] K. Nakashima et al., The novel zinc finger-containing transcription factor [17] X. Jiang et al., Tissue origins and interactions in the mammalian skull vault, osterix is required for osteoblast differentiation and bone formation, Cell 108 Dev. Biol. 241 (1) (2002) 106–116. (1) (2002) 17–29. [18] J.A. Helms, D. Cordero, M.D. Tapadia, New insights into craniofacial [42] G.K. Hunter, Role of osteopontin in modulation of hydroxyapatite formation, morphogenesis, Development 132 (5) (2005) 851–861. Calcif. Tissue Int. 93 (4) (2013) 348–354. [19] A. Abzhanov et al., Regulation of skeletogenic differentiation in cranial dermal [43] S.T. Choi et al., Osteopontin might be involved in bone remodelling rather than bone, Development 134 (17) (2007) 3133–3144. in inflammation in ankylosing spondylitis, Rheumatology (Oxford) 47 (12) [20] D.M. Noden, R.A. Schneider, Neural crest cells and the community of plan for (2008) 1775–1779. craniofacial development: historical debates and current perspectives, Adv. [44] P.V. Hauschka et al., Osteocalcin and matrix Gla protein: vitamin K-dependent Exp. Med. Biol. 589 (2006) 1–23. proteins in bone, Physiol. Rev. 69 (3) (1989) 990–1047. [21] E.T. Pineda, R.M. Nerem, T. Ahsan, Differentiation patterns of embryonic stem [45] L.W. Fisher et al., Matrix sialoprotein of developing bone, J. Biol. Chem. 258 cells in two- versus three-dimensional culture, Cells Tissues Organs 197 (5) (20) (1983) 12723–12727. (2013) 399–410. [46] K. Gkioni et al., Mineralization of hydrogels for bone regeneration, Tissue Eng. [22] M.P. Lutolf, P.M. Gilbert, H.M. Blau, Designing materials to direct stem-cell Part B: Rev. 16 (6) (2010) 577–585. fate, Nature 462 (7272) (2009) 433–441. [47] Y. Mikuni-Takagaki et al., Matrix mineralization and the differentiation of [23] M. Ravi et al., 3D cell culture systems: advantages and applications, J. Cell. osteocyte-like cells in culture, J. Bone Miner. Res. 10 (2) (1995) 231–242. Physiol. 230 (1) (2015) 16–26. [48] U.E. Pazzaglia et al., Osteoblast-osteocyte transformation. A SEM densitometric [24] H. Etchevers, Primary culture of chick, mouse or human neural crest cells, Nat. analysis of endosteal apposition in rabbit femur, J. Anat. 224 (2) (2014) 132– Protoc. 6 (10) (2011) 1568–1577. 141. [25] U.A. Gurkan et al., Simple precision creation of digitally specified, spatially [49] P. Chen et al., Biotunable acoustic node assembly of organoids, Adv. Healthc. heterogeneous, engineered tissue architectures, Adv. Mater. 25 (8) (2013) Mater. 4 (13) (2015) 1937–1943. 1192–1198. [50] S. Tasoglu et al., Magnetic levitational assembly for living material fabrication, [26] N.M. Le Douarin, G.W. Calloni, E. Dupin, The stem cells of the neural crest, Cell Adv. Healthc. Mater. 4 (10) (2015) 1469–1476. 1422. Cycle 7 (8) (2008) 1013–1019.