Collagen microencapsulation recapitulates mesenchymal condensation and potentiates chondrogenesis of human mesenchymal stem cells – A matrix-driven in vitro model of early skeletogenesis

Biomaterials 213 (2019) 119210

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Collagen microencapsulation recapitulates mesenchymal condensation and potentiates chondrogenesis of human mesenchymal stem cells – A matrixdriven in vitro model of early skeletogenesis

T

Yuk Yin Li, Kwok Lim Lam, Abigail Dee Chen, Wei Zhang, Barbara Pui Chan∗ Tissue Engineering Laboratory, Biomedical Engineering Programme, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region

A R T I C LE I N FO

A B S T R A C T

Keywords: Collagen microencapsulation Mesenchymal stem cells Mesenchymal condensation Chondrogenesis Early skeletogenesis In vitro model

Mesenchymal condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in nanofibrous collagen meshwork. We hypothesize that collagen microencapsulation of MSCs mimics the mesenchymal cell condensation process. Specifically, human MSCs at different concentrations were microencapsulated in collagen for different time points before evaluation for early skeletogenesis markers. A transient upregulation of mesenchymal condensation markers including peanut agglutinin, fibronectin, integrins α5 and αv, an enhanced nuclear localization of SOX9 and binding interactions with COL2A1, and other changes in chondrogenic, hypertropic and osteogenic marker were demonstrated. Collagen microencapsulation upregulated both the chondrogenic and the osteogenic transcription factors and the encapsulated hMSCs hold the potential to differentiate towards both chondrogenic and osteogenic lineages. We also hypothesize that collagen microencapsulation potentiates MSC chondrogenesis. Particularly, chondrogenic differentiation of hMSCs were induced at different time post-encapsulation before evaluation for chondrogenesis outcomes. Sustained SOX9, ACAN and COL2A1 expression were noted and the timing to induce supplement chondro-inductive factors matters. This study reports an extracellular matrix-based in vitro model of mesenchymal condensation, an early stage in skeletogenesis, contributing to rationalizing development-inspired tissue engineering.

1. Introduction Cell condensation is a process, frequently happened during development, where dispersed cells are condensed with high cell density to differentiate into a particular cell or tissue type, to list a few, cartilage, bone, muscle, tendon, kidney and lung [1]. This process represents the first major stage of selective gene activation in tissue morphogenesis including skeletogenesis, odontogenesis, ligamentogenesis, etc [1–3]. In the context of the skeletal system, chondrogenesis describes the process that results in the formation of the cartilage template, which eventually leads to bone formation by endochondral ossification [4]. The process of chondrogenesis occurs in stages beginning with mesenchymal condensation followed by chondrocyte differentiation and maturation [5]. Mesenchymal condensation stage is characterized by a high rate of proliferation and formation of dense cell-cell contacts and molecularly marked by cell-cell adhesion molecules such as neural cadherin (N-

cadherin) and neural cell adhesion molecule (N-CAM), and cell surface markers for peanut agglutinin (PNA) before differentiating into chondrocytes [5]. This process is also characterized by the stage-specific changes from pre-cartilaginous extracellular matrix (ECM) containing fibronectin and collagen type I (COL1A1) to cartilaginous ECM containing collagen type II (COL2A1) and aggrecan (ACAN) as chondrogenic cells differentiate [6]. Terminal differentiation of chondrocytes is associated with a collagen type X (COL10A1)-rich matrix [5] before bone formation, characterized by the expression of SP7, alkaline phosphatase (ALP), osteopontin and calcium phosphate deposition. Nevertheless, MSCs cultured in regular growth medium was found to express COL10A1 [7,8], which may be important for the establishment of a hematopoietic niche at the chondro-osseous junction as demonstrated by mice deficient in COL10A1 resulting in hematopoietic defects [9]. Specific transcription factors regulate the differentiation pathway of

∗ Corresponding author. Tissue Engineering Laboratory, Biomedical Engineering Programme, Department of Mechanical Engineering, Room 711, Haking Wong Building, Pokfulam Road, The University of Hong Kong, Hong Kong Special Administrative Region. E-mail address: [email protected] (B.P. Chan).

https://doi.org/10.1016/j.biomaterials.2019.05.021 Received 15 January 2019; Received in revised form 28 April 2019; Accepted 10 May 2019 Available online 18 May 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Collagen microencapsulation of hMSCs recapitulates early events of mesenchymal condensation. A: Schematic diagram illustrating the collagen microencapsulation process and the experimental approach; B: Phase contrast images showing the gross appearance of hMSC-collagen microspheres with different initial cell densities (2.5 × 105, 5 × 105, 1 × 106 cells/ml) over culture time (up to 10 days) (scale bar: 100 μm); C: H&E staining showing the morphology of hMSCs in the microspheres with different initial cell densities over culture time; D: Line chart showing the temporal change in the diameter and hence size of the hMSCcollagen micropsheres with different initial cell densities; E: Immunofluorescence staining of the major mesenchymal condensation marker namely PNA in hMSCcollagen microspheres with an initial cell density of 5 × 105 cells/ml over culture time (up to 4 days), and a line chart showing the temporal change in the mean intensity of the PNA staining (red: PNA; blue: nuclei stained with DAPI; scale bar: 20 μm); F: Immunofluorescence staining of other mesenchymal condensation markers including (i) fibronectin (green), (ii) integrin α5 (green), (iii) integrin β1 (green) and (iv) integrin αv (green) over culture time (up to 4 days) (blue: nuclei stained with DAPI; scale bar: 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2

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that collagen is the major ECM present during mesenchymal condensation, motivate us to hypothesize that collagen microencapsulation of MSCs mimics the mesenchymal condensation process during early skeletogenesis. Specifically, we aim to investigate whether collagen microencapsulation of hMSCs recapitulates the events of mesenchymal condensation process in accordance to the temporal and spatial markers for mesenchymal condensation and chondrongenesis and whether this matrix-drive microencapsulation process potentiates chondrogenesis, hypertrophy and osteogenesis. In brief, hMSCs at different concentrations (0.25, 0.5 and 1 million cells/ml) were microencapsulated to form MSC-collagen microspheres and the microencapsulation process was followed for different time points (0, 1, 2, 3, 4, 7 and 10 days) postencapsulation and the major markers for mesenchymal condensation, chondrogenesis, hypertrophy and osteogenesis were measured at both gene and protein levels (Fig. 1A). In separate experiments, the MSCcollagen microspheres with the highest cell density were exposed to chondro-inductive conditions at different time (0, 1 and 3 days) postencapsulation before evaluating the outcomes of chondrogenic differentiation at different time (7, 14 and 21 days) post-differentiation before evaluating the chondrogenesis, hypertrophy and osteogenesis markers (Fig. 5A). This study reports the establishment of an ECMbased in vitro model of mesenchymal condensation and early skeletogenesis, contributing to the development of rationalized and optimized differentiation protocols for development-inspired cartilage tissue engineering.

mesenchymal progenitor cells into chondrocytes and eventually replaced by osteoblasts [5,10].Transcription factors SRY (Sex Determining Region Y)-Box 9 (SOX9) and runt-related transcription factor-2 (RUNX2) play essential roles in the differentiation pathway. SOX9 activates chondrocyte-specific marker genes, such as COL2A1 and ACAN [11,12]. It is highly expressed in all osteochondroprogenitor cells and all differentiated chondrocytes, but not in hypertrophic chondrocytes [13–15]. Although RUNX2 is required for chondrocyte maturation and osteoblast differentiation [16], it is present in osteochondroprogenitor cells during mesenchymal condensation [17–19]. SOX9 is able to inhibit RUNX2 function during osteoblastic and chondrocyte maturation [20], therefore regulation of SOX9 and RUNX2 functions during mesenchymal condensation may be important in determining osteochondroprogenitor cell fate. Recently, developmentally inspired designs have attracted much attention in deriving differentiation protocols for cartilage [21]. This suggests the importance to develop in vitro models recapitulating events of early skeletogenesis for testing, screening, evaluating and optimizing developmentally inspired designs such as different types of cells, matrices or biomaterials, and tissue growth-stimulating signals for tissue engineering and regenerative medicine. Taking mesenchymal condensation for chondrogenesis as an example, a number of in vitro models have been reported, namely micromass cultures of limb mesenchymal cells [22,23], pellet or aggregate cultures of MSCs [21,24–27], seeding MSCs onto porous scaffolds such as eletrospun silk protein matrices [28] and freeze-dried collagen scaffold [21], and encapsulating MSCs within hydrogel materials such as alginate [29–31], polyethylene glycol [31–33] and gelatin [31,34]. All these models are able to mimic the high local cell density observed in mesenchymal condensation [35]. However, micromass cultures [36,37] and pellet cultures [21,25] lacks ECM as that in the case of mesenchymal condensation and requires an extremely high cell density (e.g. 107 cell/ml) that the condensation induced may interfere cell proliferation and fate determination by imposing too tight cell-to-cell contacts [25]. On the other hand, cell seeding onto porous scaffolds such as electrospun silk matrix [28] and freeze-dried collagen [21] do not readily condense the porous scaffolds due to their rigidity. Similarly, cell microencapsulation in hydrogel such as alginate [29–31], polyethylene glycol [31–33] and gelatin [31,34] does not condense the structure because cells do not express specific receptors towards these inert materials and hence cannot specifically interact with the hydrogel, unless functionally modified by matrix proteins such as RGD peptides [32]. As a result, having appropriate cell-cell interactions through high local cell density and appropriate cell-matrix interactions through presence of suitable ECM are both pre-requisites for successful skeletogenesis. During mesenchymal condensation and early skeletogenesis, it is thought that the local cells rearrangements occurred are mediated by ECM-driven movements rather than cell migration or localized proliferation [38–40]. Therefore, inclusion of ECM components when developing in vitro models to mimic the mesenchymal condensation for early skeletogenesis studies including chondrogenesis, maturation and osteogenesis is important. We have previously developed a collagen microencapsulation technique to entrap living cells including but are not limited to MSCs [41], chondrocytes [42], osteoblasts [43], intervertebral disc cells [44], HEK293 [45], embryonic stem cells (ESCs) [46] and hESC-derived cardiomyocytes [47], in a reconstituted collagen nanofibrous meshwork, leading to the formation of cell-collagen microspheres or microtissues. This platform involves an ECM-driven and cell density-dependent volume contraction and hence cell condensation process [41]. Stem cells including MSCs [48,49] and ESCs [46] have been microencapsulated and cultured in these 3D cell-collagen microspheres and are able to be differentiated towards chondrogenic lineage [48,49], forming tiny cartilage micro-tissues, which have been shown to be able to repair injured cartilage in rabbits with hyaline cartilage in a cell density dependent manner [50]. These findings, together with the fact

2. Materials and methods 2.1. Culture of human MSCs Human MSCs were obtained from the Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine (Scott & White Hospital, Bryan, TX, USA). hMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)-low glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Culture medium was replaced every 3–4 days. Cells at passage 6 were used for microencapsulation and subsequent differentiation experiments. 2.2. Microencapsulation of hMSCs in collagen microspheres Human MSC-collagen microspheres were prepared as described previously [41]. Briefly, hMSC suspensions with final cell density of 0.25, 0.5 and 1 million cells/ml were mixed with neutralized rat tail type I collagen solution (BD Biosciences) at a final concentration of 2 mg/ml in an ice-bath. Droplets of 5 μl were pipetted into a 100 mm diameter Petri dish (Sterlin, Stone, UK) covered with UV irradiated parafilm. After 30 min incubation at 37 °C, the gelated MSC-collagen microspheres were ready for subsequent experiments. 2.3. Histological, immunofluorescence and immunohistochemical staining At specific time points, microspheres were fixed with 4% PBS-buffered paraformaldehyde for 20 min and cut into 18 μm frozen sections. Routine H&E staining was used to reveal the cell morphology and Alcian blue staining was used to reveal the GAG-rich region. Major cellular and ECM markers for chondrogenesis were characterized. Unless otherwise specified, all primary antibodies for immunofluorescent staining were purchased from Abcam (Cambridge, UK). To evaluate the presence of mesenchymal condensation marker, rhodamine-conjugated PNA (25 μg/ml, Vector Laboratories) was used. Transcription factors governing chondrogenesis and osteogenesis were evaluated via immunofluorescence staining using antibodies against SOX9 and RUNX2 (Cell Signaling Technology, Inc.). Traditional 2D monolayer culture was used as a reference group where hMSCs were seeded on type I collagen coated dish (5 μg/cm2). After incubation for 3

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Master Mix (Applied Biosystems) or Power SYBR® Green PCR Master Mix (Applied Biosystems) under standard thermal conditions. Quantification Method with Ct values from monolayer as the experimental calibrator. For experiment with TGF-β3 supplementation, microspheres with cell density of 1 × 106 cells/ml were used with day 0 as the experimental calibrator (control), while 6 genes (SOX9, ACAN, COL2A1, COL1A1, COL10A1 and RUNX2) were examined.

specific time points, cells were fixed and stained for SOX9 and RUNX2. After permeabilization with 0.5% Tween 20 for 10 min, antigen retrieval was performed with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 95 °C for 20 min. After overnight incubation with primary antibodies, sections were incubated with Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) for SOX9 or Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes) for RUNX2 for 1 h. Another osteogenic transcription factor SP7 was examined using Alexa Fluor 488 goat anti-rabbit IgG after antigen retrieval with sodium citrate and overnight primary antibody incubation. To evaluate the ECM markers, antibodies against fibronectin (R&D Systems), ACAN (Santa Cruz Biotechnology, Inc.), COL10A1, and COL2A1 were used. After permeabilization, antigen retrieval was performed with 0.2% hyaluronidase at 37 °C for 30 min. After overnight incubation with primary antibody, sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG for 1 h. To examine cell-matrix interactions, antibodies against integrins α5 and β1 were used. After permeabilization, sections were incubated with the appropriate primary antibody overnight. Alexa Fluor 488 goat anti-mouse IgG was used. Finally, sections were mounted with Fluoro-gel II mounting medium with DAPI (Electron Microscopy Sciences, Hatfield, PA). Images were taken using multiphoton laser confocal scanning microscopy (LSM710, Carl Zeiss). Images from each set of experiment staining for same primary antibody were acquired at same detector setup and scanning setup with objective ×40. For the major ECM marker for chondrogenesis, immunohistochemistry using antibody for COL2A1 (Calbiochem) was performed. Sections were treated with 0.5% pepsin in 5 mM HCl at 37 °C for 30 min. After overnight incubation with primary antibody, sections were incubated with secondary antibody for 30 min. A Vectastain ABC kit (Vector Laboratories) and DAB substrate system (Dako) were used for color development.

2.5. Chromatin immunoprecipitation (ChIP) assays In order to verify whether collagen microencapsulation of hMSCs triggers chondrogenic commitment and transcriptional activation with protein-DNA binding, ChIP assays using the Pierce™ Agarose ChIP Kit were conducted according to the manufacturer's protocol (ThermoScientific Cat#26156). In brief, hMSCs at 5 × 105 cells/ml final density were encapsulated in collagen microspheres (3D) or cultured as monolayers on collagen (5 μg/cm2) coated dishes (2D) at 37 °C for 30 min before being harvested by collagenase treatment and trypsinization for 5 min respectively for subsequent ChIP assays. About 2 × 106 cells per ChIP reaction were cross-linked in 1% formaldehyde for permeabilization. The cross-linked cells were lysed by membrane extraction buffer with protease inhibitors (PI). After centrifugation, the nucleus pellet was resuspended in Micrococcal Nuclease (MN) digestion buffer with 2.5U of MN, and then incubated at 37 °C for 15 min. Later, the mixture was centrifuged and the supernatant was removed. After adding the nuclear lysis buffer with PI for resuspension, it was kept on ice for 15 min. Following centrifugation for 5 min, 10% of the supernatant as an input sample was transferred to a new tube as a positive control without antibody treatment. The remaining sample was immunoprecipitated by SOX9 (ab3697, 1:40) or normal rabbit IgG (IgG: negative control) at 4 °C for overnight and then treated with the protein A/G plus agarose for isolating the antibody-DNA-protein complexes. The precipitated DNAs from SOX9, the IgG negative control and the input sample (Input: Positive control) were purified by DNA spin column. The PCR reaction was performed including a step of 94 °C hot start for 5 min followed by 35 cycles of 94 °C for 45 s, 57 °C for 45 s and further extension at 72 °C for 10 min. The primer sets amplifying SOX9 binding sites on both the COL2A1 promoter and the intron1 are listed in Table 2. The sizes of the PCR products were 269bp and 155bp, respectively [51].

2.4. Gene expression of chondrogenic and osteogenic markers by real-time RT-PCR The expression levels of the major chondrogenic and osteogenic marker genes, including SOX9, ACAN, COL2A1, COL1A1, COL10A1, RUNX2, SP7, and ALP were used to evaluate whether the encapsulated hMSCs tend to commit to the chondrogenic or the osteogenic lineage (Table 1). At specific time points, around 30–200 microspheres were digested with 3 mg/ml collagenase (Sigma) and 1 mg/ml hyaluronidase (Sigma) at 37 °C around 30 min. The digested microspheres were then homogenized with 1 ml TRI reagent (Molecular Research Center) and the total RNA was extracted following the manufacturer's protocol. RNA concentration was determined using a NanoDrop™ 2000 spectrophotometer (NanoDrop, DE, USA) and was transcribed into cDNA using TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed using TaqMan® Gene Expression

2.6. Induction of chondrogenic differentiation In order to investigate whether hMSCs respond to chondrogenic inductive signal after microencapsulation in collagen and the effect of timing for chondrogenic differentiation, separate batches of microencapsulation followed by chondrogenic differentiation was conducted. Specifically, after 1-h, 1-d or 3-d post-encapsulation, the microspheres

Table 1 Primers used for Real-time RT-PCR analysis. Reagent: Power SYBR Gene COL2A1 COL10A1 ALP GAPDH Reagent: TaqMan Gene SOX9 ACAN COL1A1 RUNX2 SP7 GAPDH

®

Green PCR Master Mix Forward Primer

®

Reverse Primer

5′-AGCCCTGCCGGATCTGTGT-3′ 5′-CAGATTTGAGCTATCAGACCAACAA-3′ 5′-CGCACGGAACTCCTGACC-3′ 5′-GAGTCAACGGATTTGGTCGT-3′ Gene Expression Master Mix Assay ID according to Applied Biosystems Hs00165814_m1 Hs00153936_m1 Hs_00164004_m1 Hs_00231692_m1 Hs_01866874_s1 NM_002046.3 (Accession Number)

4

5′-CTGAGGCAGTCTTTCACGTCTTCA-3′ 5′-AAATTCAAGAGAGGCTTCACATACG-3′ 5′-GCCACCACCACCATCTCG-3′ 5′-TTGATTTTGGAGGGATCTCG-3′

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Table 2 Primers for detection SOX9 binding sites on COL2A1-promoter and intron1. Region

Forward primer

Reverse primer

COL2A1_promoter COL2A1_Intron 1

5′-GCTCCTTTCTACCGCTTTCC-'3 5′-TTCCAGATGGGGCTGAAAC-'3

5′-CTCTCTGGGAGTCACGCTTC-'3 5′-ATTGTGGGAGAGGGGGTCT-'3

Fig. 2. Collagen microencapsulation transiently upregulated SOX9 nuclear co-localization of SOX9 and interactions with COL2A1 of hMSCs. A: Immunofluorescence staining of SOX9 in hMSCs under 2D monolayer culture (a), in cell suspension (b) and at different time points (c, d, e: 0, 30 min and 24 h, respectively) after 3D collagen microencapsulation (SOX9 (green), DAPI (blue) and F-actin (red), scale bars: 20 μm for (a) and 10 μm for others). B: Interactions between SOX9 and SOX9-binding sites of COL2A1; ChIP showing the binding interactions between SOX9 and SOX9-binding sites on the promoter and intron1 of COL2A1 in hMSCs under 2D monolayer culture on type I collagen coated dish and 3D collagen microencapsulation; IgG was used as negative control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.7. Data analysis and statistics

were cultured in chondrogenic differentiation medium consisting of DMEM-high glucose supplemented with 10 ng/ml recombinant human TGF-β3 (Merck, Darmstadt, Germany), 100 nM dexamethasone (Sigma), 6 mg/ml insulin (Merck), 100 mM 2-phospho-L-ascorbate (Fluka, St. Louis, MO), 1 mM sodium pyruvate (Gibco), 6 mg/ml transferring (Sigma), 0.35 mM L-proline (Merck), and 1.25 mg/ml BSA (Sigma). To characterize the temporal changes of the hMSC phenotypes, at d 7, 14, and 21 post-induction, samples were harvested for histological, cytochemical, and immunofluorescence analyses, as well as real time PCR on gene expression analysis of major chondrogenic and osteogenic markers as described earlier.

All quantitative data were expressed as the mean ± standard error unless otherwise specified. Skewed data was transformed using natural logarithm for statistical analysis. Continuous variables on the expression level of genes among different groups were analysed by appropriate comparison tests including analysis of variance (ANOVA) and Kruskal-Wallis tests for the expression levels of genes among different groups. The significance level was set at 0.05 and SPSS 19.0 (SPSS Inc., Chicago, IL) was used for the analysis.

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Fig. 3. Expression of chondrogenic markers in hMSCs post-encapsulation. hMSCs at different initial cell densities (2.5 × 105, 5 × 105 and 1 × 106 cells/ml) at different time points (day 0, 1, 2, 3, 4, 7 and 10) post-encapsulation. A: SOX9 protein and gene expression; Left panel: Immunofluorescence staining of SOX9 (green) and DAPI (blue); Right panel: Box plot showing the real time PCR measurement of SOX9 gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3); B: ACAN protein and gene expression; Left panel: Immunofluorescence staining of ACAN (green) and DAPI (blue); Right panel: Box plot showing the real time PCR measurement of ACAN gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3); C: COL2A1 protein and gene expression; Left panel: Immunohistochemistry of COL2A1 (brown); Right panel: Box plot showing the real time PCR measurement of COL2A1 gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3. Results

rapidly before reaching a constant size. Two-way ANOVA showed that both cell densities (P < 0.0001) and time (P < 0.0001) significantly affected the microsphere size. The hematoxylin and eosin (H&E) staining of 3 different cell densities showed the MSCs maintained a rounded morphology upon 1-h post-encapsulation and became elongated after 1-d post-encapsulation (Fig. 1C).

3.1. Microencapsulation recapitulates the early events of mesenchymal condensation 3.1.1. Cell-density dependent volume shrinkage and contraction The hMSC-collagen microspheres showed cell density-dependent contraction and volume shrinkage, leading to densely packed cells. Microspheres with higher initial cell density (Fig. 1B) contracted more. The contraction was a function of time post-encapsulation and cell density (Fig. 1D). Microspheres with higher cell density contracted

3.1.2. Transient upregulation of PNA Microspheres with 5 × 105 cells/ml density were chosen to examine the cellular condensation and cell-matrix interaction during the first 4 days of post-encapsulation. The encapsulated hMSCs highly expressed 6

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3.3.2. Downregulation of ACAN expression Immunofluorescence staining of ACAN was positive at early time points (1-h, 1-d, and 2-d post-encapsulation), with most intense staining at 1-h post-encapsulation, particularly for the low and intermediate cell density groups (Fig. 3B left panel). The high cell density group showed negative staining of ACAN after the first hour post-encapsulation (Fig. 3B). Gene expression of ACAN showed significant decrease over time in all cell density groups (Fig. 3B right panel). Two-way ANOVA showed that both the cell density factor (P < 0.0001) and the time factor (P < 0.0001) significantly affected the ACAN expression.

cell surface molecules for PNA, the marker for mesenchymal condensation (Fig. 1E). The intensity of the PNA staining was continuously increased with a peak at 4 h, after which the intensity was decreased (Fig. 1E). 3.1.3. Transient upregulation of fibronectin, integrins α5 and αv Fig. 1F shows the labelling patterns of fibronectin, integrins α5, αv and β1 in microspheres during the first 4 days of post-encapsulation. Fibronectin mainly located at pericellular region initially (1st row, Fig. 1F). The extracellular region showed increased expression of fibronectin over time and the most intense expression was found at 48 h. However, the fibronectin expression significantly decreased after 72 h and the location of expression changed from extracellular to pericellular. Integrins α5 showed positive expression overtime but the expression was downregulated after 24 h post-encapsulation (2nd row, Fig. 1F). Integrins β1 and αv showed little expression in early time points but intensive expression after 24 h (3rd and 4th Fig. 1F).

3.3.3. Upregulated gene but not protein expression of COL2A1 collagen The gene expression of the major ECM marker for chondrogenesis COL2A1 significantly increased in all cell density groups over time (Fig. 3C right panel). For the low cell density group, there was significant increase, over 20 fold, in COL2A1 in the early time points but the expression level significantly reduced to basal level after day 2. For the intermediate cell density group, the expression level significantly increased to over 20 fold shortly after microencapsulation and then further increased to over 100 fold over time but the level reduced to around 20 fold at day 10 post-encapsulation. For the high cell density group, the expression level increased to over 20 fold on day 1 postencapsulation and gradually increased to ∼80 fold on day 7 and thereafter. Two-way ANOVA showed that COL2A1 expression was significantly different among different cell density groups (P = 0.003) but marginally for different time points (P = 0.131). Given a significant upregulation of COL2A1 gene expression, there is no corresponding increase in the protein expression as shown from the basal level expression in the immunohistochemical staining (Fig. 3C left panel).

3.2. Microencapsulation enhanced nuclear localization of SOX9 and binding interactions with COL2A1 3.2.1. SOX9 nuclear localization hMSCs under 2D monolayer culture showed positive expression of the master chondrogenic transcription factor SOX9 throughout the cell cytoplasm with little nuclear localization (Fig. 2A). hMSCs in cell suspensions also showed high level of SOX9 expression in the form of intensively stained patches throughout the cytoplasm (Fig. 2A). After 3D microencapsulation in collagen microspheres, hMSCs showed increasing level of nuclear localization of SOX9 over time as shown by the highly co-localizing signals at the nucleus region in the confocal images (Fig. 2A).

3.4. Microencapsulation-induced changes in hypertrophic and osteogenic markers

3.2.2. Interactions between SOX9 and SOX9-binding sites of COL2A1 Results of the ChIP assays demonstrated that both 2D and 3D hMSCs, immunoprecipitated by SOX9 antibody, showed a 269bp amplicon within the COL2A1-promoter region (Fig. 2B), demonstrating the interaction of SOX9 with its binding site on the region. However, 3D microencapsulation group showed stronger interactions with the binding sites on COL2A1. Specifically, hMSCs with 3D microencapsulation showed a relatively higher signal intensity than those with 2D collagen-coated monolayer cultures. Moreover, in the COL2A1intron1 region, only the 3D microencapsulated hMSCs showed a 155bp amplicon, strongly suggesting microencapsulation-induced SOX9binding activity on the COL2A1-intron1 enhancer (Fig. 2B).

3.4.1. Upregulation of RUNX2 expression Immunofluorescence staining showed that the hypertrophic and osteogenic transcription factor RUNX2 was highly expressed in the first few days (1-h, 1-d and 2-d post-microencapsulation) (Fig. 4A left panel). In particular, nuclear localization was noted in some cells shortly after microencapsulation in the low and intermediate density groups but not the high cell density group (Fig. 4A left panel), suggesting that cell-cell interaction prevents activation of RUNX2. The staining intensity declined at later time points and the expression was no longer localizing within the nuclei (Fig. 4A left panel). The level of RUNX2 gene expression in hMSCs upon microencapsulation in all cell density groups was upregulated throughout the 10-d post-encapsulation period (Fig. 4A right panel). In the low cell density, there was several fold higher than that of the monolayer control while in the intermediate and the high cell density groups, there was a temporal increase in the level of gene expression up to over 10 fold higher than that of the monolayer control (Fig. 4A right panel). Two-way ANOVA showed that RUNX2 expression was significantly affected by both the cell density factor (P < 0.0001) and the time factor (P < 0.0001).

3.3. Microencapsulation-induced changes in chondrogenic markers 3.3.1. Transient upregulation of SOX9 expression Immunofluorescence staining of SOX9 was higher in the first few days (1-h, 1-d and 2-d post-microencapsulation) in all cell density groups and declined at later time points particularly in the lower cell density groups (Fig. 3A left panel). The level of SOX9 gene expression in hMSCs, upon microencapsulation in different cell density groups, was several fold higher than that of the monolayer control. Among different cell density groups, the level of SOX9 expression varied and the intermediate cell density group (5 × 105 cells/ml) showed greater fold change (up to 4 fold) than other groups but this difference was only obvious at the early time points (Fig. 3A right panel). At later time points, the level of SOX9 expression was decreased to ∼1.5 fold for the rest of the 10 day incubation period. Two-way ANOVA showed that both the cell density factor (P < 0.0001) and the time factor (P < 0.0001) significantly affected the SOX9 expression in hMSCs. This initial upregulated expression in both protein and gene upon microencapsulation was only transient and could not be maintained over the 10-d post-encapsulation time.

3.4.2. Upregulation of SP7 expression Immunofluorescence staining of the bone specific transcription factor SP7 showed upregulated protein expression throughout the 10-d post-encapsulation period in all cell density groups (Fig. 5B left panel) although more intensive staining with nuclear co-localization was only noted in the first few time points (1-h, 1-d and 2-d post-microencapsulation) (Fig. 4B left panel). The level of SP7 gene expression in hMSCs upon microencapsulation in all cell density groups was upregulated throughout the 10-d post-encapsulation period (Fig. 4B right panel). However, the high cell density showed the least increase as compared to the low and intermediate cell density groups. Specifically, the high cell density group showed up to 25 fold increase as compared with that of the monolayer control, while in the low and intermediate 7

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Fig. 4. Expression of hypertrophic and osteogenic markers in hMSCs post-encapsulation. hMSCs at different initial cell densities (2.5 × 105, 5 × 105 and 1 × 106 cells/ml) at different time points (day 0, 1, 2, 3, 4, 7 and 10) post-encapsulation. A: RUNX2 protein and gene expression; Left panel: Immunofluorescence staining of RUNX2 (pink) and DAPI (blue); Right panel: Box plot showing the real time PCR measurement of RUNX2 gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3); B: SP7 protein and gene expression; Left panel: Immunofluorescence staining of SP7 (green) and DAPI (blue); Right panel: Box plot showing the real time PCR measurement of SP7 gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3); C: COL10A1 protein and gene expression; Left panel: Immunofluorescence staining of COL10A1 (green) and DAPI (blue); Right panel: Box plot showing the real time PCR measurement of COL10A1 gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3); D: Box plot showing the real time PCR measurement of COL1A1 gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3); E: Box plot showing the real time PCR measurement of ALP gene expression of hMSCs after microencapsulation, normalized to that in monolayer cultures (Data are presented in median with 95% CI, n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

cell density groups, the SP7 expression was up to 50 fold and over 100 fold higher than that of the monolayer control, respectively (Fig. 4B right panel). All cell density groups showed a temporal increase up to 7d post-encapsulation and then downregulate afterwards (Fig. 4B right panel). Two-way ANOVA showed that SP7 expression was significantly affected by both the cell density factor (P < 0.0001) and the time factor (P < 0.0001).

mesenchymal condensation mimicking stage, still hold the potential to be differentiated towards the chondrogenic lineage, encapsulated hMSCs were exposed to chondrogenic induction medium at different time points (day 0, 1 and 3) post-encapsulation followed by an evaluation of a panel of chondrogenic, hypertrophic and osteogenic markers at different time points (7-, 14- and 21-d) post-differentiation (Fig. 5A).

3.4.3. Upregulation of COL10A1 expression Immunofluorescence staining showed that the hypertrophic marker COL10A1 was upregulated throughout the post-encapsulation incubation period (Fig. 4C Left panel). There was a cell density dependent increase in the extracellular deposition of COL10A1 that the intermediate and the high cell density groups showed an earlier and more extensive staining of COL10A1 at the extracellular space than that of the low cell density group (Fig. 4C Left panel). There was a significant upregulation of COL10A1 gene expression in all cell density groups over the 10-d post-encapsulation period (Fig. 4C right panel). The temporal change of the COL10A1 expression was different among different cell density groups. Specifically, the low cell density group significantly upregulated COL10A1 expression shortly after the microencapsulation but it declined to around 20 fold soon after (Fig. 4C, Right panel). In the intermediate and the high cell density groups, the level of COL10A1 expression increased over time to over 100 fold and maintained thereafter (Fig. 4C right panel). Two-way ANOVA showed that COL10A1 expression was significantly affected by both the cell density factor (P < 0.0001) and the time factor (P < 0.0001).

3.5.1. Morphology of chondrogenically differentiating hMSCs The H&E staining shows the morphology of the differentiating hMSCs with cartilage-like tissues (Fig. 5B). Some differentiating hMSCs located in lacunae-like structures especially at 7-d post-differentiation (Fig. 5B). From the magnified views of the H&E staining, no obvious change in the size of the chondrogenically differentiating hMSCs was noted over time (Fig. 5B). Alcian blue staining showed that GAGs were prominent in later time points (14 and 21 days) for all 3 conditions (Fig. 5C). 3.5.2. Sustained upregulation of chondrogenic markers SOX9 was expressed as early as day 7 post-differentiation in the group where chondrogenic differentiation commenced at 3-d post-encapsulation and its expression was constantly expressed in this group, whereas prominent SOX9 expression was observed at 21 days in other groups (Fig. 5D upper panel). Fig. 5D, lower panel shows the level of SOX9 gene expression in different groups. There was temporal increase in SOX9 expression in all groups (Fig. 5D). However, there was only slight increase in the level of SOX9 expression after 21 days of chondrogenic differentiation in the groups where induction commenced at day 0 and day 1 post-encapsulation (Fig. 5D lower panel). In the group when induction commenced at day 3 post-encapsulation, there was around 4 fold of change in the SOX9 expression (Fig. 5D, lower panel). Two-way ANOVA showed that SOX9 expression was significantly affected by both the time when chondrogenic induction commenced (P < 0.0001) and the time post-differentiation (P < 0.0001). Intense ACAN staining was mainly found at day 14 and 21 for the groups where chondrogenic induction commenced at 1-d and 3-d post-encapsulation, while weak ACAN staining was observed for the remaining group (Fig. 5E upper panel). Comparing with the control group, ACAN gradually increased with time in all 3 groups, and the group in which chondrogenic induction commenced at 3-d post-encapsulation showed the greatest augmentation with ∼7 fold of change (Fig. 5E lower panel). Two-way ANOVA showed that ACAN expression was significantly affected by both the time when chondrogenic induction commenced (P < 0.0001) and the time post-differentiation (P < 0.0001). COL2A1 protein constantly expressed in the day 0 and day 1 groups but not the day 3 group (Fig. 5F upper panel). Fig. 5F lower panel shows the level of COL2A1 gene expression among different groups. Two-way ANOVA showed that COL2A1 expression was affected by the chondrogenic induction factor (P < 0.001) and the time factor (P = 0.027). Dunnett t post-hoc tests showed that, comparing with the control group, COL2A1 expression significantly increased in the groups where chondrogenic induction commenced at day 0 and day 1 post-encapsulation,

3.4.4. Downregulation of osteogenic markers COL1A1 and ALP Fig. 4D showed that, at the initial time points, the level of COL1A1 gene expression was slightly upregulated within 2 fold of change as compared to that of the monolayer control but it was downregulated at later time points post-encapsulation particularly in the high cell density group. Two-way ANOVA showed that COL1A1 expression was significantly affected by both the cell density factor (P < 0.0001) and the time factor (P < 0.0001). Fig. 4E showed that the level of expression of another osteogenic marker ALP was downregulated for over 5 fold in all cell densities over time. Two-way ANOVA showed that ALP expression was significantly affected by both the cell density factor (P < 0.0001) and the time - factor (P < 0.0001). 3.5. Chondrogenic induction of microencapsulated MSCs shortly after collagen microencapsulation resulted in sustained expression of chondrogenic and hypertrophic but not osteogenic markers Collagen microencapsulation recapitulated the initial events of mesenchymal condensation that provided the encapsulated hMSCs with enhanced potential for subsequent chondrogenic differentiation, as shown by the transient nuclear activation of SOX9. However, the encapsulated hMSCs did not readily committed towards chondrogenic lineage, as shown by the downregulated SOX9 and ACAN expression. In order to investigate whether the encapsulated hMSCs, after the initial 9

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Fig. 5. Temporal change (day 7, 14 and 21) of the expression of chondrogenic, hypertrophic and osteogenic markers in hMSC-encapsulated collagen microspheres after chondrogenic induction at different time points (day 0, 1 and 3) post-encapsulation. A: Chondrogenic differentiation of hMSC-collagen microspheres and experimental approach; B: H&E staining showing the morphology of the encapsulated hMSCs in collagen microspheres; C: Alcian blue staining showing the GAG-rich area of the hMSC-encapsulated collagen microspheres; D: Immunofluorescence labelling of SOX9 protein (green, upper panel) and box plot showing the SOX9 gene expression (lower panel); E: Immunofluorescence labelling of ACAN protein (green, upper panel) and box plot showing the ACAN gene expression (lower panel); F: Immunofluorescence labelling of COL2A1 (green, upper panel) and box plot showing the COL2A1 gene expression (lower panel); G: Immunofluorescence labelling of COL10A1 protein (green, left panel) and box plot showing the COL10A1 gene expression (right panel); and H: Immunofluorescence labelling of RUNX2 protein (green, left panel) and box plot showing the RUNX2 gene expression (right panel). Real time PCR derived gene expression data are presented as median with 95% CI of 3 independent experiments. DAPI (blue) was stained as a nuclear counter stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

with ∼18 and 10 fold, respectively (P < 0.001 for both), after 21 days of differentiation (Fig. 5F lower panel). When chondrogenic induction commenced at day 3 post-encapsulation, COL2A1 expression showed milder increase for ∼2 fold over 21 days of differentiation without statistical significance (P = 0.659).

hematopoesis and assembly of the ECM [9,52,53]. Recently, collagen I coated membranes have been found to facilitate chondrogenesis of hMSCs [54]. Collagen was used as the starting material of microencapsulation and was continually remodelled and gradually replaced by new ECM during chondrogenesis as previously shown [49]. The encapsulated hMSCs showed a slight deceasing trend in the expression of COL1A1 throughout the process. The expression of fibronectin persists during the early development [55,56]. Moreover, fibronectin fibril assembly has been found important to induce mesenchymal condensation [6]. In the current model, fibronectin expression at both protein (Fig. 1) and gene (Supplementary Information 1) level was upregulated upon microencapsulation throughout the 10 day follow up period. Strong extracellular deposition of fibronectin was found as the cells were actively condensing over the first 2 days post-encapsulation while the expression was found at the pericellular region thereafter. hMSCs showed extracellular deposition of fibronectin that should be newly synthesized from the encapsulated MSCs. The current ECM-based microencapsulation model enables testing, screening and optimization of different parameters including the type of ECM in regulating the biomimetic mesenchymal condensation process. The encapsulated MSCs expressed a complex pattern of ECM receptors including integrin α5, which highly expressed in the early stage, and integrin β1 and αv, which highly expressed in the later stage, suggesting temporal change in cellmatrix interactions during the microencapsulation process. Integrin α5 is known to bind fibronectin and its presence is gradually downregulated, suggesting the interaction between MSCs and fibronectin was gradually replaced by other cell-matrix interactions, probably through integrins β1 and αv, which have been shown important for cartilage development. In particular, previous studies demonstrated that integrin β1 expression was important for mesenchymal condensation by regulating the migration of mesenchymal cells [57,58]. In addition, Shekaran et al. (2014) [59] demonstrated that integrin β1 expression in mesenchymal condensation is essential for skeletal development using knockout mice. As for integrin αv, it is expressed in both chondrocytes [4] and MSCs [8]. Friedl et al. (2007) [60] showed a significant correlation between integrin αv and SOX9 gene expression upon mechanical stimulation of MSCs. In addition, we previously demonstrated integrin αv may play a role in cell adhesion and to support chondrogenesis [61].

3.5.3. Upregulation of hypertrophic marker COL10A1 COL10A1 was highly expressed mainly at the extracellular space throughout the differentiation duration in all 3 groups (Fig. 5G left panel). Fig. 5G right panel shows the level of COL10A1 gene expression among different groups and there were close to 100 fold of upregulation of COL10A1 expression in all groups although the group where chondrogenic induction commenced at day 3 post-encapsulation showed slightly less upregulation at day 21 post-differentiation. Two-way ANOVA showed that COL10A1 expression was significantly affected by the time post-differentiation (P < 0.0001) but not the time when chondrogenic induction commenced (P = 0.351). 3.5.4. Upregulation of RUNX2 gene expression without osteogenic commitment Fig. 5H left panel shows that there was no RUNX2 protein expression in all groups. However, there was significant upregulation of the level of RUNX2 gene expression among different groups (Fig. 5H right panel). Comparing with the control group, RUNX2 generally increased with time in the groups where chondrogenic induction commenced at day 1 and day 3 post-encapsulation, with ∼5 fold and ∼15 fold at 21-d post-differentiation, respectively while in the remaining group, RUNX2 expression peaked at 14-d post-differentiation, with ∼8 fold of change (Fig. 5H right panel). Two-way ANOVA showed that RUNX2 expression was significantly affected both by the induction commencement time factor (P < 0.0001) and the time post-differentiation factor (P < 0.0001). 4. Discussion 4.1. hMSC-collagen microencapsulation recapitulates early events of mesenchymal condensation The current study demonstrates that microencapsulation of hMSCs mimics the mesenchymal cell condensation particularly by recapitulation of the early events such as volume reduction, cell density increase, expression of condensation markers, major chondrogenic and osteogenic transcription factors and ECM deposition. Apart from a time and cell density-dependent contraction and the increased local cell density of the cell-collagen microspheres, the key marker for mesenchymal condensation PNA showed persistent expression during the microencapsulation process. More importantly, unlike other existing in vitro models for early skeletogenesis, the current microencapsulation model seems to be more closely fitted with the ECM-driven cellular rearrangements that better mimics the mesenchymal condensation during development [38–40]. In particular, ECM components in early stages of skeletogenesis including collagen and fibronectin were present in the microencapsulation model. The importance of collagen for early skeletogenesis has been suggested long by knockout mice exhibiting lethal phenotype associated with severe skeletal defects, deficiencies in

4.2. Collagen microencapsulation stimulates simultaneous expression and nuclear localization of both chondrogenic and osteogenic transcription factors As an in vitro model mimicking mesenchymal condensation, collagen microencapsulation also potentiates hypertrophy and osteogenesis of hMSCs. Nuclear localization is critical for the activity of some transcription factors including SOX9 [62] and RUNX2 [63]. SOX9 is essential for early skeletogenesis as it is highly expressed in all osteochondroprogenitors and chondrocytes [13]. Inactivation of SOX9 during early development at the limb bud, either through missing expression of SOX9 in the undifferentiated mesenchymal cells or inactivating after mesenchymal condensation, completely aborted the formation of cartilage and bone [20], demonstrating the significance of SOX9. The nuclear localization of SOX9 is important as it plays a crucial 11

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cell fate [77]. Our in-house study showed that hMSCs upon collagen microencapsulation upregulated the key transcription factor of heat shock stress response (HSF-1) and a few other HSPs including HSP72 (Supplementary Information 4). Nevertheless, collagen microencapsulation only potentiates chondrogenesis as no real chondrogenesis was observed in the collagen microencapsulated structures, as shown by the declined SOX9 expression, absence of ACAN and COL2A1 deposition. The current collagen microencapsulation mimics the early stages of the endochondral ossification particularly the mesenchymal condensation that potentiates the subsequent chondrogenesis but presence of the right molecular signals are necessary for chondrogenesis to continue. We have verified that, by exposing the collagen microencapsulated hMSCs to chemically defined chondrogenic medium containing TGF-β3, the expression of SOX9 sustained, the chondrogenic ECM markers ACAN and COL2A1 are highly expressed and deposited in the extracellular space, demonstrating successful chondrogenesis and cartilage-like tissue formation. This work also demonstrated that the timing of supplementation of TGF-β3 is important. Another parameter influencing the outcome of chondrogenesis is cell density, corroborating with our previous in vitro [48] and in vivo [50] data. We found that high cell density favoured chondrogenesis and hypertrophy while low cell density favoured osteogenesis. For MSC differentiation, cell-cell interaction may become an important factor at high cell density, whereas cell-matrix interaction may become a critical factor at low cell density [78]. Previous studies demonstrated that type I collagen promotes osteogenesis of hMSCs [79] via integrin β1-mediated ERK activation [80]. In addition, low cell density allows cell spreading which favours osteogenesis [81]. Therefore, our type I collagen based microsphere at low cell density may enhance osteogenesis of MSCs. With high cell density, our contractible microsphere allows a close cell-cell interaction analogous to mesenchymal condensation. Proper spatiotemporal expression of cell adhesion molecules, N-cadherin and N-CAM is required for cellular condensation and thereby chondrogenesis [82,83].

roles on SOX9-dependent transcription for chondrogenic genes including collagen and aggrecan under cooperation to some coactivators such as p300, HADC and most recently, long non-coding RNA are also reported to involve an indirect regulation of SOX9 functions promoting the chondrogenesis [64–66]. RUNX2 is modulated by association with a variety of cofactors in the nuclear matrix [63] while its nuclear localization contributes to regulating the expression of bone-specific genes, including ALP [67] and osteocalcin [68]. In the current microencapsulation platform, encapsulated hMSCs showed simultaneous nuclear localization of SOX9 and RUNX2 for a short period of time postencapsulation as shown by the whitish colour of the co-localizing signals (Supplementary Information 2). This suggests that the encapsulated hMSCs may have the possibility to commit towards osteochondral progenitors upon microencapsulation. This is in line with the skeletogenesis where both SOX9 and RUNX2 are expressed in the condensed mesenchyme [69]. 4.3. Collagen microencapsulation potentiates but not induces chondrogenesis Comparing with the 2D controls, the microencapsulation process that entrapping MSCs in 3D collagen meshwork upregulated the SOX9 expression and stimulated activation of SOX9 by enhancing its nuclear localization. Collagen microencapsulation resulted in the activation of SOX9 through an enhanced but transient nuclear localization, as well as an increased binding to its target gene COL2A1 promoter and intron. This SOX9 activation may be due to a sudden change in the cytoskeletal organization and cell shape, from an elongated and spread morphology in 2D culture to a rounded or spherical morphology in 3D culture. Previous study restricting the cytoskeletal organization and cell shape of hMSCs on micropatterns with different areas, resulted in an upregulation and activation of SOX9 [70]. It is also noted that SOX9 nuclear localization was resulted from encapsulation of hMSCs in 3D agarose gel (Supplementary Information 3), further suggesting that it is the changes in cytoskeletal organization and cell shape, rather than the specific cell-matrix interactions that stimulates SOX9 activation. Moreover, MSC chondrogenesis can be regulated by varying the integrin ligand density and thereby cytoskeletal organization [71]. The ChIP analysis confirms that collagen microencapsulation potentiates chondrogenesis as enhanced nuclear localization of SOX9 in association and enhanced DNA-protein interactions between SOX9 and SOX9binding sites on the promoter and intron 1 of COL2A1 were found only in the 3D collagen microencapsulation group but not the monolayer culture on collagen-coated culture dish. This suggests that SOX9 and COL2A1-enhancer interaction might be induced via an alteration of epigenetic structures presumably leading to an ultimate transactivation of COL2A1 expression. In the 3D collagen encapsulation, an initial concentration of 2 mg/ml was used but the volume reduction during the cell condensation process was over 100 fold that could lead to a very high collagen density of 20 mg/mm3 [72], orders of magnitude higher than the 2D coating. It is therefore possible that significantly higher SOX9 expression and higher nuclear localization were resulted at very high matrix ligand density in the 3D model. The epigenetic mechanisms of the collagen microencapsulation-associated SOX9 activation and target gene enhancer interactions warrants further investigation but a recent study has demonstrated 3D encapsulation-induced hypoacetylation of SOX9 enhancing its penetration to the nucleus and binding to the enhancer sites of genes [73], unravelling the pivotal involvement of the epigenetic modification of 3D encapsulation. Furthermore, nuclear localization of SOX9 induced by mechanical stimuli can promote association of SOX9 with COL2A1 enhancer [74], probably through ROCK pathway [75,76]. Another possible explanation of the SOX9 activation could be the responses to the collagen microencapsulation associated with cellular stresses particularly HSP70. It has been reported that collagen conformation elicited cellular stresses may elevate HSP70, an interactive partner of SOX9, and hence associate with chondrogenesis

4.4. Collagen microencapsulation potentiates hypertrophy and osteogenesis From our data, encapsulation potentiates hypertrophy as both gene and protein expression of the hypertrophy markers RUNX2 and COL10A1 are upregulated significantly. However, hypertrophy morphology of differentiating hMSCs was not observed and therefore these cells should hold the potential to mature into hypertrophic chondrocytes. COL10A1 is predominantly associated with hypertrophic chondrocytes. However, studies reported COL10A1 is present in normal articular cartilage at the superficial zone [84] and is expressed by cultured articular chondrocytes [85]. The possibility that COL10A1 may play a role in the establishment of a hematopoietic niche at the chondro-osseous junction has been reported as demonstrated by the fact that mice deficient in COL10A1 resulted in hematopoietic defects [9]. Therefore, without supplementing any chondrogenic maturation signals or growth factors, the encapsulated MSCs might not readily differentiate into hypertrophic chondrocytes. From the osteogenic marker results, expression of RUNX2 and its nuclear activation as well as SP7 indicate that the microencapsulation potentiates osteogenesis. However, from the negative results of osteogenic markers COL1A1 and ALP, we speculate that the encapsulated MSCs did not readily differentiate into osteogenic lineage but held the potential to do so. This further indicates the potential of using the encapsulation model as an in vitro model mimicking early stage of mesenchymal condensation, which potentiates the subsequent commitment towards different lineages depending on the type of signals present. We further verify that when appropriate osteogenic differentiation signals present, the encapsulated MSCs readily differentiated into osteogenic lineage as shown by the positive expression of ALP and other genes as well as deposition of calcium (Supplementary Information 5). 12

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Conclusion [12]

The current study demonstrates that the microencapsulation recapitulated the early events of mesenchymal condensation, forming a structure mimicking that of the condensed mesenchyme. In addition, the microencapsulation process spontaneously and transiently upregulated and activated the key chondrogenic and osteogenic transcription factors SOX9 and RUNX2 and hold the potential to commit towards both the chondrogenic and the osteogenic lineages as shown by the upregulated markers. Nevertheless, without supplementation of appropriate inductive signals for chondrogenesis and osteogenesis, the encapsulated MSCs did not differentiate and mature into chondrogenic and osteogenic lineages, respectively. Successful chondrogenesis with sustained SOX9 expression, upregulated ACAN expression and deposited COL2A1 were only noted after supplementing chondrogenic inductive medium. Moreover, both the time to expose to the inductive signals and the cell density are important factors influencing the outcomes of chondrogenesis. This in vitro model can be used to study cellular and molecular interactions and functional evaluation of genes important for mesenchymal condensation and subsequent chondrogenesis and osteogenesis during endochondral ossification and early skeletogenesis, contributing to rationalizing development-inspired tissue engineering.

[13]

[14]

[15]

[16] [17] [18]

[19] [20]

[21]

Conflicts of interest BPC holds patents related to this work.

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Acknowledgments

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The authors thank Mr. MA Lam for his assistance in the osteogenesis experiments, the Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White for providing the hMSCs, and funding support from the Research Grant Council (GRF 17100714 & 17164116) of the Hong Kong Special Adminstrative Region and the University Research Committee (Seed funding for basic science 104004595) of the University of Hong Kong.

[24]

[25]

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Appendix A. Supplementary data

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Supplementary data to this chapter can be found online at https:// doi.org/10.1016/j.biomaterials.2019.05.021.

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