Effects of Hydroxyapatite and Hypoxia on Chondrogenesis and

Article pubs.acs.org/journal/abseba

Effects of Hydroxyapatite and Hypoxia on Chondrogenesis and Hypertrophy in 3D Bioprinted ADMSC Laden Constructs Ying Wang,†,‡ Shaohua Wu,†,‡ Mitchell A. Kuss,†,‡ Philipp N. Streubel,§ and Bin Duan*,†,‡,∥ †

Mary & Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, South 42nd Street & Emile Street, Omaha, Nebraska 68198, United States ‡ Division of Cardiology, Department of Internal Medicine, University of Nebraska Medical Center, South 42nd Street & Emile Street, Omaha, Nebraska 68198, United States § Department of Orthopedic Surgery and Rehabilitation, University of Nebraska Medical Center, South 42nd Street & Emile Street, Omaha, Nebraska 68198, United States ∥ Department of Surgery, College of Medicine, University of Nebraska Medical Center, South 42nd Street & Emile Street, Omaha, Nebraska 68198, United States ABSTRACT: Hydrogel-based cartilage tissue engineering strategies require the induction and long-term maintenance of adipose derived mesenchymal stem cells (ADMSC) into a stable chondrogenic phenotype. However, ADMSC exhibit the tendency to undergo hypertrophic differentiation, rather than forming permanent hyaline cartilage phenotype changes. This may hinder their implementation in articular cartilage regeneration, but may allow the possibility for bone and bone to soft tissue interface repair. In this study, we examined the effects of hydroxyapatite (HAp) on the chondrogenesis and hypertrophy of ADMSC within bioprinted hyaluronic acid (HA)-based hydrogels. We found that a small amount of HAp (∼10% of polymer concentration) promoted both chondrogenic and hypertrophic differentiation of ADMSC. Increased HAp contents promoted hypertrophic conversion and early osteogenic differentiation of encapsulated ADMSC. Subsequently, ADMSC-laden, stratified constructs with nonmineralized and mineralized layers (i.e., HA based and HA-HAp based) were 3D bioprinted. The constructs were conditioned in chondrogenic medium in either a normoxic or hypoxic environment for 8 weeks to assess the effects of oxygen tension on ADMSC differentiation and interface integration. We further implanted the bioprinted constructs subcutaneously into nude mice for 4 weeks. It was found that hypoxia partially inhibited hypertrophic differentiation by significantly down-regulating the expression of COL10A1, ALP, and MMP13. In addition, hypoxia also suppressed spontaneous calcification of ADMSC and promoted interface integration. This study demonstrates that both HAp content and hypoxia are important to mediate chondrogenesis, hypertrophy, and endochondral ossification of ADMSC. An optimized recipe and condition will allow for 3D bioprinting of multizonal grafts with integrated hard tissue and soft tissue interfaces for the treatment of complex orthopedic defects. KEYWORDS: heterogeneous structure, osteochondral, fibrocartilage, bioinks, tissue engineering

1. INTRODUCTION

lineages in the same construct to mimic a bone-to-tendon/ ligament or bone-to-cartilage interface. For example, the rotator cuff tendons attach to bone via fibrocartilaginous insertions, which have four typical zones, i.e., tendon, nonmineralized fibrocartilage, mineralized fibrocartilage, and bone.10,11 The complexity of the transition between mineralized and unmineralized tissues requires heterogeneous differentiation of ADMSC, posing a significant challenge for effective interface regeneration.12 In particular, the damaged rotator cuff tissue tends to form a fibrovascular scar rather than a fibrocartilage transition in the bone−tendon interface.13,14 Therefore, it is of

Adipose derived mesenchymal stem cells (ADMSC) are considered to be an attractive cell source in clinical applications due to their high availability, high proliferation capacity, and multipotency.1−4 Although the osteogenic and chondrogenic differentiation of ADMSC have been well established and widely used, the fate of ADMSC in three-dimensional (3D) cultures, such as the hydrogel-based matrix and micromass culture systems, is not completely predictable or controllable. For example, under chondrogenic induction, ADMSC exhibit the tendency to undergo hypertrophic differentiation, rather than forming permanent hyaline cartilage phenotype changes.5−7 Consequently, the neocartilage matrix undergoes endochondral ossification after implantation.8,9 Another challenge is to regulate ADMSC differentiation toward multiple © XXXX American Chemical Society

Received: February 11, 2017 Accepted: March 20, 2017 Published: March 20, 2017 A

DOI: 10.1021/acsbiomaterials.7b00101 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

dissolved in phosphate buffered saline (PBS) or cell culture medium with cell suspension (6 × 106 cells/mL) and 0.05% w/v 2-hydroxy1(4-(hydroxyethox)pheny)-2-methyl-1-propanone (Irgacure 2959; CIBA Chemicals). For HA-HAp hydrogels, HAp with different concentrations (0%, 10%, 50%, and 100% of polymer weight, and denoted as HA-HAp0, 10, 50, 100, respectively) were added and mixed with the hydrogel. Gel precursors were loaded into the 3D bioprinter (3D Bioplotter, EnvisionTEC, Germany), extruded based on the disc shaped design (8 mm × 1 mm), and subsequently exposed to an OmniCure S2000 UV lamp (Lumen Dynamics, 0.45 Mw/cm2) for 80 s at room temperature. The cell-hydrogel constructs were washed with PBS once for 1 min and maintained at 37 °C and 5% CO2 2.2. 3D Bioprinting. HA based constructs were 3D bioprinted using the 3D Bioplotter (EnvisionTEC, Germany). The precursors with or without cells were loaded in the deposition syringes (Nordson EFD) with a 18G tip. A pressure of 1.8 bar was applied to the syringe, and a deposition speed of 3.5−4 mm/s was used. The printings were conducted at room temperature. The disc shaped design was used and printed to evaluate the effects of HAp concentration on ADMSC differentiation. For zonal interface bioprinting, dual syringes were applied. The layer with HAp was first printed using a HA-HAp50 hydrogel and the construct exposed to UV light for 80 s for crosslinking. Then, the HA layer without HAp was printed onto the HAHAp50 hydrogel layer using HA-HAp0 hydrogel. The size is 8 mm × 1 mm for each layer. The construct was further cross-linked for another 80 s. 2.3. Cell and Cell-Laden Construct Culture. ADMSC were obtained from Lonza and cultured in stem cell growth medium containing DMEM/F12 medium (Invitrogen), 10% FBS, and 1% P/S. Cells were used at passages 3 to 6. For chondrogenic differentiation, the ADMSC-laden bioprinted constructs were conditioned in chondrogenic differentiation medium consisting of growth medium with 100 nM dexamethasone, 0.2 mM ascorbic acid, 1 mM sodium pyruvate (Sigma), 1× insulin-transferrin-selenite (ITS+Premix, BD Biosciences), and 10 ng/mL transforming growth factor β1 (Sigma).30 The constructs were cultured for 56 days before characterization or further in vivo implantation. 2.4. Mechanical Properties Test. Uniaxial compressive tests for each hydrogel, with and without cells, were performed at day 56 using an MTS machine (BOSE ElectroForce 3200) at a loading speed of 0.6 mm/min. The bulk compressive moduli were calculated from the slope of the initial linear region (10%−15% strain for compressive test) of the respective stress−strain curves. 2.5. Biochemical Analysis. A dimethylmethylene blue (DMMB) assay was performed to measure the sulfated glycosaminoglycan (sGAG) production in the hydrogels after 28 and 58 days of culture.29 The constructs (n = 5) were digested with 300 μg/mL papain in 50 mM phosphate buffer (pH 6.5), containing 5 mM cysteine and 5 mM EDTA for 16 h at 60 °C. sGAG concentration was calculated by calibrating against a standard curve obtained with shark chondroitin sulfate (Sigma). The total collagen content was determined using a hydroxyproline assay.30 Samples were hydrolyzed with 4.8 N HCl. Dried hydrolyzates were treated with chloramine-T reagent for oxidation. Ehrlich’s aldehyde reagent was reacted with samples at 65 °C for 20 min in order to generate chromospheres. The amount of hydroxyproline was measured with a microplate reader at 550 nm (n = 5). 2.6. RNA Isolation and Quantitative Real Time Polymerase Chain Reaction (PCR). Total RNA was extracted from cell-laden constructs using QIA-Shredder and RNeasy mini-kits (QIAgen) according to the manufacturers’ instructions. Total RNA was synthesized into first strand cDNA in a 20 μL reaction using an iScript cDNA synthesis kit (BioRad Laboratories). Real-time PCR analysis was performed in a StepOnePlus Real-Time PCR System (Thermo Scientific) using SsoAdvanced SYBR Green Supermix (BioRad). cDNA samples were analyzed for the gene of interest and for the housekeeping gene 18S rRNA. The level of expression of each target gene was calculated using the comparative Ct (2−ΔΔCt) method. 2.7. In-Vivo Subcutaneous Implantation. Eight-week old female athymic nude mice were purchased from Jackson Laboratory.

significant relevance to replicate the microenvironment for musculoskeletal tissue development and maturation, and to precisely control ADMSC differentiation. Various biomaterials and conditions have been studied to encapsulate and induce chondrogenic and osteogenic differentiation of ADMSC. Hyaluronic acid (HA) is one of the most promising matrices to support the chondrogenesis of ADMSC. Three-dimensional (3D) porous HA-based scaffolds have been reported to have better proliferation and chondrogenic differentiation of ADMSC compared to those of micromass cultures.15 Increased cross-linking-density HA-based hydrogels have been shown to decrease cartilage matrix content and promote hypertrophic differentiation.9,16 The addition of hydroxyapatite (HAp) promotes osteogenic differentiation of MSC, which has led to the implementation of HA-HAp based scaffolds for osteochondral regeneration.17 However, it is unclear as to how HAp affects differentiation of ADMSC into mineralized fibrocartilage and hypertrophy during chondrogenesis. A hypoxic environment or low oxygen tension (normally ≤5%) is known to replicate the physiological avascular microenvironment that occurs during musculoskeletal tissue development.18 Hypoxia has been shown to promote the chondrogenesis of ADMSC and suppress hypertrophy via factors like hypoxia-inducible transcription factor 1 alpha (HIF1α).5,7,19,20 The hypoxic condition has to date not been widely adopted in musculoskeletal engineering applications, and it is unclear how hypoxia affects interface regeneration between mineralized and unmineralized tissues. 3D bioprinting has been widely used in musculoskeletal regeneration, especially for bone, cartilage, and osteochondral tissues.21−23 This technology enables the precise design and control of the architecture of scaffolds, while allowing for the incorporation of living cells and bioactive factors.24,25 In addition, 3D bioprinting supports multiple biomaterials with multiple cell types and allows the fabrication of heterogeneous constructs with zonal structure21 or multiple tissue components.26 Although constructs with mineralized and unmineralized interfaces, like osteochondral, have been successfully bioprinted with the incorporation of MSC,27,28 the interface itself is not well characterized, and its function has not yet been established. In the present study, we encapsulated human ADMSC within bioprintable HA-based hydrogels consisting of various amounts of HAp and determined how hypertrophy and chondrogenesis of ADMSC were influenced. Subsequently, we 3D bioprinted ADMSC-laden, stratified constructs with two zonal structures (i.e., HA based and HA-HAp based). The constructs were conditioned in chondrogenic medium in either normoxic or hypoxic environments for 8 weeks to assess the effects of oxygen tension on ADMSC differentiation and interface integration. We further implanted the bioprinted constructs subcutaneously into nude mice after an eight week in vitro culture to determine in vivo chondrogenesis, hypertrophy, and matrix mineralization.

2. MATERIALS AND METHODS 2.1. Polymer Modification and Hydrogel Preparation. Photocross-linkable HA (NovaMatrix, ∼1200 kDa) and gelatin (Gel, from bovine skin, Sigma) were synthesized as previously reported through the reaction of methacrylic anhydride (Sigma) with 0.5% HA or 10% Gel in deionized water.29 For hydrogel preparation, methacrylated HA (Me-HA, 5%w/v) and methacrylated Gel (Me-Gel, 2%w/v) were B


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Figure 1. Fabrication and mechanical characterization of acellular HA/Gel based hydrogels with varying HAp concentration. (A) Gross view of fabricated HA/Gel based hydrogels after photo-cross-linking (from left to right: HA-HAp0, 10, 50, 100); (B) representative load−displacement curves; (C) stiffness; and (D) compressive strength with varying HAp content.

Figure 2. Dynamic changes of total collagen content (A), sGAG content (B), and biomechanics (C and D) of HA/Gel based hydrogels with varying HAp contents after in vitro culture. (C) Representative load−displacement curves and (D) stiffness of different ADMSC-laden hydrogels after 56 days of culture. (n = 5−6; bars that do not share letters are significantly different from each other; ** indicates p < 0.01; # indicates p < 0.05 compared to HA-HAp0 and 100 at day 56.) and six mice in total). For the surgery, the mice were deeply anesthetized, and one dorsal incision was made lateral to the spine. Two subcutaneous pockets were made from this single incision, one on each side of the mouse, using blunt dissection. The scaffold implants were inserted into the subcutaneous pockets. The skin incision was closed with staples, and mice were monitored closely to

Mice were housed in compliance with the University of Nebraska Medical Center (UNMC) guidelines. All surgical procedures were reviewed and approved by the UNMC Animal Care and Use Committee. The cell laden constructs (with zonal structure) were conditioned in normoxic or hypoxic environments in chondrogenic medium for 56 days, and then, six samples of each group were subcutaneously implanted in the nude mice (two samples per mouse C


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Figure 3. Histological and immunohistochemical staining of ADMSC-laden hydrogels with varying HAp contents after 56 days of culture. (A) Alcian blue staining for sGAG; (B) ALP; (C) Col I; (D) Col II; and (E) Col X. (Scale bar: 0.5 mm for A and C−E; 1 mm for B). ensure full recovery from anesthesia. All animals were sacrificed 4 weeks after implantation. 2.8. Histological and Immunohistochemical Staining. Alkaline phosphatase (ALP) staining was performed after 56 days of in vitro culture using the alkaline phosphatase leukocyte kit (Sigma) according to the manufacturer’s protocol. After in vitro and in vivo culture, the bioprinted constructs were fixed, paraffin embedded, sectioned (5−8 μm), and stained for negatively charged sGAG using Alcian blue. For immunohistochemical staining of Col I, II, and X, the Vectastain ABC kit and the DAB Substrate kit for peroxidase (Vector Laboratories) were used. Briefly, the sections were deparaffinized, blocked by using 1% BSA overnight, and incubated with primary antibodies overnight at dilutions of 1:1000, 1:100, and 1:1000 for collagen I (Col I, mouse monoclonal to Col I, abcam), collagen II (Col II, rabbit polyclonal to Col II, abcam), and collagen X (Col X, mouse monoclonal to Col X, abcam) antibodies, respectively. All procedures followed the manufacturers’ protocols. The slides were imaged under a Zeiss Discovery v8 stereo microscope (Zeiss) . 2.9. Statistical Analysis. All quantitative data were expressed as the mean ± standard deviation (SD). Pairwise comparisons between groups were conducted using ANOVA with Scheffé posthoc tests. A p value of 0.05) (Figure 1D). 3.2. Effects of HAp on ECM Content and Compressive Property Changes. We encapsulated ADMSC within various hydrogels and conditioned the constructs in chondrogenic medium for up to 56 days. We determined the dynamic changes of sGAG and collagen contents at 28 and 56 days of culture (Figure 2A,B). At day 28, there was no statistically significant difference for GAG and collagen content among hydrogel groups. With increasing culture time, total GAG and collagen content significantly increased for HA-HAp0, 10 and 50, but not for HA-HAp100. Interestingly, HA-HAp10 the showed highest sGAG and collagen production. Figure 2C,D shows the compressive mechanical properties after 56 days of culture. Compared to acellular hydrogels, the compressive moduli for all three groups significantly increased (p < 0.05), indicating cellular remodeling and strengthening. The HA-

3. RESULTS 3.1. Characterization of HA Based Hydrogels with HAp Particles. Figure 1 displays the gross morphologies of various acellular HA-HAp hydrogels and their compressive, D


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Figure 4. Real-time PCR analysis of chondrogenic (upper panel) and hypertrophic (lower panel) biomarkers of ADMSC-laden hydrogels with varying HAp contents after 56 days of culture. Relative gene expression is presented as normalized to 18S and expressed relative to HA-HAp0. (n = 3; bars that do not share letters are significantly different from each other.)

Figure 5. Three-dimensional bioprinted, stratified constructs with zonal structures and comparative ECM contents and gene expressions. (A) Gross view of 3D bioprinted hydrogel with zonal structure (upper layer, HA-HAp0; bottom layer, HA-HAp50); quantitative analysis of collagen (B) and sGAG (C) content of ADMSC laden stratified constructs after conditioning in normoxia and hypoxia for 56 days (n = 5; ** indicated p < 0.01); (D) real-time PCR analysis. Relative gene expression is presented as normalized to 18S and expressed relative to constructs in a normoxic environment. (n = 3; * indicates p < 0.05; ** indicates p < 0.01.)

E


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Figure 6. (A) Images of the harvested 3D bioprinted ADMSC-laden hydrogel implants 4-week after subcutaneous in vivo implantation following 8week in vitro culture in normoxia or hypoxia and (B−F) histological or immunohistochemical staining of Alcian blue (B), von Kossa (C), Col I (D), Col II (E), and Col X (F) for the explants. (Scale bar, 1 mm for B−F; 0.5 mm for the inset in B).

HAp0 group had the highest compressive modulus (21.7 ± 5.5 kPa), while there was no statistical difference among the groups with HAp (12.2 ± 2.7 kPa for HA-HAp10; 11.0 ± 2.3 kPa for HA-HAp50; and 10.9 ± 2.8 kPa for HA-HAp100). 3.3. Effects of HAp on Chondrogenic and Hypertrophic Differentiation of ADMSC. Figure 3 depicts various staining images for the assessment of chondrogenic and hypertrophic differentiation of ADMSC in hydrogels with different HAp concentrations. After 8 weeks in culture, sGAG was expressed more intensely in HA based hydrogels compared to that of HA hydrogels with HAp as shown by Alcian blue staining (Figure 3A). In addition, the encapsulated ADMSC showed more spreading morphology and larger lacunae. ALP staining showed that without HAp, HA based hydrogels expressed very limited ALP, whereas the ALP expression was increased with increasing HAp concentrations (Figure 3B), indicating spontaneous osteogenic differentiation of ADMSC, even in chondrogenic medium condition. IHC staining showed that HA-HAp10 had a high intensity of Col I and Col II expression (Figure 3C,D). Staining against Col X increased with increasing HAp content (Figure 3E). We further examined and compared the gene expression of encapsulated ADMSC in four different hydrogels after 56 days of chondrogenic induction. All of the chondrogenic related gene expressions (i.e., Col II, Sox9, and aggrecan) were significantly upregulated in the HA-HAp10 group (Figure 4). However, with increasing HAp content, Col II, Sox9, and aggrecan expressions gradually decreased. ADMSC in HAHAp100 hydrogels showed the lowest expression. In contrast, hypertrophic and osteogenic related gene expressions (Col X, Runx2, and ALP) were significantly upregulated with increasing HAp content. HA-HAp100 group showed the highest expression. These results were consistent with the results of histological staining. 3.4. Three-Dimensional Bioprinting of Stratified Constructs with Zonal Structures. We 3D printed stratified constructs with two zones by using the HA-HAp0 hydrogel for the top layer and the HA-HAp50 hydrogel for the bottom layer (Figure 5A). These two zones were clearly identified by their

colors, and they were integrated together by the photo-crosslinking process without delamination. We further bioprinted the constructs with the encapsulation of ADMSC and conditioned the constructs in either a normoxic (5% CO2 and 21% O2) or hypoxic (5% CO2 and 5% O2) environment in chondrogenic medium for 56 days. Hypoxia decreased the total collagen content in the constructs, whereas oxygen tension had limited effects on GAG deposition (Figure 5 B,C). For gene expression, the hypoxic environment did not affect Col II and Sox9 expression but significantly upregulated aggrecan expression (Figure 5D). For hypertrophic and osteogenic differentiation related gene expression, Col I and ALP were greatly downregulated in the hypoxia group. However, there was no significant difference in the expression level of Col X and Runx2 between the normoxia and hypoxia groups. Hypoxia also significantly upregulated the expression of HIF1α and MMP13, which are late markers of hypertrophic chondrocytes, whereas hypoxia had no statistical effect on MMP2 expression, which is an enzyme nominally expressed in chondrocyte constructs. 3.5. In Vivo Effects of Hypoxic Conditioning on ADMSC Hypertrophy in Stratified Constructs. We evaluated the effects of oxygen tension on the chondrogenesis and hypertrophy of ADMSC within bioprinted, stratified constructs in vivo. We conditioned the constructs in a normoxic or hypoxic environment for 8 weeks, then subcutaneously implanted the constructs into nude mice for an additional 4 weeks. All of the explanted constructs maintained the intact structures with two bioprinted layers (Figure 6A). However, the gross view and histological staining images showed that the two bioprinted layers were partially separated by the infiltration of host cells/tissues (Figure 6). Alcian blue staining showed that both the normoxia and the hypoxia groups were positive for sGAG, exhibiting similar intensity. The separation distance for constructs in the hypoxic condition was smaller in comparison to their counterparts in the normoxic condition (Figure 6B). The top layers, without HAp, in both constructs were negative to von Kossa staining, whereas the bottom layers, with HAp, were positive, indicating the mineralization of the F


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dynamically changed by secreting new collagen and sGAG and digesting existing ones. Such dynamic remodeling resulted in a significant increase of compressive moduli by up to 2-fold over 8-week culture. Our results indicate that we can control matrix contents and biomechanics to mediate the chondrogenic, fibrochondrogenic, and hypertrophic differentiation of ADMSC. Low oxygen tension is generally believed to mimic the physiological conditions within cartilage tissue and promote the chondrogenic potential of MSC and chondrocytes.19,47 Furthermore, hypoxia has also been shown to promote chondrogenesis and suppress the hypertrophy of MSC by increasing AKT and p38 phosphorylation, along with the subsequent stabilization of HIF-1α48−50 However, a recent study showed that hypoxia substantially enhanced MSC hypertrophy, leading to elevated tissue calcification in the hydrogels with high HA concentration.51 In addition, hypoxia was also reported to promote Col X production by upregulating p38 MAPK signaling.52 These indicate that the effects of hypoxia are dependent on microenvironments. It is also unclear how hypoxia affects MSC differentiation at interfaces with and without mineral components. In this study, we implemented a 3D bioprinting technique to generate a dual-layered construct with controlled HAp concentration. This model allows us to examine the influence of HAp and hypoxia on ADMSC chondrogenesis and hypertrophy in a heterogeneous construct with a defined in vitro setting and a physiological relevant microenvironment. In our study, we found that hypoxia decreased the total collagen content but had only minor effects on GAG deposition, which is consistent with another study reported by Meretoja et al.19 Real time PCR results showed that hypoxia significantly downregulated Col I expression and had no effects on Col II and X expression. In addition, hypoxia suppressed some gene expression of hypertrophic markers (like ALP, MMP13, but not Runx2 and Col X) and promoted maturation of chondrogenic differentiation of ADMSC. These results indicate that hypoxia can partially suppress hypertrophy and enhance chondrogenesis. In comparison, Zhu et al. reported that hypoxia substantially enhanced MSC hypertrophy within HA based hydrogels with a high concentration of HA (5% w/v).51 One possible explanation is that the addition of a small amount of gel might balance the cell adhesion and cell-matrix crosstalk for ADMSC and promote chondrogenesis.53,54 Our results showed that hypoxia also suppressed the hypertrophy of ADMSC within the 3D bioprinted zonal constructs with HAp by decreasing Col I and X expression. In both normoxic and hypoxic environments, calcification was only localized in the zonal region with HAp after 28-day in vivo subcutaneous implantation. Several studies demonstrated that HA hydrogels supported spontaneous calcification of MSC, even in hypoxia.9,51 Our results proved that our hydrogel recipe in the nonmineralized zone might also suppress tissue calcification in vivo. In addition, hypoxia significantly reduced the calcification of HAp incorporating region. O’Reilly et al. developed a computational model and demonstrated that a sustained hypoxic environment limited angiogenesis to the osseous region of an osteochondral defect and improved cartilage repair.55 Our results confirmed that hypoxia may be beneficial for the hard tissue and soft tissue interface regeneration by maintaining the chondrogenesis of the cartilage region and promoting interface integration. However, similar to chitosan constructs, as reported by Sheehy et al.,56 the current

hypertrophy of the chondrogenically induced ADMSC (Figure 6C). The hypoxia group showed less intense von Kossa staining, indicating less calcium phosphate deposition in comparison to that in the normoxic group.31 In addition, the hypoxia group had a lower intensity of Col I and Col X expression and a higher intensity of Col II expression, compared to the normoxia group (Figure 6D−F). The top layer (HA-HAp0) expressed more Col II than the bottom layer (HA-HAp50) (Figure 6E). For Col I and Col X expression, there was no obvious interface between the top layer and bottom layer (Figure 6D and F).

4. DISCUSSION Hydrogel based cartilage tissue engineering strategies require induction and long-term maintenance of ADMSC into a stable chondrogenic phenotype. Unfortunately, ADMSC tend to undergo hypertrophic differentiation, which is characterized by cell volume increase, upregulation of Runx2 and ALP, and more secretion of Col X and MMP13.32,33 On the other hand, the hypertrophic, cartilaginous grafts promote the transition from engineered hypertrophic cartilage into bone by replicating the endochondral bone formation at an early stage of development34,35 and have been used to treat large bone defects.36,37 Therefore, it is of significant importance to control the microenvironment in order to regulate cell-material interactions and thus mediate the ADMSC fate. The objective of this study is to unveil the effects of HAp and hypoxia on chondrogenic and hypertrophic differentiation of ADMSC within bioprinted HA based hydrogels. Additionally, constructs with a zonal structure, consisting of HAp containing and nonHAp containing regions, were 3D bioprinted to mimic the softto-hard tissue interface and to investigate the heterogeneous differentiation of ADMSC. Both HA and Gel have been used to encapsulate ADMSC for cartilage tissue engineering.38,39 Levett et al. demonstrated that Me-Gel alone did not support complete chondrogenesis in vitro.40 With the addition of Me-HA, the hydrogels provided a more supportive environment for the maintenance of the chondrocyte phenotype. We implemented the Me-HA/Me-Gel hydrogel with the addition of HAp for the encapsulation of ADMSC. In contrast to some studies that showed HAp stiffened hydrogels,41 our results showed that the addition of HAp decreased hydrogel stiffness. This is probably because the presence of HAp particles creates material voids and interferes with the photo-cross-link efficiency, rather than playing a load bearing role. These results have been replicated elsewhere.42 Interestingly, we found that the addition of a small amount of HAp significantly promoted collagen and sGAG deposition, along with chondrogenic differentiation of ADMSC. However, the induced ADMSC showed a more fibrochondrogenic phenotype, with more Col I expression, compared to constructs without HAp. The fibrochondrogenic differentiation of ADMSC is probably due to the synergistic effects of the incorporation of HAp with Me-Gel, along with decreased mechanical properties.43,44 With increasing HAp concentration, the encapsulated ADMSC showed more of the hypertrophic phenotype, with increasing expression of Col X, Runx2, and ALP, and stronger Col X staining. ADMSC within HA-HAp100 hydrogels expressed the highest hypertrophic and early osteogenic differentiation biomarkers. Previous studies focused more on the effects of initial stiffness on chondrogenic and hypertrophic differentiation of MSC.45,46 Our results showed that the stiffness of the ADMSC laden constructs was G


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HA based hydrogel is probably not an ideal choice for endochondral ossification, due to the lack of spontaneous calcification capacity. 3D bioprinting enables the implementation of multiple biomaterials and cell types to fabricate heterogeneous architectures within the same constructs.57,58 On the basis of this concept, the osteochondral constructs with cartilage and bone layers,59 cartilage constructs with a zonal structure,60 and tendon-to-muscle interfaces61 have been bioprinted. A better understanding of the regulation of MSC chondrogenesis, hypertrophy, and ossification is essential to ensure the heterogeneous differentiation of MSC, in order to promote the interface regeneration. We demonstrated that both HAp concentration and hypoxia mediated the differentiation of MSC. A small amount of HAp might induce fibrocartilage and/ or calcified fibrocartilage formation in the interface, like tendon/ligament-to-bone, while a large amount of HAp promoted endochondral ossification. For hypoxia, the optimal timing, duration, and exposure pattern should be further determined to maximize the interface integration. One limitation of this study is that we bioprinted and photo-crosslinked the mineralized zone first and then conducted nonmineralized zone printing and photo-cross-linking, rather than in situ cross-linking during the whole printing process. This is probably the main reason why the interface was not closely integrated and had infiltration of host tissues. Another limitation is that the in vivo cartilage formation and hypertrophic calcification of bioprinted constructs were conducted in the subcutaneous space of the nude mice. This animal model is useful to preliminarily assess the in vivo response of the different hydrogel zones and the interface in the same bioprinted constructs. Other clinically relevant models, like the articular joint surface in large animals and autologous MSC, will be more powerful to assess chondrogenesis and hypertrophic calcification.

Bin Duan: 0000-0002-5647-3793 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Mary & Dick Holland Regenerative Medicine Program start-up grant, Mary & Dick Holland Regenerative Medicine Program Cartilage Tissue Engineering and Regeneration pilot research grant, and Nebraska Research Initiative funding. We thank Dr. Mehrdad Negahban and Mr. Wenlong Li for the assistance of mechanical property testing. We thank Janice A. Taylor and James R. Talaska of the Advanced Microscopy Core Facility at the University of Nebraska Medical Center (UNMC) for providing assistance with confocal microscopy. Support for the UNMC Advanced Microscopy Core Facility was provided by the Nebraska Research Initiative, the Fred and Pamela Buffett Cancer Center Support Grant (P30CA036727), and an Institutional Development Award (IDeA) from the NIGMS of the NIH (P30GM106397).

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ABBREVIATIONS ADMSC, adipose derived mesenchymal stem cells; HA, hyaluronic acid; HAp, hydroxyapatite; HIF-1α, hypoxiainducible transcription factor 1 alpha; Gel, gelatin; Me-HA, methacrylated hyaluronic acid; Me-Gel, methacrylated gelatin; DMMB, dimethylmethylene blue; sGAG, sulfated glycosaminoglycan; ALP, alkaline phosphatase; Col I, collagen I; Col II, collagen II; Col X, collagen X; ECM, extracellular matrix; Runx2, runt related transcription factor 2; MMP, matrix metalloproteinase

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5. CONCLUSIONS In summary, this work examined the effects of HAp on chondrogenesis and hypertrophy of ADMSC within bioprinted HA/Gel based hydrogels. We found that a small amount of HAp in the hydrogel promoted both chondrogenic and hypertrophic differentiation of ADMSC. Increased HAp contents promoted hypertrophic conversion and early osteogenic differentiation of encapsulated ADMSC. The increased hypertrophy of ADMSC in HA/Gel bioinks was an outcome of combinatorial effects of HAp concentration, dynamically altered biomechanics, and ECM remodeling. We further 3D bioprinted zonal constructs with mineralized and nonmineralized layers to determine the effects of hypoxia on ADMSC differentiation in different zones in vitro and in vivo. Hypoxia partially suppressed hypertrophic differentiation and calcification of ADMSC, and promoted interface integration. The results of this work will be important in the design and optimization of hydrogels and culture conditions to mediate chondrogenesis, hypertrophy, and endochondral ossification of ADMSC, and to 3D bioprint multizonal grafts with integrated hard-tissue and soft-tissue interfaces for the treatment of complex orthopedic defects.

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Effects of Hydroxyapatite and Hypoxia on Chondrogenesis and

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