Injectable Stem Cell Laden Open Porous Microgels That Favor

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Injectable stem cell-laden open porous microgels that favour adipogenesis: in vitro and in vivo evaluation Pengfei Xia, Kunxi Zhang, Yan Gong, Guifei Li, Shifeng Yan, and Jingbo Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13065 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 23, 2017

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ACS Applied Materials & Interfaces

Injectable stem cell-laden open porous microgels that favour adipogenesis: in vitro and in vivo evaluation Pengfei Xia a, Kunxi Zhang a, Yan Gong a, Guifei Li a, Shifeng Yan a, Jingbo Yin a, * a

Department of Polymer Materials,Shanghai University, 99 Shangda Road, Shanghai

200444, PR China. Email: [email protected] Key Words: open porous microgels, spheroidal stem cell shape, high stem cell viability, favour adipogenic differentiation, adipose tissue engineering Abstract: Microgels, with large surface area per volume, show great advantages in adipose tissue engineering due to their injectability and similarity with natural extracellular matrix. However, to date, no studies have tried applying microgels to adipose tissue regeneration. Herein, based on double bonded poly-(L-glutamic acid)-g-2-Hydroxyethyl

methacrylate

(PLGA-g-HEMA)

and

maleic

anhydride-modified chitosan (MCS), an open porous microgel with high hydrophilicity and great injectability is successfully prepared (microgels diameter of 200-300 µm, pore diameter of 38 µm, and porosity of 88.3%). The storage modulus of 30 mg/ml of microgel dispersions is 2000 Pa, which is similar to that of the native adipose tissue. The spheroidal stem cell shape and extensive cell-cell connections can be formed in the present microgels to promote adipogenic differentiation and realize adipose tissue regeneration. After injection in vitro, the microgels can maintain high stem cell viability up to 14 days. The extensive Oil Red O staining is observed after adipogenic induction for 14 days. After 12 weeks post-implantation, adipose tissues can be regenerated well. Blood vessels are formed in the neo-generated tissues. The degradation rate of microgels roughly matches with the adipose tissue formation rate. The study offers an applicable microgel system to boost the adipose tissue regeneration. 1. Introduction Repair of facial adipose tissue defects resulting from trauma has become a very essential rehabilitation process for patients.1-3 In clinical practice, materials such as 1 ACS Paragon Plus Environment


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collagen and hyaluronic acid are usually used to fill adipose tissue defects, while the rapid absorption and the inflammatory reaction limit their application.4 Autologous adipose tissue transplantation is efficient, however, maintaining the long-term functionality of the transplanted adipose tissue is a challenge.5 Adipose tissue engineering shows a great potential towards the repair of damaged adipose tissue.1-3 Scaffolds such as sponges, foams, and injectable hydrogels are widely applied in the adipose tissue regeneration.3,

6-7

When compared to the sponges and foams, the

injectable hydrogels exhibit an advantage in repairing irregularly shaped tissue defects.8 What’s more, their injectability can make them injected with minimum invasiveness.9 However, there are two disadvantages existed in the current injectable hydrogels. Firstly, the cellular viability is difficult to maintain in the interior of hydrogels. It is reported that the nutrients diffusion is greatly affected by the gel depth. 10

The poor nutrients diffusion leads to the low cell viability. Secondly, the majority

of cell-hydrogel interaction is encapsulation effect, which blocks the cell-cell connections, thus causing an obstacle in tissue regeneration.11, 12 When compared to the injectable hydrogels, microscale hydrogels (microgels) with better nutrients diffusion can address the first disadvantage to maintain high cell viability.10 Microgel beads have attracted extensive research interest due to their well-defined shapes and large surface area per volume.13, 14 At present, microgels have been widely used in artificial enzyme, drug delivery, and microreactor.15-17 The applications of microgels in tissue engineering are mainly focused on cell culture and the repair of limb ischemia model.18-20 While microgels used in tissue regeneration are rarely reported. On the one hand, materials used for preparing the microgels are natural materials such as hyaluronic acid and synthetic materials such as poly-(ethylene glycol).14,

21

However, natural materials usually possess an

inflammatory reaction, while synthetic materials show its disadvantage in degradation. On the other hand, most reported cell-laden microgels are nonporous and homogeneous gel beads.18,

22-23

The encapsulation effect still exist in the current

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microgels. Then, what kind of microgels are suitable for tissue regeneration, especially for adipose tissue regeneration? Towards adipose tissue regeneration, an ideal microgel has to meet four particular requirements. Firstly, materials with excellent biocompatibility and biodegradability should be chosen to fabricate the microgels. In our previous studies, Poly-(L-glutamic acid) (PLGA) and chitosan (CS) had been proved to possess the advantages of excellent biodegradability and biocompatibility.24 Secondly, the microgels should make stem cell shape spheroidal to promote adipogenic differentiation. More and more researchers have demonstrated that adipogenic differentiation is favoured in scaffolds where stem cells are on the spheroidal shape, while flattened cell adhesion is of benefit to osteogenesis.

25-28

Thirdly, the microgels should possess an open porous

structure to avoid the encapsulation effect to form the cell-cell connections. The extensive cell-cell connections are of great benefit to realize tissue regeneration.

11, 12

Finally, when being injected, the microgels should remain an excellent shape stability to exhibit a great injectability. Therefore, the microgels could provide protection for cells from mechanical insult. 29, 30 Herein, based on double bonded poly-(L-glutamic acid)-g-2-Hydroxyethyl methacrylate (PLGA-g-HEMA) and maleic anhydride-modified chitosan (MCS), we propose a simple strategy to fabricate an open porous microgels with high hydrophilicity and great injectability to meet the abovementioned unmet need using water-in-oil (W/O) emulsion method and freeze-drying technique. The well-designed microgels are expected to make stem cells spheroidal to favour adipogenic differentiation and form cell-cell connections to realize tissue regeneration. The open porous structure and hydrophilicity of microgels were tested. The injectable performance of microgels was tested to reflect the microgels’ ability to protect cells during injection. Rheological experiments were carried out to determine the injected concentration of microgels. In vitro and in vivo degradation of microgels were evaluated to prove that the rate of microgel degradation and adipose tissue formation could be roughly matched. After cell seeding, the stem cell shape and cell-cell 3 ACS Paragon Plus Environment


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connections were observed. The stem cell viability was evaluated. At the same time, Oil red O staining and the expression of adipogenic genes were detected after 14 days of adipogenic induction. Finally, the induced and non-induced microgel-cell dispersions were injected into the nude mice to evaluate the formation of adipose tissues to verify the advantage of the open porous microgels. 2. Results 2.1 Preparation of the open porous microgels The PLGA-g-HEMA was synthesized through esterification reaction between PLGA and HEMA (Figure 1). Proton peaks at 6 and 6.5 ppm were attributed to the CH2=C- groups of HEMA (Figure 2a).

31

The proton peak at 5 ppm was attributed to

the α-carbon of PLGA. The grafting ratio of HEMA was 71%, which was calculated from the area ratio of proton peaks at 6-6.5 and 5 ppm. The FTIR spectrum of PLGA-g-HEMA further showed the characteristic absorption band at 1450 cm-1 was attributed to the stretching vibration of CH2=C- groups (Figure 2c). As shown in Figure 1, synthesis of MCS was carried out in a one-step reaction between maleic anhydride and chitosan. The maleic anhydride could be grafted to CS backbone through esterification and amidation reaction. The proton peaks of double bonds appeared at 5.8, 6, 6.6, and 6.8 ppm (Figure 2b). 32 The proton peaks of the CS backbone (at ca position of Figure 2b) appeared at 4.5 ppm. The grafting ratio of maleic anhydride was 56%, which was calculated from the area ratio of proton peaks at 5.8-6.8 and 4.5 ppm. The FTIR spectrum of MCS further showed the characteristic absorption band at 810 cm-1 was attributed to the stretching vibration of double bonds (Figure 2c). Microgels could be crosslinked by PLGA-g-HEMA and MCS through double bonds. Compared to two macromolecule precursors, The FTIR spectrum of microgels showed that the intensity of characteristic absorption band at 1450cm-1 and 810 cm-1 decreased (Figure 2d). 32, 33 Figure 2e showed the prepared microgels could remain excellent shape stability in PBS, and microgels at the dry state were also exhibited in Figure 2f, illustrating that the microgels had been prepared successfully. 4 ACS Paragon Plus Environment

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2.2 Characterization of the open porous microgels 2.2.1 Open porous structure of microgels An open porous structure was of great importance to realize the cell-cell connections and better facilitate nutrients diffusion. 11, 12, 34, 35 The porous structure of microgels was obtained by ice crystals (as a porogen). The size of ice crystals was regulated by different freezing temperature at -20 oC, -80 oC, and -196 oC. The effect of freezing temperature on porous structure was studied at a controlled initial concentration of 6%. When compared to microgels obtained at -80 oC and -196 oC, the pore diameter and porosity of microgels prepared at -20 oC were largest (Figure S1). At the same time, the effect of initial concentration on porous structure was studied at a controlled freezing temperature of -20 oC. Microgels with the initial concentration of 5% showed a poor shape stability (Figure S2a, a1). Microgels with the initial concentration of 7% showed a small pore diameter and porosity (Figure S2c, c1). Therefore, the optimal pore diameter and porosity of microgels could be obtained by the freezing temperature of -20 oC and initial concentration of 6% (Figure S2b, b1 and Figure 3a). A CLSM image further showed that the microgels at the swelling equilibrium state still possessed an open porous structure (Figure 3b). A mercury porosimeter was used to detect the pore diameter and porosity of microgels with the initial concentration of 6%. The optimal pore diameter and porosity of microgels was 38 µm and 88.3% (Figure 3c). The 6% of microgels were chosen for subsequent characterization and evaluation. 2.2.2 Hydrophilicity of microgels The swelling behavior of microgels was studied to illustrate the hydrophilicity of microgels.

36, 37

Microgels at the dry and swelling equilibrium state were observed by

a phase contrast microsocorpy. As shown in Figure 3d, and e, the diameter of microgels increased obviously after the dried microgel was immersed in PBS at 37 oC. The diameter of microgels was further determined by a laser particle analyzer. Result showed the diameter of microgels at the dry and swelling equilibrium state was 230 µm and 340 µm (Figure 3f). After being calculated, the swelling ratio of microgels 5 ACS Paragon Plus Environment


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was 223%. Besides, the microgel at swelling equilibrium state was transparent (Figure 3e), illustrating that the microgels possess a high hydrophilicity. 2.2.3 Injectability of microgels Two images were taken to observe the injectability of microgels. The microgels could pass through the needle and quickly return to their original shape. After the microgel was injected, no fragments could be observed (Figure 3g, h), indicating a great injectability of microgels. 38 Therefore, the microgels can provide protection for cells during injection. 29, 30 2.2.4 Rheological performance of microgels An appropriate injection concentration of microgels should be determined. The G’ and G’’ of microgel dispersion were detected. In Figure 3i, the G’ and G’’ exhibited a plateau in the strain range of 0.01-0.1%. The plateau region was a linear viscoelastic region. 39 As the concentration of microgel dispersions increased from 25 to 35 mg/ml, the G’ improved from 1000 Pa to over 3500 Pa (Figure 3j). For all the dispersions, the G’ was always larger than G’’ in the strain range of 0.01-5%, illustrating that the microgels were well cross-linked. When the strain increased to over 5%, the microgels collapsed. Notably, the G’ of 30 mg/ml of microgel dispersions (2000 Pa) was similar to that of the native adipose tissue.

40

Therefore, 30 mg/ml of microgel

dispersions were chosen for adipose tissue regeneration. 2.2.5 In vitro degradation of microgels In tissue regeneration, degradation of 3D matrixes was necessary.

41

The in vitro

degradation of microgels was evaluated by examining the weight loss and morphology collapse. As shown in Figure S3 and S4, the morphology of microgels collapsed, and the weight was lost gradually. After 4 weeks of degradation, the microgels could still remain shaped stability. After 12 weeks of degradation, the microgels collapsed totally (Figure S3) and the remaining weight (%) was around 20% (Figure S4). 2.2.6 Bovine Serum Albumin (BSA) absorption in microgels A BSA absorption experiment was performed to illustrate the mechanism of 6 ACS Paragon Plus Environment

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cell-microgel interaction. A strong green fluorescence was shown in Figure S5a after fluorescein isothiocyanate (FITC) labeled BSA solutions were absorbed into the dried hydrophilic microgel, but the green fluorescence intensity significantly decreased after the microgel was washed with deionized water for 1, 2, and 3 times (Figure S5b-d), indicating that the microgel could prevent protein adsorption well to weaken the cell-microgel interaction. 2.3 Characterization of hASCs in the open porous microgels 2.3.1 Cell morphology and survival The stem cell shape in the microgels were observed at 1, 7, and 14 days after seeding. As shown in Figure 4a-c, hASCs remained spheroidal over a culture period of 14 days. What’s more, microgels could load a large number of spheroidal cells because of the high hydrophilicity and large porosity (Figure 4d-f).

29

The large

number of cells in the open porous structure formed extensive cell-cell connections (Figure 4d-f). After the microgel-hASCs were injected in vitro, the stem cell viability in microgels was evaluated. The dead cells maintained at a very low level up to 14 days post-injection due to the open porous structure which could significantly facilitate nutrients diffusion 34, 35 and great injectability which could provide protection for cells from mechanical insult (Figure 4g-i). 29, 30 2.3.2 Adipogenic differentiation of hASCs in the open porous microgels To evaluate whether the spheroidal stem cell shape facilitate adipogenic differentiation of hASCs, the intracellular lipid accumulation was detected in the induced and non-induced group after 7 and 14 days. Qualitative Oil Red O images were shown in Figure 5. In the induced group, red lipids were shown in the spheroidal hASCs after 7 days of adipogenic induction (Figure 5a), and extensive red lipids were exhibited at 14 days after adipogenic induction (Figure 5d). 42 In the non-induced group, the lipid accumulation was not found in any spheroidal hASCs (Figure 5b, e). A quantitative PCR assay was applied to further evaluate the expression of the master adipogenic gene. In the induced group, the expression of C/EBP α, in particular for 7 ACS Paragon Plus Environment


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the expression of LPL, increased over time. After 7 and 14 days of adipogenic differentiation, the gene expression levels in the induced group were always higher than those in the non-induced group (Figure 5c, f). 42 2.4 In vivo adipose tissue regeneration In view of the excellent adipogenic differentiation and extensive cell-cell connections enabled by the open porous microgels, 30 mg/ml of microgel-hASC dispersions were injected into the nude mice to evaluate the formation of adipose tissue. The subcutaneous injection surgery was shown in Figure 6a1-a3. After the nude mice were sacrificed, light yellow adipose tissues were exhibited (Figure 6b1, c1, d1, e1, f1, and g1). The H&E and Oil red O staining were conducted to observe the neo-generated adipose tissues. At 4 weeks post-implantation, both induced and non-induced group showed no obvious adipose tissues formation (Figure 6b2, b3, e2, and e3). The H&E staining of induced group at 8 weeks exhibited adipose tissues had been formed locally (Figure 6c2). The Oil red O staining also showed some red intracellular lipid accumulation (Figure 6c3). When the implantation time was increased to 12 weeks, the ring-like morphology and the vacuole structure were shown in the induced group (Figure 6d2), and the Oil red O staining further demonstrated the adipose tissues formation (Figure 6d3). 35 While non-induced group at 8 and 12 weeks showed no adipose tissues (Figure 6f2, f3, g2, and g3). Besides that, H&E staining also demonstrated capillaries were formed in both induced and non-induced group at 12 weeks post-implantation (Figure 6d2, g2, blue arrows). Immunohistochemical staining of CD31 and vWF were futher applied to observe the formation of blood vessels in the induced group. At 4 weeks, capillary characteristics were not exhibited (Figure 7 a1, a2). Capillaries could be observed locally at 8 weeks (Figure 7 b1, b2, red arrows). With the formation of adipose tissue, obvious capillaries were formed with a diameter of around 50 µm (Figure 7c1, c2, red arrows). 20, 43 At the same time, the microgels were biodegraded in the nude mice. At 4 weeks post-implantation, microgels could be found in induced and non-induced group. The microgels maintained their shaped stability (Figure 6b2, e2, green arrows). The H&E 8 ACS Paragon Plus Environment

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staining of adipose tissues at 8 weeks revealed that the microgels collapsed and small fragments could be seen in induced and non-induced group (Figure 6c2, f2, green arrows). When the implantation time was prolonged to 12 weeks, with the formation of adipose tissue, the microgels in induced group had almost been degraded (Figure 6d2, green arrows). Though there were no adipose tissues in the non-induced group, microgels were also rarely found (Figure 6g2, green arrows). The rate of microgels degradation and adipose tissue formation were roughly matched. 3. Discussion Towards adipose tissue defects, use of filler materials and autologous adipose tissue transplantation are applied as the major repair strategy but received limited effect. 4, 5 Adipose tissue engineering shows a great potential towards the repair of adipose tissue defects.

1-3

Injectable hydrogels are widely used in the field of adipose tissue

regeneration because they can repair irregularly shaped defects and can be implanted with minimum invasiveness.

8

However, the low cell viability and the encapsulation

effect which blocks the cell-cell connections in the injectable hydrogels cause damage to the tissue regeneration. 10-12 The present microscale hydrogel (microgels) with large surface area per volume and open porous structure can overcome the limitations of currently practiced injectable hydrogels. The well-designed microgel system is greatly expected to show excellent and advanced applications in adipose tissue regeneration. To the best of our knowledge, no studies have reported so far that microgels are utilized in the field of adipose tissue regeneration. In order to achieve the excellent target of adipose tissue regeneration, the open porous microgels should meet three requirements: making stem cell shape spheroidal to favour adipogenic differentiation,

25-28

maintaining high stem cell viability, and

forming extensive cell-cell connections to realize adipose tissue regeneration.

29, 30

Adipogenic differentiation was greatly influenced by cytoskeletal tension of stem cells. Several studies showed that the non-adhesive stem cells on the spheroidal shape could cause an inhibition of F-actin polymerization and RhoA-ROCK pathway to reduce the contractility of cytoskeleton, thus favouring adipogenic differentiation. 25-28 9 ACS Paragon Plus Environment


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It was reported that a hydrophilic matrix could effectively prevent protein adsorption to weaken the cell-matrix interaction, thus suppressing cell adhesion to make stem cell shape spheroidal.

44-45

Microgels were transparent at the swelling equilibrium

state (Figure 3e) and exhibited a high swelling ratio of 223% (Figure 3f), indicating that the microgels possess a high hydrophilicity. The high hydrophilicity of microgels caused an excellent protein-resistant property (Figure S5), which weakened cell-microgel interaction to make hASCs spheroidal (Figure 4a-c). Furthermore, Extensive intracellular lipid accumulation was exhibited at 14 days after adipogenic induction (Figure 5d). The expression of C/EBP α and LPL significantly increased over an adipogenic induction period of 14 days (Figure 5c, f), indicating that the spheroidal stem cell shape in the open porous microgels led to an excellent adipogenic differentiation. Besides the spheroidal stem cell shape which facilitated adipogenic differentiation, a sufficient dose of live cells and extensive cell-cell connections were required to successful tissue regeneration.

11, 12

The nutrients diffusion in microgels and

mechanical insult during injection had great impact on stem cell viability. The microgels still possessed an open porous structure at the swelling equilibrium state (Figure 3b). The open porous structure could make nutrients diffused very well. 34, 35 Besides, great injectability of the microgels indicated microgels’ great protection for cells from mechanical insult (Figure 3g, h).

29, 30

Therefore, hASCs could maintain

high viability up to 14 days post-injection (Figure 4g-i). Furthermore, the open porous structure was beneficial to provide necessary space to form cell-cell connections. 11, 12 As shown in Figure 4d-f, a large number of live cells in the open porous structure formed extensive cell-cell connections. At last, H&E and Oil Red O staining showed a growing tendency in the formation of adipose tissues (Figure 6b2-d2, b3-d3). Neo-generated adipose tissues were formed at 12 weeks post-implantation (Figure 6d2, d3). In further consideration of the long-term functionality of neo-generated adipose tissues, the formation of blood vessels in neo-generated adipose tissue was of great importance. 10 ACS Paragon Plus Environment

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The

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immunohistochemical staining of CD31 and vWF clearly exhibited newly formed blood vessels in the adipogenic induced group at 8 and 12 weeks post-implantation (Figure 7, red arrows).

20, 43

At the same time, well-designed microgels for adipose

tissue regeneration should match the rate of degradation to tissue regeneration. Fast degradation rate limited their ability to provide support, while slow degradation rate would prevent adipose tissue regeneration. 11 The H&E staining illustrated that the degradation rate of microgels could roughly match with the adipose tissue regeneration rate (Figure 6b2-d2). Correspondingly, in vitro degradation of microgels showed a similar degradation period up to 12 weeks (Figure S3, 4). The microgel system holds great promise to be an adopted cell delivery system in adipose tissue regeneration. 4. Conclusion Collectively, the present work provides new insights into the excellent open porous microgels with high hydrophilicity and great injectability. The optimal pore diameter and porosity was 38 µm and 88.3%, respectively. The hydrophilic microgels made the stem cell shape spheroidal to favour adipogenic differentiation. The extensive cell-cell connections enabled by open porous structure of microgels were formed to realize the adipose tissue regeneration. Besides, the open porous structure greatly promoted nutrients diffusion, and great injectability provided protection for cells, thus causing a high stem cell viability. The rate of microgels degradation and adipose tissue formation were roughly matched. Based on these advances, adipose tissues could be well regenerated at 12 weeks post-implantation. Blood vessels were also formed to remain the long-term survival and functionality of neo-generated adipose tissue, indicating an excellcent microgel system towards adipose tissue regeneration. 5. Experimental Section 5.1

Materials

PLGA (viscosity averaged molecular weight [Mη] =3×104) was synthesized by the ring-opening polymerization in our laboratory. CS (weight-averaged molecular weight (Mw) =4×104, degree of deacetylation (DD) =95%) was purchased from Jinan 11 ACS Paragon Plus Environment


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Haidebei Marine Bioengineering Corp. (Shandong, China). Maleic anhydride, p-tolunesulfonic acid monohydrate, dichloromethane, formamide, and 2-hydroxyethyl methacrylate (HEMA) were purchased from Aladdin Corp. (Shanghai, China). Dimethyl sulfoxide (DMSO), petroleum ether, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP), ammonium persulfate (APS), span 80, and tetramethylethylenediamine (TEMED) were purchased from Sinopharm chemical reagent Corp. (Shanghai, China). Bovine Serum Albumin (BSA) and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (United States). 5.2

Synthesis of PLGA-g-HEMA

The PLGA (1 g) was firstly dissolved in DMSO. The EDC/DMAP was added into the solution as an esterification catalyst. HEMA (1 g) was then added. After being dialyzed and lyophilized, the PLGA-g-HEMA was obtained for further use. 5.3

Synthesis of MCS

The CS (1 g) was dissolved in the formamide and p-toluene sulfonic acid. The maleic anhydride (4 g) was then added under the protection of N2 gas. After being precipitated, dialyzed, and lyophilized, the MCS was fabricated for further use. 5.4

Preparation of the open porous microgels

PLGA-g-HEMA (0.08 g) and MCS (0.04 g) were dissolved in a 0.4M NaOH water solution (1.9 ml) firstly. A 10 wt% APS water solution (0.1 ml) was then added to form water phase. The initial concentration of water phase was 6%. A W/O emulsion composed of water phase (4 ml), span 80 (3 ml), and petroleum ether (30 ml) was then produced. The W/O emulsion was frozen rapidly at -20 oC, -80 oC, and -196 oC. After the span 80 and petroleum ether were removed, the frozen water phase was lyophilizated to form an open porous microsphere precursor. The porous microsphere precursors were then dispersed into dichloromethane and TEMED under the protection of N2 gas to be corss-linked to form an open porous microgel. At the same time, the 5% and 7% of initial concentration were also obtained to prepare the microgels to determine the optimal pore diameter. Microgels with diameter of 12 ACS Paragon Plus Environment

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200-300 µm were obtained by standard sieves. After being lyophilizated, open porous microgels were stored for further use. 5.5

Characterization of MCS and PLGA-g-HEMA

5.5.1 1H Nuclear magnetic resonance (1H NMR) spectroscopy 1

H NMR spectra were recorded on an AV 500 NMR from BRUKER to confirm the

reaction efficiency of HEMA to the PLGA and maleic anhydride to the CS. The MCS (15 mg) was dissolved in the deuterium oxide (D2O, 0.6 ml). The PLGA-g-HEMA (15 mg) was dissolved in the trifluoroacetic acid-d (TFA-d, 0.6 ml). 5.5.2 Fourier transform infrared (FTIR) spectroscopy FTIR spectra of PLGA, CS, PLGA-g-HEMA, and MCS were obtained with a FTIR spectrophotometer (AVATAR 370, Nicolet, USA) in the wavenumber region of 4000– 500 cm-1. 5.6

Characterization of open porous microgels

5.6.1 FTIR spectroscopy The FTIR spectrum of microgels was recorded with the FTIR spectrophotometer in the wavenumber region of 4000–500 cm-1. 5.6.2 Morphology The surface morphology of microgels at the dry state was observed through a scanning electron microscopy (SEM, HITACHI SU-1500, Japan). The microgels were coated with gold using a sputter coater (DeskII, Denton Vacuum Inc). To determine whether the microgels at the swelling equilibrium state still possessed an open porous structure, microgels were immersed in PBS for 8 h at 37 oC. After being labeled with hoechst 33258 dye (Sigma-Aldrich, USA), the microgels were observed using a confocal laser scanning microscopy (CLSM, Nikon Y-FL, Japan). To visualize the swelling behavior of microgels. Microgels at the dry and swelling equilibrium state were observed through a phase contrast microsocorpy (LEICA DM2500M, Germany). 5.6.3 Pore diameter and porosity A mercury porosimeter (Quantachrome PoreMaster33GT, USA) was used to 13 ACS Paragon Plus Environment


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characterize the pore diameter and porosity of open porous microgels. 5.6.4 Diameter and swelling behavior To determine the diameter and swelling behavior of microgels, a laser particle analyzer (MASTERSIZER 2000, MALVERN INSTRUMENTS ITD, UK) was applied to measure the diameter of microgels at the dry (dd) and swelling equilibrium state (ds). The swelling ratio could be calculated using the following formula: Swelling ratio (%) = (ds3- dd3)/dd3×100%. 5.6.5 Injectable performance The injectability of microgels was observed with a high speed digital microscopic system (KEYENCE, VW-9000, Japan). Microgels were labeled by toluidine blue dye, and then be injected using a 30 G needle. 5.6.6 Rheological performance Rheological experiments were carried out on a rheometer (AR2000, TA Instruments, USA). Dried microgels were dispersed into PBS to form 25 mg/ml, 30 mg/ml, and 35 mg/ml of microgel dispersions. All the microgel dispersions were tested at 37 oC. A linear viscoelastic region of the open porous microgels was determined firstly through stress sweep test from 0.01% to 100% at a controlled frequency of 0.1 Hz. The storage modulus (G’) and loss modulus (G’’) were then recorded within the linear viscoelastic region. Subsequently, the frequency sweep test was carried out in a frequency range of 0.01 - 1 Hz at a controlled strain of 0.1%. 5.6.7 In vitro degradation Microgels (W0 = 100.0 mg) immersed in 0.1 M PBS were placed on an orbital shaker at 10 rpm and 37 oC. The PBS was replaced every day. At every time point, microgels were harvested, washed, and lyophilized. The remaining weight of microgals (Wt) was recorded. The morphology of microgels was observed by SEM. Weight (%) = Wt / W0 ×100%, where W0 and Wt were the weights of dried microgels before and after degradation. 5.6.8 BSA absorption in microgels FITC labeled BSA solutions (0.2mg/ml) were prepared based on a previous report. 14 ACS Paragon Plus Environment

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46

After FITC labeled BSA solutions were absorbed into the dried hydrophilic

microgels, the microgels were washed with deionized water once an hour. The confocal laser scanning microscopy was used to observe the BSA absorption in microgels. 5.7

Cell seeding and in vitro culture

5.7.1 Cell seeding The human adipose stem cells (hASCs) harvest protocol were approved by the Research Ethical Committee of the Hospital. Fresh lipoaspirates were washed with PBS and digested with 0.1% collagenase type I (Sigma, USA) at 37 °C. The resulting pellet was cultured in low glucose Dulbecco’s modified Eagle’s medium (LG-DMEM, HyClone, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, USA) and 1% penicillin–streptomycin (HyClone, USA). The cells were cultured at 37 oC in a humidified atmosphere with 5% CO2. Dried microgels (1 ml) were sterilized by

60

Co γ irradiation (at 5 mrad). The

third-passage hASCs labeled with 3, 3’-dioctadecyloxacarbocyanine perchlorate (Dio) fluorescent dye (Molecular Probes, USA) were collected and suspended at a cell density of 4×107 cells/ml. 1 ml of cell suspensions were evenly dropped into the 1ml of microgels. After hASCs were automatically absorbed into the dried spongy open porous microgels, microgel-hASCs were immersed in 3ml of LG-DMEM at 37 oC. 5.7.2 Cell morphology The cell morphology in microgels was observed by SEM and CLSM at 1, 7, and 14 days after seeding. In CLSM observation, microgels were labeled with hoechst 33258 dye in 0.1 M PBS. 5.7.3 Cell survival The cell survival in microgels was studied using a live/dead assay at 1, 7, and 14 days after microgel-hASCs were injected using a 30 G needle. Microgel-hASCs were washed with PBS and stained with fluorescein diacetate/ propidium iodide (FDA/PI) (Invitrogen). The stem cell viability was observed by CLSM. Live cells showed green fluorescence, while the nuclei of dead cells showed red fluorescence. 15 ACS Paragon Plus Environment


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5.8

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Adipogenic differentiation of hASCs in the open porous microgels

Microgel-hASCs were placed in either a 3 mL of adipogenic inducing medium (the LG-DMEM

medium

supplemented

with

10

µM

insulin,

0.5

mM

3-isobutyl-1-methylxanthine, 200 µM indomethacin, and 1 µM dexamethasone (all from Sigma-Aldrich, USA)) as a induced group or a 3 mL of LG-DMEM as a non-induced group. Both the induced and non-induced group were evaluated at 7 and 14 days after adipogenic induction. To observe lipid droplet, Oil red O staining was used. Microgel-hASCs were immersed in a 4% neutral buffered formalin solution for 24 h, and in a 60% Oil red O solution for 30 min. After being washed with 0.1 M PBS and rinsed with isopropanol, microgel-hASCs were observed through the phase contrast microsocorpy. A quantitative PCR analysis was applied to further evaluate the expression levels of adipogenic genes. The PCR was performed using a quantitative real-time amplification system (MxPro-Mx3000P; Stratagene, La Jolla, CA). Adipogenic genes, including CCAAT/enhancer-binding protein alpha (C/EBP α) and lipoprotein lipase (LPL) were detected at 1, 7, and 14 days after adipogenic induction. Primers for Real-Time Polymerase Chain Reaction were seen in Table 1. Relative expression levels of adipogenic genes were calculated by normalizing the quantified cDNA transcript level to that of the GAPDH. The relative adipogenic gene expression of the induced and non-induced group at 1 day were set as 1. 5.9 In vivo evaluation of microgel-hASCs 5.9.1 Subcutaneous injection of microgel-hASCs dispersion After being immersed in LG-DMEM or adipogenic inducing medium for 2 weeks, induced and non-induced microgel-hASC were collected and injected into the nude mice using an 18 G needle. The institutional review committee of Shanghai Jiao Tong University School of Medicine approved all animal study protocols. After 4, 8, and 12 weeks post-implantation, mice were sacrificed to harvest the neo-generated adipose tissues for further evaluation. 5.9.2 Histological evaluation 16 ACS Paragon Plus Environment

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Hematoxylin and eosin (H&E) staining and Oil red O staining were carried out to observe the neo-generated adipose tissues. All the the neo-generated adipose tissues were fixed in neutral buffered formalin and embedded in paraffin. 5.9.3 Immunohistochemical evaluation The immunohistochemical staining of anti-CD31 and anti-von Willebrand Factor (vWF, all form Abcam, Cambridge, UK) were applied to observe the formation of neo-generated blood vessels. 5.10 Statistical analysis All the experimental data were expressed as mean ± standard deviation (SD). Statistical differences were performed through One-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant.

Supporting Information The effect of freezing temperatures and initial concentrations on open porous structure of microgels were exhibited. In vitro degradation of the microgels was presented. BSA absorption in microgels was studied. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements: The work was supported by the Science and Technology Commission of Shanghai Municipality (Grant no. 15JC1490400) and the National Natural Science Foundation of China (Grant nos. 51373094, 51503119, 51473090).

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Figure 1. Schematic illustration of the preparation of PLGA-g-HEMA, MCS, and open porous microgels. After cell seeding, spheroidal cells were maintained in the 24 ACS Paragon Plus Environment

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open porous microgels. Nutrients could be diffused very well. 30 mg/ml of microgel-hASC dispersions were injected to evaluate the formation of adipose tissue after 14 days of adipogenic induction in vitro.



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Figure 2. 1H NMR spectra of (a) PLGA-g-HEMA and (b) MCS. (c, d) FTIR spectra

of

PLGA,

CS,

PLGA-g-HEMA,

MCS,

and

microgels.

(e)

Phase contrast microscope image of microgels in 0.1 M PBS. (f) SEM image of

microgels. (Scale bar: 300 µm)


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Figure 3. Characterization of microgels. (a) SEM and (b) CLSM images of microgel. (c) Pore diameter and porosity of microgels. (d, e) Phase contrast microsocorpy images of microgels at the dry and swelling state. (f) Particle diameter and swelling ratio of microgels. (g, h) Injectable performance of microgels. (i, j) Rheological performance of microgel dispersions with different concentration. (Scale bar: 100 µm).



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Figure 4. (a, b, c) SEM images of microgel-hASCs at 1, 7, and 14 days after cell seeding. (d, e, f) CLSM images of microgel-hASCs at 1, 7, and 14 days after cell seeding, where green fluorescence were Dio-labeled hASCs, and blue fluorescence represented microgels labeled with hoechst 33258 dye. (g, h, i) Live/Dead staining at 1, 7, and 14 days after injection. Black arrows indicated spheroidal hASCs. (Scale bar: 100 µm).


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Figure 5. Adipogenic differentiation of hASCs in vitro. (a, d) Oil Red O staining of adipogenic induced group. (b, e) Oil Red O staining of non-induced group. (c, f) The expression of adipogenic genes, including LPL and C/EBP α. Green and blue arrows indicated the spheroidal hASCs in microgels. (Scale bar: 50 µm). (n=6 for all the experimental group, *p

Injectable Stem Cell Laden Open Porous Microgels That Favor

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