Biocompatible interface modified tissue engineering chamber reduces

May 29, 2019 - Adipose flap expansion using tissue engineering chamber (TEC) presents a promising candidate for soft tissue regeneration by activating...
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Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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Biocompatible Interface-Modified Tissue Engineering Chamber Reduces Capsular Contracture and Enlarges Regenerated Adipose Tissue Zijin Qin,†,∥ Qiang Chang,†,‡,∥ Chen Lei,†,§ Yunfan He,† Zhiyong Huang,§ Malcolm Xing,*,‡ and Feng Lu*,† †

Department of Plastic and Cosmetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China Department of Mechanical Engineering, University of Manitoba, Winnipeg, Manitoba, Canada § Department of Plastic and Aesthetic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China

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S Supporting Information *

ABSTRACT: Adipose flap expansion using a tissue engineering chamber (TEC) presents a promising candidate for soft tissue regeneration by activating in situ adipose tissue regeneration. However, foreign body reaction (FBR) and capsular contracture caused by a silicone chamber limit large tissue reconstruction. Here, a hydrophilic and biodegradable film made of poly(ethylene glycol) diacrylate (PEG-da) with methacrylated gelatin (gelatinMA) was presented between the host tissue and silicone chamber to tune the local wound and to prevent initiation of FBR. After a 60 day investigation, 6.1-fold-regenerated fat tissue was obtained from the PEG−gelatin group, whereas only 3-fold tissue was harvested from a silicone group. Histological staining demonstrated that the structure of the neo-formed adipose tissue in both groups was similar to mature adipose tissue. Noticeably, a more distinct and denser fibrous capsule was observed in the silicone group compared to the PEG−gelatin group. Immunohistochemistry of CD206 and TGF-β expression indicated less M2 macrophage infiltration and a minor inflammation reaction with PEG−gelatin assistance. Less collagen deposition and myofibroblast activation in the PEG−gelatin group were demonstrated via α-SMA and type I collagen staining. All these demonstrated that a biocompatible membrane supplement can attenuate capsule formation and contracture leading to a larger tissue regeneration through the TEC technique, which could lead to new perspectives to the relationship between materialsmattered FBR and tissue regeneration. KEYWORDS: poly(ethylene glycol), methacrylated gelatin, tissue engineering chamber, adipose tissue regeneration, foreign body reaction

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INTRODUCTION

material type of the chamber. Previous reports revealed that neo-formed adipose tissues harvested over a long period were always surrounded by a thick-layer fibrous capsule, exerting strong contractile centripetal force to the inside soft adipose tissue and limiting further tissue expansion.3,4 The contractile centripetal force produced by active myofibroblasts distributing in the capsule is up to 40−80 N which assumes the main responsibility for significant volume loss or regeneration failure.5 The insights into interfacial interaction between host tissue and TEC may pave the way to alleviate the capsule formation as well as contracture and induce larger engineered adipose tissue regeneration for clinical practices.

Large soft-tissue defect reconstruction remains the most challenging procedure for plastic surgeons.1 The existing methods like musculocutaneous flap and autologous fat transfer have their drawbacks such as additional scars and potential morbidity at donor sites. Currently, techniques that could induce a large volume of soft tissue regeneration in situ will be crucial to advance large soft-tissue defect reconstructions. A tissue engineering chamber (TEC), a chamber made of a rigid inert material, can induce in situ adipose tissue regeneration to restore the defects spontaneously. TEC raises local aseptic inflammation, promotes chemotactic migration of surrounding adipose precursor cells to the turbulent site, and activates proliferation and differentiation of stem cells to mature adipocytes.2 However, the regenerated soft tissue could not fulfill the chamber, regardless of the chamber size and © 2019 American Chemical Society

Received: August 8, 2018 Accepted: May 29, 2019 Published: May 29, 2019 3440

DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

Article

ACS Biomaterials Science & Engineering The thick fibrous capsule is believed to result from ubiquitous and nonspecific foreign body reaction (FBR) caused by the implanted silicon chamber which is inert yet less biocompatible. FBR is a sequence of continuous events starting from nonspecific protein absorption, followed with macrophage adhesion and giant cell formation, eventually leading to fibroblast activation and collagen production along with collagenous encapsulation.6 Previous studies have proved that intrinsically biocompatible materials, like allogeneic and xenogenous acellular dermis (AlloDerm, Strattice) plating on implanted devices acting as an adjunct between breast tissue and prosthesis, can significantly decrease the occurrence of capsular contracture to lower than 0.4%, which is up to 30% in primary breast augmentation without such additives.7−9 However, the extravagant expenditure and introduction of exogenous immunogen should be considered when employing acellular dermis to implants.4 Compared with exogenous biomaterials, integrating synthesis biomaterials with TEC to advance the adipose tissue regeneration may develop the new perspective to the relationship between fibrotic capsule and tissue growth. In this study, for the first time, a hydrophilic PEG−gelatin membrane was integrated to the interior surface of the silicone TEC (Scheme 1), and the efficiency of reduction of the FBR

the CH2 group and N−H stretching of amide (II) were centered at 2960 and 3305 cm−1, respectively, and the peaks observed ∼1645, 1539, and 1234 cm−1 could be attributed to the CO stretching vibrations, N−H bending vibrations (amide II), and N−H bending (amide III), respectively, which indicated the backbone chain of gelatin. For the PEG-da monomer, the peak at 1645 cm−1 could belong to double carbon bond stretching by the presence of a terminal acrylate group, and the peak around 1721 cm−1 could be assigned as CO symmetric stretching. The ∼2866 cm−1 peak indicated C−H stretching vibrations, and other characteristic peaks observed were ∼1099 cm−1 (symmetric stretching of the C− O−C group) and 1455 cm−1 (bending vibration of the CH2 group). It was clear that some characteristic peaks of monomer remained at the spectrum of PEG−gelatin hydrogel after gelation, e.g., symmetric stretching of the C−O−C group (1099 cm−1) and C−H stretching vibrations (2866 cm−1) at the PEG-da spectrum. The spectrum revealed that the shoulder peak (∼1645 cm−1) corresponding to the CC almost disappeared after total cross-linking of gelatin−MA and PEGda, which indicated most of the CC bonds reacted to hydrogel network formation. Adhesion of proteins to the materials is influenced by the surface energy (surface tension), which is affected by the hydrophilicity of the film surface.13 The surface hydrophilicity was assessed with a water contact angle system. The hydrophilicity visualized with contact angle (CA) of the smooth silicone chamber and PEG−gelatin membrane was shown, respectively (Figure 1A and B). For a smooth silicone

Scheme 1. PEG−Gelatin Membrane Anchored to the Inner Surface of the Silicone TEC and Followed with Flap Harvest and Implant Procedures

was demonstrated. Hydrogel based on poly(ethylene glycol) diacrylate (PEG-da) and gelatin can improve the surface characteristics at the aspect of hydrophilicity and biocompatibility to minimize unspecific protein fouling, control FBR, and decrease collagen deposition and eventual capsulation.10 This work may offer a feasible strategy to a large volume of adipose tissue regeneration in TEC and its future clinical practices.

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RESULTS AND DISCUSSION Characterization of the PEG−Gelatin Membrane. The FBR is defined as the nonspecific host’s response to exogenous materials, which comprises a complex cascade of immune modulators.11 To reduce FBR, we synthesized a hybrid composite combining natural gelatin and PEG-da. PEG could repel unspecific protein and cell attachment, while gelatin increases the biofunctionality and biocompatibility of the synthetic composite.12 ATR-FTIR analysis of lyophilized samples was carried out to represent the chemical structure of the different hydrogels: PEG-da, gelatin, gelatin−MA, and PEG−gelatin (Figure S1). First, methacrylate groups were functionalized on gelatin side amine groups to synthesize gelatin−MA for gelation with PEG-da. The spectrum of gelatin−MA showed absorption bands similar to the gelatin backbone: the C−H stretching of

Figure 1. Hydrophilic surface and porous structure of the PEG− gelatin membrane. (A) The CA of the silicone chamber. (B) The CA of the PEG−gelatin chamber. (C) The CAs of the silicone chamber and PEG−gelatin chamber were significantly different (*p < 0.05). (D) SEM images of the PEG−gelatin membrane demonstrated the porous structure.

chamber, the angle was 113.6 ± 4.9°, and the angle was 72.9 ± 7.4° for the PEG−gelatin membrane (Figure 1C). Smaller CA of the PEG−gelatin membrane indicated that the membrane was more hydrophilic than the control silicone chamber. The porous structure of the PEG−gelatin membrane was assessed with a scanning electron microscope, and the film exhibited a 3441

DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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ACS Biomaterials Science & Engineering typical porous structure with a mean of 64 ± 5 μm of the infiltrate pore diameter (Figure 1D). Previous reports revealed that the protein-resistance effect is associated with interfacial tension and energy by a hydrophilic polymer coating.10 Water molecules would reside or penetrate into the polymer network to form a hydration layer based on a hydrogen bond along with a PEG−gelatin chain distributing and stretching inside. When proteins approach the chamber surface, the compression of the PEG−gelatin chain and hydration layer would create a thermodynamically unfavorable dehydration entropic effect and produce a remarkable expulsion force.14,15 The in vitro enzymatic degradation study was implemented to reveal the degraded kinetics and stability of the PEG− gelatin membrane. Gelatin−MA, like its predecessor, collagen, maintains its susceptibility to collagenase degradation, whereas hydrogels based on the PEG derivate are not susceptible to enzymatic dissociation.16 In the present study, degradation profiles of the PEG−gelatin membrane were conducted with physiological condition (PBS) or with enzymatic condition (collagenase II, 300 U/mL).17 The percent of mass remaining of collagenase-II-treated and PBS-treated films is presented in Figure 2. The weight remaining of collagenase-II-treated films

than the silicone group since day 14 (*p < 0.05). There was a sharp increase in the PEG−gelatin group on day 14, which decreased slightly to 0.513 ± 0.096 mL at day 21 and finally remained steady until the end of the experiments (Figure 3F). Morphology results were presented by H&E staining. A small amount of seroma had accumulated in the PEG−gelatin group on day 14 (Figure 4C). While in the control group, a large amount of inflammatory provisional matrix was surrounding the adipose tissue (Figure 4A). By day 60, both groups developed well-organized adipose tissue encapsulated by a connective capsule layer (Figure 4B and D), and a much denser and thicker capsule layer could be observed in the control group. Whole-mount staining showed that both groups developed a similar structure compared to normal mature adipose tissue; meanwhile, the blood vessel interacted well with adipocytes (Figure 5A and B). The capillary area was significantly larger in the PEG−gelatin group than the silicone group (2.27 ± 0.23/ 103 μm2 vs 1.29 ± 0.16/103 μm2) (Figure 5C, *p < 0.05). This indicated that PEG−gelatin membrane assistance did not influence the regenerated adipose tissue architecture and could induce a rich vascularized adipose tissue formation. Additionally, there were more small adipocytes in the PEG−gelatin group, which were a potential precursor of mature adipocytes. Taken together, the increased capillaries and the presence of a precursor cell give the explanation of the larger construct in the PEG−gelatin group. Capsular Contracture and Fibrous Tissue Formation Assessments. Capsule formation and contracture generated centripetal force on the adipose tissue and limited its further expansion as well as the microvessel number and alignment.22,23 The components in the capsule (collagen density, spiral fashion, and the amount and distribution of myofibroblasts) are in high correlation with contractile strength.24 The collagen formation in the construct was quantified by Masson’s trichrome staining. The collagen predominated and formed the whole layer of the fibrous capsule. The capsular thickness at day 60 was 62.57 ± 12.16 μm in the PEG−gelatin group (Figure 6A), which was significantly thinner than the control group (733.25 ± 80.89 μm, Figure 6B and C, *p < 0.05). Collagen also deposited among the adipocytes, mainly toward the well-vascularized connective tissue. The amount of collagen accumulated in adipose tissue was 11.42 ± 0.43% in the PEG−gelatin group and 19.30 ± 0.66% in the control group (Figure 6D, *p < 0.05). α-SMA is the specific marker of myofibroblasts which act as the main role of capsular contracture. In the PEG−gelatin group, α-SMA positive cells were visualized at not only the deep position in the capsule but also the walls of small arteries (Figure 7A). In the control group, α-SMA was mainly expressed in the slender spindle-shaped cells at day 60, located through the whole capsule, lying parallel to the surface of the capsule (Figure 7B). This is corresponding with the previous report.25 Significantly more α-SMA positive cells were located throughout the capsule in the control group (Figure 7C). Less collagen deposition, less disorganized spiral fashion, and fewer myofibroblasts all contribute to less constraining stress formation and larger tissue in the PEG−gelatin group. Inflammatory Reaction toward the Membrane. Continuous myofibroblast activation results in synthesis, deposition of collagen, and capsular contracture.26,27 Differentiation and mutation of fibroblasts always occur with the presence of TGF-β, a cytokine that participates in the whole

Figure 2. Physiological and collagenase degradation kinematics of PEG−gelatin membranes. The remaining weight of PEG−gelatin hydrogel membrane in solutions with or without collagenase II.

dropped quickly in the first 1 day and gradually decreased to 45.1 ± 4.72% after 9 days, while the weight remained nearly the same in the PBS group. The enzymatic degradation rate of the PEG−gelatin membrane is significantly faster than in PBS solution due to the inherited cleave point from collagen. Nevertheless, the rate is much slower than the pure gelatin which should contribute to the high cross-linking density with the PEG monomer.17 Volume and Morphology Analysis of the Regenerated Adipose Tissue. In order to generate a larger adipose tissue, refinement has been made upon the operating procedures, tissue engineering chamber device, extracellular matrices, exogenous cell, or growth factors of this model.18−21 Previous studies have proved that attachment of porous electrospinning polycaprolactone (PCL) mesh to the interior surface of the silicone chamber resulted in 1.3 times larger adipose tissue.4 The initial volume was 0.116 ± 0.03 mL for the PEG−gelatin group and 0.128 ± 0.026 mL for the control group. Presentation of PEG−gelatin membrane to the chamber could generate 1.8 times larger construct (0.708 ± 0.13 mL vs 0.396 ± 0.09 mL, Figure 3D and E) after 60 days. Moreover, the PEG−gelatin group had significantly larger adipose tissue 3442

DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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ACS Biomaterials Science & Engineering

Figure 3. Chamber configuration and gross analysis of the construct. (A) Photograph of vascularized adipose tissue flap harvest. (B) PEG−gelatin membrane (scale bar = 10 mm). (C) The PEG−gelatin film was attached to the chamber with 6−0 nylon sutures. Constructs harvested at day 60 were much larger in the (D) PEG−gelatin group compared with the (E) control group. (F) Volume changes of the construct. The volume increased steadily since day 45 in both groups, and full development was achieved by day 60 (*p < 0.05).

Figure 4. Histological analysis of the constructs. At day 14, abundant inflammatory cells penetrated to the provisional matrix in the control group (A), while small scattered seroma accumulated in the PEG−gelatin group (C). At day 60, both the PEG−gelatin group (B) and silicone chamber group (D) developed a mature adipose tissue encapsulated by a connective capsule layer.

(Figure 8F). During FBR, TGF-β is first secreted by platelet degranulation during acute inflammation of FBR, and then monocytes were recruited to the implant site. After differentiation of monocytes to macrophages, TGF-β is mainly secreted by the M2 macrophage which could be marketed by CD206.29 In the PEG−gelatin group, CD206 positive cells

process of FBR.28 The expression level of TGF-β in the PEG− gelatin group was 400.65 ± 20.32 pg/mL at day 1 and then slightly decreased to 291.36 ± 15.74 pg/mL on day 60. In the control group the level of TGF-β was 287.65 ± 14.60 pg/mL initially, and it then increased to 500 ± 25.3 pg/mL in the following 7 days and remained at a high level until day 21 3443

DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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ACS Biomaterials Science & Engineering

Figure 5. Visualization of the constructs from the PEG−gelatin group (A) and silicone group (B) at day 60. Integration of the vascular system and adipocytes in regenerated adipose tissue was revealed by whole mount stain, (adipocytes: BODIPY, orange; blood vessels: lectin, green). Scale bar = 200 μm. The PEG−gelatin group has more capillaries than the silicone group (C, *p < 0.05).

Figure 6. Collagen deposited throughout the capsule and among adipocytes at day 60. Masson’s trichrome staining was conducted to quantify the collagen formation in both the PEG−gelatin group (A) and control group (B). PEG−gelatin group had both thinner capsule and less collagen content (C, D) (*p < 0.05).

Figure 7. Capsular contracture assessment. The α-SMA positive cells penetrated in the walls of some small vessels and the deeper layer of the capsule in the PEG−gelatin group (A) and in the slender spindle-shaped cells throughout the capsule in the control group (B). Significantly more α-SMA positive cells appeared in the control group (C) (*p < 0.05).

usually lied at the outermost layer of the capsule (Figure 8A). By contrast, CD206 positive cells dispersed more evenly in the whole layer in the capsule of the control group (Figure 8B). By quantification, less CD206 cells were observed in the PEG− gelatin group (*p < 0.05) (Figure 8C). Galectin-3 staining was also performed to identify the M1 macrophage, and the quantities of the M1 macrophage were few in both groups (Figure 8D and E).

The relationship between inflammation toward biomaterial and adipogenesis has been well-established. Both the intensity and the polarization state of macrophage showed a materialdependent response.30 At day 60, the M1 macrophage was few in both groups. However, a previous study showed that M1 type was dominant at day 7 in poly(L-lactic acid) (PLLA) and PEG.31 Although not observed in this experiment, the M1 macrophage often expresses some pro-inflammatory cytokines 3444

DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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ACS Biomaterials Science & Engineering

Figure 8. PEG−gelatin membrane induced minor inflammation in adipose regeneration. CD206 positive cells scattered at the outermost layer of the capsule in the PEG−gelatin group (A) and dispersed more evenly in the whole layer in the capsule of the control group (B). By quantification, less CD206 cells were observed in the PEG−gelatin group (C). Galectin-3 positive cells were few in both PEG−gelatin (D) and the control group (E). Scale bar = 50 um. TGF-β expression at a lower level in the PEG−gelatin group (F) (*p < 0.05),.

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after infiltration. As for the M2 type, the surface topography does not seem to exert a relevant effect on the polarization of the macrophage in the silicone group, and it always displays a dominant M2 macrophage subtype.32 By comparison, the continuing implantation of PEG−gelatin only recruits few M2 macrophages, presenting a minor anti-inflammatory response and tissue repair.33 TGF-β is one of the cytokine networks that participates in wound healing and in the FBR. In the presence of TGF-β, quiescent fibroblasts rapidly developed into protomyofibroblasts, which possess limited contractility, and then into differentiated myofibroblasts, leading to large contractile force generation. Due to the few amounts of M2 macrophages in the PEG−gelatin group, early secretion of TGF-β might be produced by platelets.34 Nevertheless, more M2 macrophage might secrete more TGF-β in the silicon chamber group. The response came stronger and persisted longer.35 The minor and shorter response leads to fewer myofibroblasts and less collagen deposition with the PEG−gelatin supplement. Less collagen deposition inside the regenerated tissue resulted in inapparent effective stiffness, which would promote the ingrowth of neovessels and the storage of lipid.36 Continuing exploration is emphasizing the specific mechanisms of balance between capsule formation and adipose tissue regeneration in the chamber. Sufficient knowledge of the relationship between capsule formation and adipogenesis has profound enlightenment for a larger volume of adipose tissue regeneration to reconstruct soft tissue defects in plastic and reconstructive surgery.

MATERIALS AND METHODS

Materials. Poly(ethylene glycol) diacrylate (PEG-da, molecular weight = 700 Da), type A gelatin (∼300 bloom), ammonium persulfate (APS), methacrylic anhydride (MA), and N,N,N′,N′tetramethylethane-1,2-diamine (TEMED) were purchased from Sigma-Aldrich and used without further purification. PEG−Gelatin Membrane Preparation. Gelatin−MA was synthesized following the previous protocol.37 Briefly, gelatin solution was prepared with phosphate-buffered saline (PBS) with concentration at 10 wt %, after totally dissolved at 60 °C under magnetic stirring; MA was dropwise added to the obtained gelatin solution; and the modification process was reacted for 1 h at 60 °C. The reaction was stopped by dilution with PBS, and then dialysis against distilled water with dialysis tube (10 kDa) was continued for 4 days at 40 °C. The water was changed three times every day. The solution was lyophilized and stored at −20 °C before use. Gelatin−MA foam (50 mg/mL) and PEG-da (200 mg/mL) were fully dissolved in deionized water under stirring. Gelatin−MA and PEG-da were mixed at the ratio of 1:4, and the 500 μL compound was added between two coverslips with the presence of a 7 μL initiator (APS, 200 mg/mL) and 1 μL of TEMED to form a supportive hydrogel. Then the coverslips were removed carefully, and the obtained membrane was attached to the internal side of the chamber. The original dimension of the membrane was 25 × 25 × 0.8 mm, and the membrane was cut to 22 × 18 × 0.8 mm to cover the whole chamber inner surface. SEM Characterization of Film Surfaces. A scanning electron microscope (Hitachi Company, Tokyo, Japan) was used to detect the surface characteristics of the film. The film was dehydrated and sputtered with gold before imaging. The acceleration voltage is 20 kV, and a computerized camera was used for image capture (Canon Inc., Tokyo, Japan). Attenuated Total Reflection Spectroscopy Analysis (ATR). ATR assessment of PEG-da, gelatin, gelatin−MA, and PEG−gelatin freeze-dried hydrogel was carried out. The ATR spectra were conducted in a Nicolet iS10 spectrometer equipped with a Smart iTR auxiliary (Thermo Scientific Inc., USA) by recording with spectral wavelength ranging from 700 to 4000 cm−1. Contact Angle Measurement. Contact angle measurement is the ideal method to provide information on surface hydrophilicity and wettability.38 The CA was measured in triplicate with a Goniometer (Sheng Ding, China) on five different locations of silicone chamber and PEG−gelatin hydrogel membrane. A 5 μL distilled water droplet

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CONCLUSION This study used the TEC model to successfully generate a larger adipose tissue with the assistance of the PEG−gelatin membrane to switch the hydrophilicity and biocompatibility of the interface between silicone and host tissue. Our results revealed that the PEG−gelatin membrane could reduce the extent of FBR and capsular contracture, leading to greater adipose tissue regeneration. This strategy would contribute a new perspective to the relationship between FBR and adipose tissue regeneration with the TEC model. 3445

DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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ACS Biomaterials Science & Engineering was dropped on the membrane surface; a CCD camera of the CA setup was used to capture the images, and the static CA was measured immediately. In Vitro Degradation Test. The in vitro of PEG−gelatin films was performed with or without collagenase. Films were immersed in a culture dish containing 5 mL of DPBS with or without collagenase II (300 U/mL, GIBCO, China) and set in an incubator at 37 °C for 1, 3, 5, 7, and 9 days. The hydrogel residues were washed multiple times with DPBS and dehydrated under vacuum at designed time. The percent corruption was determined by the ratio of dried weight after and before treatment. In Vivo Test. All tests were endorsed by the Nanfang Hospital Animal Ethics Committee and were directed by the guidelines of the National Health and Medical Research Council (China). Tissue Engineering Chambers (TECs). Silicone chambers were produced by the clinical research center of Nanfang Hospital (Guangzhou, China). The internal diameter of the cylindric chamber is 6 mm with a tallness of 22 mm, together to shape a 2.48 mL chamber. An opening in the wall considers the passage of veins and fat flap. Surgical Techniques and Study Design. Sprague−Dawley rats (male, 300−350 g) were anesthetized by a general method (10% chloral hydrate, 0.3 mL/100 g, intraperitoneal injection). The groin skin was depilated and then was purified with chlorohexidine and alcohol. After uncovering the skin, a fat flap (8 × 5 × 1.5 mm) was dissected free of the surrounding tissue. It was based on the superficial epigastric vessels (Figure 2A). The fat flap was inserted into the silicone chamber appropriately from the side entrance to avoid vasospasm; afterward, both ends of the chamber were totally sealed with the same silicone patches and medical wax and then moored to the surrounding muscle with a 7−0 nylon suture (Figure 2C). The injuries were closed with 4−0 sutures. In the experimental group (n = 30), the PEG−gelatin film was cut to a specific dimension to cover the whole chamber inner surface (Figure 2B) and then attached to the chamber with 6−0 nylon sutures (Figure 2C). The untreated silicon chamber group served as control (n = 30). Rats were sacrificed at 7, 14, 21, 45, and 60 days, respectively (n = 6 per time point), and the whole device including chambers, fluid, and the tissue samples were harvested. Tissue Volume Analysis. The liquid displacement was employed to measure the volume of the collected tissue. Briefly, the collected tissue was submerged in a measuring glass of saline. The volume of harvested tissue was determined equally to the volume change of the displaced saline (1 g/mL). Histological Examination. Tissues from both experiment and the control group were fixed in paraformaldehyde (4%), dried out, and embedded in paraffin. Tissue cubes were sequentially separated (5 μm areas) along the longitudinal pivot, then mounted onto a 3aminopropyl-triethoxysilane-treated glass slide. At that point, they were recolored utilizing hematoxylin and eosin. Masson’s trichrome staining was carried out following the existing protocol.4 Whole-Mount Staining. Adipose tissue from the PEG−gelatin group was processed using the whole-mount stain to assess the structure difference compared with normal adipose tissue. Tissues were sliced to around 1 mm thickness and brooded 60 min with the accompanying reagents: BODIPY 558/568-conjugated phalloidin (Molecular Probes, Eugene, Ore.) for adipocytes visualization and Alexa Fluor 488-conjugated isolectin GS-IB4 (Molecular Probes, Eugene, Ore.) for endothelial cells visualization. The sample was washed to remove the remaining reagents and photographed by a confocal microscope (Leica TCS SP2; Leica Microsystems GmbH, Wetzlar, Germany). TGF-β Expression Level. Chamber liquid was extricated through a syringe at various time points and stored at −20 °C until use. Samples were thawed and incubated at 4 °C, and cytokine levels were quantified within 2 weeks. The TGF-β protein level was measured utilizing the ELISA kits (DUMA, Shanghai, China) as per the producer’s guidance. Plates were read on a Multiskan microplate reader within 15 min (Thermo Fisher, Massachusetts, USA) at 450

nm absorbance. The enzyme-linked immunosorbent assay (ELISA) examination was performed in copy. Immunohistochemistry for α-SMA, Collagen, and CD206. Immunohistochemistry was utilized for affirming the cell types and ECM types from the examples of both groups. The sections were deparaffinized sodium citrate buffer (10 mmol/L) by microwave (10 min), followed with 3% H2O2 in methanol for 10 min to recover antigenicity. Sections taken from each time point were inspected utilizing the goat antirat α-SMA antibody (1:200), rabbit antirat collagen type I antibody (1:200), rabbit antirat CD206 antibody (1:200), and mouse antirat galectin-3 antibody (1:200) (Santa Cruz Biotechnology, San Diego, CA). Then they were incubated with secondary antibody and stained with diaminobenzidine. Slides were scored by two independents with microscopy imaging (Olympus Corp., Tokyo, Japan). The intensity of collagen type I was introduced as mean optical thickness (MOD), which was determined by integrated optical density (IOD)/area. Statistical Analysis. Statistical analyses were conducted with SPSS software (version 23.0, SPSS, Inc.). Data are shown as mean ± standard deviation. Results were compared using analysis of variance, with the post-hoc LSD test as appropriate. P value less than 0.05 was considered statistically significant.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00930. The spectrum of gelatin, gelatin−MA, PEG-da, and PEG−gelatin hydrogel (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Qiang Chang: 0000-0002-0091-7420 Malcolm Xing: 0000-0002-3547-0462 Author Contributions ∥

Zijin Qin and Qiang Chang contributed equally to this work and should be the cofirst authors.

Funding

This work was supported by the National Nature Science Foundation of China (81471881, 81372083, 81171834, and 81671931) and China Postdoctoral Science Foundation (F319NF0002). Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447

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DOI: 10.1021/acsbiomaterials.8b00930 ACS Biomater. Sci. Eng. 2019, 5, 3440−3447