Chitosan-based thermosensitive composite hydrogel enhances the therapeutic efficacy of human umbilical cord MSC in TBI rat model

Materials Today Chemistry 14 (2019) 100192

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Chitosan-based thermosensitive composite hydrogel enhances the therapeutic efficacy of human umbilical cord MSC in TBI rat model M. Yao a, b, *, d, Y. Chen a, d, J. Zhang a, F. Gao a, S. Ma a, ***, F. Guan a, b, c, ** a

School of Life Science, Zhengzhou University, 100 Science Road, Zhengzhou, 450001, PR China Center of Stem Cell and Regenerative Medicine, First Affiliated Hospital of Zhengzhou University, 40 University Road, Zhengzhou, 450052, PR China c Henan Provincial People's Hospital, No. 7 Weiwu Road, Zhengzhou, 450003, Henan, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 July 2019 Received in revised form 11 August 2019 Accepted 22 August 2019 Available online xxx

Recently, stem cellebased therapy shows great promise in treating traumatic brain injury. However, the low rate of cell engraftment and survival are two major barriers for efficacy. To improve the therapeutic effect, a new thermosensitive hydrogel based on chitosan, hydroxyethyl cellulose, hyaluronic acid, and b-glycerophosphate (CS-HEC-HA/GP) was developed in this study. This CS-HEC-HA/GP hydrogel exhibits a faster gelation process and better biocompatibility to human umbilical cord mesenchymal stem cells (hUC-MSC) versus CS/GP or CS-HEC/GP hydrogels. The suitable rheological behavior similar to brain tissue supports that the CS-HEC-HA/GP hydrogel might be a preferable neural scaffold. In addition, CS-HEC-HA/GP hydrogel loaded with hUC-MSC could enhance the retention, survival, and migration of encapsulated hUC-MSC, improve survival and proliferation of endogenous neural cells probably by secreting neurotrophic factors and inhibiting apoptosis, and thereby accelerate remodeling of brain structure and neurological function recovery in TBI rats. Thus, this hydrogel shows enormous potentials in stem cellebased neural tissue repair and regeneration. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Thermosensitive hydrogel Human umbilical cord mesenchymal stem cells Traumatic brain injury Neurological function recovery

1. Introduction Traumatic brain injury (TBI) is a common neurotrauma with high mobility and mortality, for which there is no effective treatment in clinic [1]. It is widely acknowledged that the neurons in the hippocampus, which are critical for learning and memory, are extremely vulnerable to TBI. Once injured, the neurons will suffer massive degeneration and necrosis, ultimately leading to the dysfunction of cognition and other related neural deficits [2e4]. Therefore, several approaches such as pharmacological drug treatment, neural repair and regeneration, or other rehabilitative adjuvant therapy are urgently needed to improve outcomes for TBI patients.

* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (M. Yao), [email protected] (S. Ma), [email protected] (F. Guan). d Minghao Yao and Yihao Chen contributed equally to this work and are co-first authors. https://doi.org/10.1016/j.mtchem.2019.08.011 2468-5194/© 2019 Elsevier Ltd. All rights reserved.

In recent years, it is found that stem cellebased therapy can effectively promote the neural repair in TBI through cell replacement and/or main paracrine effects including induction of differentiation, neurotrophy, and immunoregulation [5e9]. However, after stem cell implantation, all the injuries result from the mechanical damage, the rapid flow of cerebrospinal fluid, and inflammatory reaction at the lesion lead to an insufficient cell migration and survival, which restrict their therapeutic effect [10]. Therefore, it is very critical to establish an optimized microenvironment for improving the therapeutic efficacy of stem cells transplant. Hydrogels are three-dimensional cross-linked polymeric networks, and have been used extensively in tissue repair and regeneration [11,12]. They have the appropriate structural properties, including high moisture, elasticity, porous structure, and good permeability to oxygen and metabolites, which make them perfect to mimic the natural extracellular matrix. Especially, injectable hydrogels, such as chitosan (CS)ebased thermosensitive hydrogels, exhibit many advantages, including minimally invasive implantation, simple encapsulation of cells and/or biomolecules, and enhanced patient compliance [13,14]. Injectable hydrogels could enable the accurate filling of any irregular tissue defects by

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injection or spray-based approaches [15]. Herein, the authors used CS and hyaluronic acid (HA) to develop a novel biocompatible hydrogel as an artificial neural scaffold aimed to provide an optimized microenvironment for stem cell therapy in TBI. Compared with bone marrow stem cells, human umbilical cord mesenchymal stem cells (hUC-MSC), which derived from Wharton jelly of umbilical cord, have more advantages such as enriched sources, convenient collection, no adverse effects on the donor, low demand in human leukocyte antigen (HLA) matching consistency, and low risk of virus infection after transplantation [16]. CS, the cationic (1-4)-2-amino-2-deoxy-b-D-glucan, partly acetylated to the typical extent close to 0.25 is industrially produced in medical/ pharmaceutical grade from marine chitin, and can form an injectable thermoresponsive hydrogel at the presence of b-glycerophosphate (GP) [17]. GP is an organic compound naturally found in the body, which is usually used as a phosphorus supplement in the treatment of unbalanced phosphate metabolism and its venous administration has been approved by FDA [18]. As a major component of the extracellular matrix, HA not only regulates cell adhesion and growth by interacting with its specific cell receptor CD44, but also involves in the neuronal migration, neurite outgrowth, and the differentiation of embryonic stem cells and neural stem cells [19,20]. Hydroxyethyl cellulose (HEC) is one of the most important commercial soluble cellulose derivatives, which could be used as a thickening agent and therefore might reduce the usage of GP [21]. In this study, the authors fabricate a thermosensitive composite hydrogel CS-HEC-HA/GP, and loaded with hUC-MSC as an injectable scaffold aiming to treat TBI. This kind of hydrogel can maintain its liquid state below 25
C and quickly transform into a hydrogel at body temperature 37
C. The physical and biological characters of this hydrogel, including gelation time, water content, degradation property, micromorphology rheological behavior, and biocompatibility were investigated in a series of tests. The therapeutic effect of CS-HEC-HA/GP thermoresponsive hydrogel loaded with hUC-MSC was also evaluated in a moderate experimental TBI model in Sprague Dawley (SD) rats. In vivo results demonstrate that this composited hydrogel could significantly promote the therapeutic effect of hUC-MSC in TBI model compared with hUC-MSC alone. 2. Materials and methods 2.1. Synthesis and characterization of CS-HEC-HA/GP hydrogel Synthesis of CS-based thermosensitive hydrogel has been described everywhere [13,14]. In brief, 2 g CS (75e85% degree of deacetylation, 200e800 cP viscosity, Sigma-Aldrich) was sterilized under UV condition for 30 min and dissolved in 9 mL 0.1 M hydrochloric acid at room temperature with stirring for overnight to obtain a clear CS solution. After that, 50% wt GP (Sigma-Aldrich) was prepared and filter sterilized. CS/GP hydrogel was formed by drop-wise adding of 1 mL 50% GP solution into 9 mL CS solution under stirring at 4
C and then incubating at 37
C. The final concentration of CS and GP was 2% and 5%, respectively. 2 g CS and 0.2 g HEC (Sigma-Aldrich) were sterilized under UV condition for 30 min and dissolved in 9 mL 0.1 M hydrochloric acid at room temperature with stirring for overnight to obtain a clear CS-HEC solution. After that, 30% GP was prepared and filter sterilized. CS-HEC/GP hydrogel was formed by drop-wise adding of 1 mL 30% GP solution into 9 mL CS-HEC solution under stirring at 4
C and then incubating at 37
C. The final concentration of CS, HEC, and GP was 2%, 0.2%, and 3%, respectively. For preparation of CS-HEC-HA/GP hydrogel, 0.2, 0.4, or 0.6 g HA (HA sodium salt from Streptococcus equi, 1.5e1.8  106 Da, SigmaAldrich) was prepared and sterilized under UV condition for

30 min, and then mixed with CS-HEC solution. Other steps are consistent with CS-HEC/GP hydrogel process. The final concentration of HA is 0.2%, 0.4%, or 0.6%. Inverted tube test was used to determine the successful gelation process and gelation time of CS/GP, CS-HEC/GP, and CS-HEC-HA/GP hydrogels. All measurements were repeated thrice at least. The water content of these hydrogels was calculated with the following formula: D (%) ¼ [(Ww  Wd)/Ww]  100 where D denotes the water content of the hydrogels, Ww denotes the wet weights of the hydrogels, and Wd denotes the dried weights after freeze-drying. The degradation performance of the hydrogels was measured by another formula; and is as follows: L (%) ¼ [(Wi  WD)/Wi]  100 where L denotes the mass loss rate after hydrogels were immersed into artificial cerebrospinal fluid (ACSF, containing 7.254 mg/mL NaCl, 0.373 mg/mL KCl, 0.15 mg/mL NaH2PO4, 0.24 mg/mL MgSO4, 2.016 mg/mL NaHCO3, 0.222 mg/mL CaCl2, and 1.800 mg/mL glucose in deionized water) for 3, 7, 14, 21, and 28 days. The initial mass of hydrogels at day 0 before immersing into ACSF was labeled as Wi, and the mass of hydrogels after immersion for 3, 7, 14, 21, and 28 days was labeled as WD. The internal morphology CS/GP, CS-HEC/GP, and CS-HEC-HA/GP hydrogels was characterized by scanning electron microscopy (SEM, FEI Quanta200, Netherlands) after lyophilizing, breakage, and gold spraying. The specific operation is as follows: The hydrogels were prepared, frozen overnight at 80
C, and lyophilized for 3 days until their contained water was completely sublimed. The lyophilized hydrogels were fractured carefully in liquid nitrogen. The fractured surfaces of the hydrogels were coated with gold for 30 s, and the internal morphology of the hydrogels was observed by an SEM. The rheological behavior of CS/GP, CS-HEC/GP, and CS-HEC-HA/ GP hydrogels was evaluated by detecting their modulus of elasticity using a rheometer platform (Leica DHR2, Germany). A strain sweep test (0e10%) at an oscillatory frequency of 10 rad/s was performed to reveal the linear viscoelastic regime, followed by a frequency sweep test performed at a strain value in the linear regime. Correlation parameters were as follows: 20 mm parallel plate, 1,000 mm gap, 37
C, 1% strain, and 100e0.1 rad/s angular frequency. 2.2. Separation and identification of hUC-MSC The hUC-MSC were derived from Wharton jelly of the neonatal umbilical cord according to the authors modified method previously reported [22]. The morphology of the primal (P0), 1st generation (P1), and 3rd generation (P3) of hUC-MSC was observed by inverted microscope. The P3 of hUC-MSC were labeled with the following antibodies: CD44, CD90, CD105, HLA-ABC, CD34, CD45, and HLA-DR (BD Bioscience, USA) before analyzed by the FACS Calibur flow cytometer (Becton-Dickinson, USA). 2.3. Cytotoxicity of GP, HEC, HA, and hydrogels on hUC-MSC To investigate the cytotoxicity of GP, HEC, HA, and extract liquid of hydrogels (CS/GP, CS-HEC/GP, and CS-HEC-HA/GP) on hUC-MSC, the cell viability was quantitatively determined by a CCK-8 kit (Dojindo Molecular Technologies, Japan) according to the manufacture's protocol. In brief, hUC-MSC at P3 were plated in 96-well

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plate at a density of 3,000 cells/well and cultured in 100 mL DMEM/ F12 complete medium (with 10% fetal bovine serum) for 24 h. Then the culture medium was replaced with the culture medium containing GP (0%, 3%, 4%, 5%, 6%, 7%), and/or HEC (0.1%, 0.2%, 0.3%, 0.4%, 0.5%), HEC þ HA (0.2% þ 0.2%), the extract liquid of hydrogels for 1, 2, and 5 days (using 48-well plate at a density of 3,000 cells/ well), rinsed with phosphate-buffered saline (PBS, pH 7.4) and incubated in DMEM/F12 with 10 mL CCK-8 for each well for 2 h at 37
C and the absorbance of the solution was measured by a microplate reader (Bio-Rad, USA) at a wavelength of 450 nm. 2.4. 3D culture of hUC-MSC within hydrogels in vitro 3D cellular culture was established to evaluate the cell distribution and survival within CS-HEC/GP and CS-HEC-HA/GP hydrogels. The hUC-MSC at P3 was suspended in the CS-HEC/GP or CS-HEC-HA/GP mixed solution at a concentration of 5  105 cells/ mL, and then placed at 37
C condition for gelling. The hUCMSCeloaded hydrogels were cleaned with PBS thrice and cultured using fresh DMEM/F12 complete medium at 37
C in a humidified atmosphere containing 95% air and 5% CO2. Cells were fed with freshly prepared culture medium every day. After culturing for 3 days, the hUC-MSCeloaded hydrogels were stained with cellular Live/Dead kit at 37
C for 20 min. The confocal laser scanning microscope (Nikon C2 Plus, Japan) in 3D scanning mode was used to examine the hUC-MSC distribution and survival within CS-HEC/GP and CS-HEC-HA/GP hydrogels. 2.5. Animal experiments All animal experiments were performed according to the protocols approved by Zhengzhou University, the Ethical Committee, and Laboratory Administration Rules of China. Sprague Dawley rats (male, 7 weeks old, 230e250 g) obtained from the Experimental Animal Center of Zhengzhou University were used throughout this study. The hUC-MSC at P3 were used for animal experiments. Sprague Dawley rats were divided into the following four groups randomly: normal saline (NS) as the control samples, single hUC-MSC (MSC), single CS-HEC-HA/GP hydrogel (Scaffold), and hUC-MSCeloaded CS-HEC-HA/GP hydrogel (Scaffold þ MSC). 2.5.1. Evaluation of the biocompatibility and biodegradation of CSHEC-HA/GP hydrogel in vivo Hematoxylin and eosin (HE) staining was performed to investigate the immune response for the transplanted scaffold. 200 mL solution of CS-HEC-HA/GP was subcutaneously injected at the dorsum of SD rats. CS-HEC-HA/GP solution formed hydrogel quickly in situ. The hydrogels and surrounding tissues were separated out on day 7, 14, 21, and 28. Subsequently, the weight of hydrogels was measured to analyze the biodegradation rate, and the tissues were sectioned for HE staining to assess the biocompatibility of CS-HECHA/GP hydrogel in vivo.

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2.5.2. Experimental timeline The experimental timeline of procedures, treatment administration, and behavioral testing are shown in Scheme 1. The day of TBI model establishment was set as day 0. The start date of transplantation was given on day 7. Firstly, a lesion cavity will form after 7 days of injury, providing space without removing additional brain tissue. Secondly, acute inflammation should have subsided by this time theoretically, allowing the environment to become stable and more supportive of donor cell survival. The termination of TBI treatment was on day 35. The MAB1281 immunofluorescence was performed on day 14 and 35. The modified neurologic severity score (mNSS) was conducted on day 1, 3, 7, 14, 21, 28, and 35. The Morris water maze (MWM) and sucrose preference test (SPT) were performed from day 28 to 35. Novel object recognition (NOR) was conducted on day 34. All SD rats were sacrificed on day 35 to determine the damaged volume, Western blot (WB), quantitative real-time polymerase chain reaction (qRT-PCR), and other immunofluorescence analysis. 2.5.3. Establishment of a moderate TBI model in SD rats The TBI animal model was established via a typical Feeney's weight-drop method on the SD rats [10]. In brief, after intraperitoneal injection of anesthesia (chloral hydrate, 10% wt, 0.35 mL/ 100 g body weight), the SD rats underwent a normal preoperative hair removal. Then, the scalp was incised sagittally along the median line, and the fascia was bluntly dissociated to expose the right skull. Next, a hole was opened with a diameter of 5 mm located at approximately 3 mm behind the anterior fontanel and 2 mm at the right side of the centerline, with the dura left intact. Next, the SD rats were fixed on stereotaxic instruments (Shenzhen Ruiwode Lift Technology Co. Ltd, China), and a craniocerebral percussion device (Shenzhen Ruiwode Lift Technology Co. Ltd, China) was adjusted so that the striker was aimed at the center of the bone hole. Subsequently, the striker was slowed to be in contact with the dura, after a decline of 2.5 mm with a free fall of the 40 g impact hammer from the height of 20 cm, causing moderate brain injury, and rapidly following the completion of the blow. Finally, routine cleaning of the wound and hemostasis were performed, and after confirming that there was no active bleeding, the bone hole was closed with bone wax, followed by scalp suturing. The respiration and heartbeat were monitored during surgery. A total of 200,000 units of penicillin were injected intraperitoneally after completion of the surgery; and the rats were kept warm until they completely woke up and the vital signs were stable. 2.5.4. Transplantation of Scaffold and hUC-MSC Seven days (day 7) after TBI, the SD rats were anesthetized, and the primary bone hole was exposed so that an in situ injection into the center of the lesion could be carried out. In brief, the tip of the syringe was placed 1.0 mm below the dura, and 50 mL graft (CSHEC-HA/GP hydrogel loaded with 1  106 hUC-MSC, CS-HEC-HA/ GP hydrogel, 1  106 hUC-MSC, or NS) was slowly injected into the site of injury with a 1 mL syringe in a process lasting approximately

Scheme 1. Experimental timeline of procedures, treatment administration, and behavioral testing. MWM, Morris water maze; NOR, novel object recognition; SPT, sucrose preference test; TBI, traumatic brain injury.

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5 min to reduce the leakage of cells along the needle tract. After injection, the needle was maintained in vivo for an additional 5 min before it was slowly pulled out. Finally, the bone hole was closed with bone wax, followed by scalp suture. The rats were kept warm until they waked up. Routine antibiotics were applied, and the survival, limb movements, and wound healing of the SD rats were regularly observed. 2.5.5. Retention, survival, and migration of transplanted hUC-MSC Seven days (day 14) and 28 days (day 35) after the hUC-MSC injection, the complete brain tissues of the SD rats were obtained, snap-frozen in liquid nitrogen, and cryo-sectioned for histological analysis. To investigate the retention and survival of the hUC-MSC in the brain tissue, and the migration of hUC-MSC from lesion to hippocampus, the cross sections of samples in Scaffold þ MSC and MSC groups were fixed with 4% paraformaldehyde and stained with antibodies specific for transplanted hUC-MSC (MAB1281, Merck, Germany). 2.5.6. Lesion volume analysis Frozen sections of the lesion site on day 35 after treatment in TBI SD rats were prepared to measure the volume of brain injury. Serial coronal sections were made at 2.0 mm before and after the lesion site, with the thickness of 20 mm for each brain slice. One piece was randomly selected from each 10 consecutive brain slices for crystal violet (CV) staining. Image J software was used to analyze the lesion area of each group. The brain injury volume formula is given below: brain injury volume (unit: mm3) ¼ average injury area  number of brain slices n  10  2%.

2.5.7. Neurological motor function and motion assessment On day 1, 3, 7, 14, 21, 28, and 35 after TBI, the neurological motor function and mood behavior of the SD rats (3e6 rats each group) was evaluated and scores by the double-blind method according to the mNSS. The criteria of mNSS scoring was to evaluate the degree of motor, sensory, balance, and reflexes in rats: from 0 points (healthy) to 18 (most severe). The NOR test was also conducted on day 34. Each rat was placed in a large opaque box (80 cm  60 cm  40 cm) to acclimate for 10 min. On day 35, the rats were moved to the box with two identical objects (building blocks) and the video recorder was turned on. The rats were allowed to explore the box for 10 min and then moved back to home cage for 1 h. In the second testing phase, one object was replaced by a novel object with different shape and color. The rats were returned to the box and the video recorder was turned on for 5 min exploration. Before each phase of NOR test, the object and box were cleaned with 75% ethanol. Cumulative time spending on each object, including sniffing or climbing, was recorded by a blinded observer. The time spent on the novel object was calculated as discrimination index; and the formula is given below: d (%) ¼ [tn/(tn þ to)]  100 where d denotes the discrimination index, tn denotes the time spending on the novel object, and to denotes the time spending on the old object. The emotion of TBI rats was evaluated by the SPT from day 28 to 35. During the first 3 days, all the rats were singly housed in individual cage, and two bottles of 500 mL of 1% sucrose water were available in each cage. After 3 days, one bottle was replaced by 500 mL of pure water. Preference of sucrose was measured in the next 4 days. Percent of sucrose consumption was considered as sucrose preference index; and the formula is as follows:

p (%) ¼ [s/(sþw)]  100 where p denotes the sucrose preference index, s denotes the sucrose consumption, and w denotes the water consumption. 2.5.8. Morris water maze The learning ability and memory recall of the SD rats from day 28 to 35 were investigated using an MWM system (diameter 1.2 m, depth 0.6 m), with a platform (diameter 10 cm) located 2 cm underwater. Before experimentation, rats were trained four times in 20-min intervals with the start position randomly changed. The platform was marked with ‘‘B” so that rats could position themselves and search for the platform. Before entering the water, the rats were placed on the platform for 10 s to become familiar with the surrounding environment. Then, the rats were repositioned far away from the platform so that they were required to search for the marked platform. If the rats were not able to find the marked platform in 60 s, they would be placed there once more for an additional 10 s to re-acclimatize themselves with the surrounding environment and were given the test once more. The water temperature ranged from 19
C to 21
C, and the entire test process was filmed with a camera. The escape latency of the rats and the length of time on the marked platform were recorded. Finally, the platform was removed, and the rats were repositioned far away at the same place mentioned above, and the swimming trails, the number of passes through the platform, and the length of time that the rats remained in the platform quadrant in 60 s were recorded. 2.5.9. Western blot On day 35, four rats in each group were randomly selected and sacrificed under anesthesia. Brain tissue protein was extracted and subjected to WB. In brief, tissues were lysed in 200 mL lysis buffer and the protein amount was examined. Then equal amount of protein from cell lysates was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After that, primary antibodies against NeuN (specific marker for mature differentiated neuron), BDNF (neurotrophic factor), and BAX (promotor of cell apoptosis, Proteintech Group, Wuhan, China) were added, followed by horseradish peroxidaseeconjugated goat anti-rabbit IgG secondary antibodies. bActin was used as an internal control. The protein expression was performed by Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA). 2.5.10. RNA extraction and qRT-PCR On day 35, four rats in each group were randomly selected and sacrificed under anesthesia. Total RNA was extracted from brain tissues and cells by using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and was reversely transcribed into cDNAs using the reverse transcription kit (Takara, Shiga, Japan). The cDNA template was synthesized through qRT-PCR using SYBR Premix Dimmer Eraser kit (Takara) by the ABI7900 system (Thermo Fisher Scientific). The relative expressions of NeuN and BDNF in the brain were calculated by the 2-DDCt method and normalized to the internal control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 2.5.11. Immunofluorescence On day 35, the brain tissues of the SD rats were obtained, snapfrozen in liquid nitrogen, and cryo-sectioned for histological analysis. To evaluate the neural cell survival and proliferation of the injected grafts, all the samples were stained with antibody for cell proliferation (Ki-67, Proteintech Group, Wuhan, China).

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2.6. Statistical analysis Statistical analysis was made by two-way analysis of variance for multiple comparisons. A p-value of <0.05 was considered to be statistically significant. Error bars represent mean ± SD of biological replicates. 3. Results and discussion 3.1. Design and optimization of CS-based thermosensitive hydrogel Chitosan-based thermosensitive hydrogels have been widely used in cartilage tissue engineering, cornea endothelium reconstruction, drug-loaded implants, and so on [23e25]. This hydrogel can maintain its liquid formation at 4
C, whereas it transforms into gel formation at body temperature 37
C, which is convenient for application in tissue regeneration. However, a high GP concentration is essential for the gelation process. Excess GP leads to the cellular dehydration because of high external osmotic pressure around cells, and thus restrict the further application of CS/GP hydrogel [17,26]. Decrease of GP content will compromise the gelation time, which is adverse to the practical injection performance. More than 30 min is needed for gelation when GP content is less than 5%. Yan et al. [26] and Naderi-Meshkin et al [27] had developed CS-based thermosensitive hydrogels with a lower GP content (4% or 3.24%) by adding a thicker HEC, thereby promoting the cytocompatibility of hydrogel. In addition, as the major component of extracellular matrix, HA not only regulates cell adhesion and growth by interacting with its specific cell receptor CD44, but also involves in the cell migration and neurite outgrowth [19,20]. Therefore, the study authors chose CS, HEC, and HA to fabricate a modified injectable CS-HEC-HA/GP hydrogel as the composite scaffold for loading hUC-MSC aimed to treat TBI. The gelation time was determined by the inverted tube test. No visible flow within 60 s was regarded as the criteria for gel formation when the vial was vertically inverted. Results indicated that CS solution with 5% GP could form hydrogel at 37
C, CS or CS-HA solution (data not shown) with 3% GP could not gel at 37
C, whereas the CS-HEC or CS-HEC-HA solution with 3% GP could form

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hydrogel (Fig. 1A). As shown in Fig. 1B, CS with 5% GP formed hydrogel quickly within 9 min, the presence of HEC accelerated the gelation process to 7 min, and HA could further reduce gelation time to 3 min. This phenomenon probably attributed to the thickening effect by HEC and the formation of hydrogen bond among HEC, HA, and CS. From Fig. 1C and D, the water content of CS-HECHA hydrogel gradually raised with the increasing of HA concentration from 0.2% to 0.6% because of the high water absorption and moisture retention of HA, whereas the stability gradually decline but all better than that of CS/GP hydrogel. The property of high water content might contribute to reduce the frictional irritation to environment [28]. High stability more than 28 days could effectively protect the loaded cells from leakage in vivo and satisfy the long-term recovery process of brain trauma. The micromorphology of CS/GP, CS-HEC/GP, and CS-HEC-HA/GP hydrogels is shown in Fig. 1EeG. Fig. 1eeg is the amplification of Fig. 1EeG, respectively. All hydrogels possess a sequential and porous network, and their pore diameters range from hundreds to tens of microns. The relatively large pore size makes these hydrogels beneficial for the permeation of nutrients, exchange of oxygen and carbon dioxide, discharge of metabolites, and so on, which might provide a friendly microenvironment for cells survival and proliferation. The linear viscoelastic behavior of hydrogels was characterized by oscillatory frequency sweep measurements; and the storage modulus was shown in Fig. 1H. All samples except CS/GP hydrogel have a storage modulus ranging from 100 Pa to 200 Pa at an angular frequency of 100 rad/s, similar to that of brain tissue (100e1,000 Pa) [29,30]. This might contribute to the survival and proliferation of endogenous nerve cell and neural differentiation of exogenous MSC. 3.2. Isolation, culture, and identification of hUC-MSC The hUC-MSC grew out from the Wharton's jelly from day 7 to 14 after isolation, and displayed a monolayer of bipolar spindle-like or fibroblast-like morphology with a whirlpool-like array (Fig. 2A). Flow cytometric results revealed that hUC-MSC was positive to stromal/MSC markers (CD44, CD105, CD90), integrin markers

Fig. 1. (A) Inversed gelation experiment of CS/GP5% (containing 5% GP), CS/GP3% (containing 3% GP), CS-HEC/GP (containing 3% GP and 0.2% HEC), and CS-HEC-HA/GP (containing 3% GP, 0.2% HEC and 0.2% HA) mixture; (B) gelation time, (C) water content, and (D) degradation rate of CS/GP5%, CS-HEC/GP, and CS-HEC-HA/GP hydrogels. “þHEC” means CS-HEC/GP, “þ0.2%” means CS-HEC-HA/GP containing 0.2% HA, “þ0.4%” means CS-HEC-HA/GP containing 0.4% HA, “þ0.6%” means CS-HEC-HA/GP containing 0.6% HA. Micromorphology of (E) CS/GP hydrogel, (F) CS-HEC/GP hydrogel, and (G) CS-HEC-HA/GP hydrogel containing 0.2% HA by SEM; e, f, and g is the amplification of E, F, and G, respectively. (H) Rheological behavior of CS/GP hydrogel, CS-HEC/GP hydrogel, and CS-HEC-HA/GP hydrogels with 0.2%, 0.4%, or 0.6% HA. Measurements were performed at 1% strain, pH 7.4, and 37
C. CS, chitosan; GP, b-glycerophosphate; HA, hyaluronic acid; HEC, hydroxyethyl cellulose.

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Fig. 2. (A) The morphology of the primary (P0), 1st generation (P1), and 3rd generation (P3) of human umbilical cord mesenchymal stem cells (hUC-MSC) observed by inverted microscope, Arrow: Wharton Jelly, scale bars ¼ 200 mm; (B) cells labeled with the following antibodies: CD44-FITC, CD105-APC, CD29-PE, CD90-FITC, HLA-ABC-FITC, CD34-APC, CD45-PE, and HLA-DR-PE analyzed by the FACS Calibur flow cytometer.

(CD29), and human leukocyte antigen HLA-ABC, whereas negative for hematopoietic endothelial markers (CD34, CD45) and leukocyte antigen HLA-DR (Fig. 2B). This is consistent with what has been reported in the literature [22,31]. 3.3. Cytotoxicity of the compositions of hydrogel for hUC-MSC The cytotoxicity of GP, HEC, HA, and extract liquid of hydrogels was evaluated by CCK-8 assay. From Fig. 3A, the cytotoxicity of GP dramatically increased once GP content exceeded 3%, whereas there was almost no difference among 4%e7%. HEC had nearly no cytotoxicity to hUC-MSC (Fig. 3B). GP with 0.2% HEC and 0.2% HA exhibited a better cytocompatibility compared with single GP (Fig. 3C). In addition, from the result of Fig. 3D, the survival rate of

hUC-MSC treated by the extract liquid of CS/GP hydrogel was 40% on day 1, 38% on day 2, and 23% on day 5, whereas the survival rate of cells treated by the extract liquid of CS-HEC/GP hydrogel was 73% on day 1, 56% on day 2, and 60% on day5, and the survival rate of cells treated by the extract liquid of CS-HEC-HA/GP hydrogel was 89% on day 1, 82% on day 2, and 72% on day 5. It is obvious that the introduction of HEC and HA could significantly improve the cytocompatibility of hydrogel. 3.4. The distribution and survival of hUC-MSC in 3D culture model The distribution and survival of hUC-MSC within the CS-HEC/GP and CS-HEC-HA/GP hydrogels on day 3 were observed by the laser scanning confocal microscope, and the results were displayed in

Fig. 3. Cytotoxicity of GP (A), HEC (B), GP þ HEC and GP þHEC þHA (C), and hydrogels extract liquid (D) for human umbilical cord mesenchymal stem cells (hUC-MSC) detected by CCK-8 assay after culturing for 2 days (*p<0.05). CS, chitosan; GP, b-glycerophosphate; HA, hyaluronic acid; HEC, hydroxyethyl cellulose.

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Fig. 4. Confocal fluorescence images of human umbilical cord mesenchymal stem cells (hUC-MSC) within CS-HEC/GP hydrogel and CS-HEC-HA/GP hydrogel on day 3. Cells stained with Calcein-AM/PI. Green labels the living cells and red labels the dead cells. CS, chitosan; GP, b-glycerophosphate; HA, hyaluronic acid; HEC, hydroxyethyl cellulose.

Fig. 5. (A) CS-HEC-HA/GP hydrogels in situ forming under skin (the arrows pointing to the hydrogels), (B) weight variation of GH hydrogels on day 7, 14, 21, and 28, (C) hydrogels in vivo, and (D) HE staining of tissues surrounding hydrogels on day 7, 14, 21, and 28. CS, chitosan; GP, b-glycerophosphate; HA, hyaluronic acid; HEC, hydroxyethyl cellulose.

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Fig. 4. Both hydrogels clearly exhibited a homogeneous distribution of hUC-MSC, whereas the CS-HEC-HA/GP hydrogel group presented an obviously higher hUC-MSC density versus the CS-HEC/GP hydrogel group. These results indicated that this injectable and thermosensitive hydrogel enhanced the homogeneous distribution of cells, and HA promotes the proliferation of hUC-MSC. Based on the physical property and cytocompatibility results, it is clear that CS-HEC-HA/GP hydrogel is a better choice as the scaffold for loading hUC-MSC compared with CS-HEC/GP and CS/GP hydrogel. Therefore, CS-HEC-HA/GP hydrogel was selected for the further study in vivo. The transplantation treatment was performed on day 7 after TBI model establishment. Seven days after injury, a lesion cavity will form and allow for transplantation without removing additional brain tissue. Afterward, the climax of acute inflammatory stage should have subsided by this time, allowing the environment to become relative stable and more supportive for cell transplantation and survival.

3.5. Biocompatibility and biodegradation of CS-HEC-HA/GP hydrogel in vivo As a bioscaffold applying in tissue engineering and regeneration, hydrogel should possesses an excellent biocompatibility and biodegradation in vivo [11,32,33]. Therefore, the biocompatibility and biodegradation of CS-HEC-HA/GP hydrogel under skin were detected and analyzed before further study in vivo. CS-HEC-HA/GP solution rapidly formed hydrogel in situ under skin (Fig. 5), and this hydrogel could be completely degraded within 28 days (Fig. 5BeC). From HE staining result in Fig. 5D, it was hardly to observe the inflammatory cells in the tissues around hydrogels, which suggest that this hydrogel has excellent histocompatibility and less immune response in vivo on day 7, 14, 21, and 28. Therefore, it is clear that this hydrogel possesses good biocompatibility and biodegradation in vivo.

Fig. 6. MAB1281 staining of the brain lesion region of traumatic brain injury (TBI) Sprague Dawley (SD) rats in MSC and ScaffoldþMSC groups on day 14 (A) and day 35 (B). MAB1281 staining of DG region in hippocampus of TBI SD rats on day 35 (C). The red color indicates positive expression of MAB1281; the nuclei were stained with DAPI and are blue in color. The arrows are pointing to the positive staining of MAB1281. MSC, mesenchymal stem cells.; DAPI, 4',6-diamidino-2-phenylindole, used to stain nuclei; DG, dentate gyrus.

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Fig. 7. (A) Images of brains on day 35 after traumatic brain injury (TBI); (B) damaged volume of NS, Scaffold, MSC, and Scaffold þ MSC transplanted groups on day 35 after TBI (*p<0.05 compared with NS, #p<0.05 compared with MSC control, mean ± SD, n ¼ 4); and (C) crystal violet staining of the injured brain tissue with the arrow pointing at the injury location. MSC, mesenchymal stem cells; NS, normal saline.

3.6. CS-HEC-HA/GP hydrogel promotes the retention, survival, and migration of injected hUC-MSC

3.7. CS-HEC-HA/GP hydrogels loaded with hUC-MSC minish the lesion volume after TBI in rats

To further observe the retention, survival, and migration of hUCMSC of ScaffoldþMSC and MSC groups after implantation, immunofluorescence staining for MAB1281 [34] was performed on the rats’ brain slice (Fig. 6). Tissue samples of lesion region in both ScaffoldþMSC and MSC groups expressed positive MAB1281 marker on day 14 (Fig. 6A) and 35 (Fig. 6B); and it is clear that the ScaffoldþMSC group exhibited more MAB1281 cells compared with pure MSC groups, which indicated that the CS-HEC-HA/GP scaffold effectively protected the retention and survival of the hUC-MSC within the brain lesion of the TBI SD rats as long as 28 days. Furthermore, lots of MAB1281 fluorescence was detected in DG region of hippocampus (Fig. 6C), which indicated that the transplanted hUC-MSC could migrate to hippocampus. The increased positive staining of MAB1281 in the ScaffoldþMSC group compared with the MSC group, suggesting more migration of hUC-MSC from the lesion location to hippocampus in the ScaffoldþMSC group.

The damaged volume of each group was analyzed on day 35. The images, CV staining, and damaged volume analysis were shown in Fig. 7. These results demonstrated that the damaged volume of TBI rats treated by MSC, and ScaffoldþMSC transplantation after 28 days were significantly minished compared with NS control. In particular, the damaged volume of ScaffoldþMSC group was remarkable smaller than the MSC group, which indicate that the combined ScaffoldþMSC transplantation could accelerate the repair of neural trauma. 3.8. CS-HEC-HA/GP hydrogel loaded with hUC-MSC promotes the neurological motor function recovery, ameliorates the depression after TBI in rats An mNSS [35] was performed to assess the extent of neurological motor function recovery in a blinded way at defined time

Fig. 8. (A) mNSS score, (B) discrimination index, and (C) sucrose preference index of the NS, Scaffold, MSC, and Scaffold þ MSC transplanted traumatic brain injury (TBI) Sprague Dawley (SD) rats. (*p<0.05 compared with NS and Scaffold control, #p<0.05 compared with the MSC control, mean ± SD, n ¼ 4). mNSS, modified neurologic severity score; MSC, mesenchymal stem cells; NS, normal saline.

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Fig. 9. Escape latency (A), time in platform quadrant (B), swimming trails (C), time in platform quadrant (D), and times crossing platform (E) of the Sham, NS, Scaffold, MSC, and Scaffold þ MSC transplanted traumatic brain injury (TBI) Sprague Dawley (SD) rats were recorded from day 30 to 35 (*p<0.05 compared with NS and Scaffold control, #p<0.05 compared with the MSC control, mean ± SD, n ¼ 4). MSC, mesenchymal stem cells; NS, normal saline.

intervals after TBI. The results showed that the motor ability of the rats gradually recovered over time as reflected by the low mNSS scores; and for all the implanted groups, there was no significantly greater recovery of the motor ability of the rats within day 21. However, Scaffold þ MSC and MSC groups displayed markedly reduced mNSS scores on day 21, 28, and 35 after TBI compared with the Scaffold and NS groups. In particular, the Scaffold þ MSC group showed better motor ability recovery compared with the pure MSC group (#p<0.05) on day 35 after treatment (Fig. 8A). From Fig. 8B, the rats in both MSC and Scaffold þ MSC groups spent significantly more time on the novel object exploring versus NS group, suggesting an improvement of identification ability after the transplantation treatment. As shown in Fig. 8C, there was an obviously higher sucrose preference index in the MSC and Scaffold þ MSC groups than NS group, indicating the anesis of depression after TBI in rats. Moreover, the Scaffold þ MSC group showed lessened depression than that of MSC only group. 3.9. CS-HEC-HA/GP hydrogel loaded with hUC-MSC improves the learning and memory ability after TBI in rats To evaluate the impairment of learning and memory in TBI rats, the MWM experiment [10] was performed on day 35 after TBI. For spatial navigation trial, TBI rats in MSC and Scaffold þ MSC groups remarkedly reduced latency compared with NS control groups from day 31 to 34 (Fig. 9A). For spatial probe trial, a significantly increase in time in platform quadrant on day 33 and 34 (training period,

Fig. 9B), time in platform quadrant on day 35 (test period, Fig. 9D), and times crossing platform on day 35 (Fig. 9C and E) was observed in MSC and Scaffold þ MSC groups versus NS control group. However, transplantation of Scaffold þ MSC presented a higher performance in the previously mentioned parameters than that of the MSC group. These results demonstrated that Scaffold þ MSC transplantation has a better effect in ameliorating the learning and memory ability of rats and promoting the recovery of neurological function after TBI than single MSC transplantation. 3.10. CS-HEC-HA/GP hydrogel loaded with hUC-MSC increases the cell survival and proliferation by secreting BDNF and inhibiting cell apoptosis Western blotting of NeuN, BDNF, and BAX was also performed to investigate the curative effect of injected hUC-MSC on brain injury repair. Fig. 10A and B shows that the Scaffold þ MSC and MSC groups exhibited a clearly higher expression of NeuN and BDNF, and a lower expression of BAX compared with the control groups, which improved the survival/proliferation of nerve cells and suppression of nerve cell apoptosis. Furthermore, the expression of BDNF in the Scaffold þ MSC group was notably higher than that of the MSC group, and this indicated that the Scaffold þ MSC group contributed to the enrichment of neurotrophic factor. The qRT-PCR characterization showed the expression of NeuN and BDNF genes of each group (Fig. 10C). The Scaffold þ MSC and MSC groups clearly exhibited higher mRNA expression levels for all the detected genes

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Fig. 10. (A) Western blotting images of NeuN, BDNF, and BAX in the brain tissue of NS1, Scaffold2, MSC3, and (Scaffold þ MSC)4 groups on day 35; b-actin was used as a protein loading control; (B) quantitative analysis of Western blotting (WB) results; (C) The mRNA expression levels of BDNF and NeuN genes in the brain tissue of NS, Scaffold, MSC, and Scaffold þ MSC groups on day 35 (*p<0.05 compared with NS control, #p<0.05 compared with MSC control, mean ± SD, n ¼ 4); (D) NeuN and Ki67 staining of the hippocampus of traumatic brain injury (TBI) Sprague Dawley (SD) rats on day 35. The green color indicates positive expression of NeuN; the red color indicates positive expression of Ki67; the nuclei were stained with DAPI and are blue in color. The arrows are pointing to the positive staining of Ki67. The scale bar is 100 mm. MSC, mesenchymal stem cells; NS, normal saline.

compared with the other groups; and the mRNA expression levels of Scaffold þ MSC group is observably higher than that of the MSC group. These results demonstrated that the transplantation of CSHEC-HA/GP hydrogel loaded with hUC-MSC promoted the survival/proliferation of endogenous neurons by suppress apoptosis and neurotrophic effect. Moreover, the immunofluorescence was also used to study the contribution of hUC-MSC to the survival/proliferation of endogenous nerve cells. The more positive expression of NeuN and Ki67 in Scaffold þ MSC and MSC groups versus other groups demonstrated that the exogenous hUC-MSC clearly promoted the survival and proliferation of endogenous nerve cells in the hippocampus of TBI SD rats on day 35 (Fig. 10D). More importantly, samples of Scaffold þ MSC group showed a higher positive expression of NeuN and Ki67 as compared with that of the pure MSC group, suggesting the excellent MSC loading and release ability of this scaffold.

4. Conclusions In summary, a new thermosensitive hydrogel CS-HEC-HA/GP composed of CS, HEC, HA, and GP, was successfully developed for optimizing the microenvironment of transplanted hUC-MSC and consequently promoted the neurological function recovery of TBI model in rats. This kind of hydrogel requires lower amount of GP, but possesses quicker gelation rate and better biocompatibility compared with CS/GP hydrogel. After injection into the lesion location of the cerebral injury SD rats for 28 days, CS-HEC-HA/GP scaffold loaded with hUC-MSC made a greater contribution to the recovery of their motor, learning, and memory abilities compared with hUC-MSC alone and other groups. In addition, the results of WB, qRT-PCR, and immunofluorescence staining suggested that

hUC-MSC laden CS-HEC-HA/GP hydrogel transplantation not only increases the retention and survival of hUC-MSC in lesion location, but also makes a positive contribution to the survival and proliferation of endogenous neural cells probably by nutrition supply and apoptosis suppression. All these results highlight the tremendous beneficial potential of this new thermosensitive CS-HEC-HA/ GP hydrogel loaded with hUC-MSC in the future translational studies for TBI. Acknowledgements The project was supported by the National Natural Science Foundation of China (NSFC 31700820), Joint Fund of the National Natural Science Foundation of China and Henan province (U1804198), China Postdoctoral Science Foundation (2017M612420), and Key Scientific Research Projects of higher education institutions in Henan province (18A180003). Competing financial interests The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtchem.2019.08.011. References [1] A.I.R. Maas, N. Stocchetti, R. Bullock, Moderate and severe traumatic brain injury in adults, Lancet Neurol. 7 (8) (2008) 728e741.

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