Thermosensitive chitosan/gelatin/β-glycerol phosphate hydrogel as a

The release profile of FA from C/G/GP hydrogel was shown in Fig. 4. 7. The C/G/GP hydrogel released ~2% of FA in the first 0.5 hours, and the cumulative concentration of FA in the PBS was 1.058 ± 0.740 μM. As shown in Fig. 4. 2, this cumulative concentration of FA in the first 0.5 hours was enough to terminate the free radical reaction induced by 100 μM H2O2. Ruel-Gariépy et al. [62] reported that almost 80% of hydrophilic compounds incorporated in the thermosensitive C/GP hydrogel were released in the first 24 hours. On the contrary, C/GP hydrogel containing hydrophobic compounds can sustain the delivery for at least 1 month [63]. In our study, the C/G/GP hydrogel showed sustained slow release of FA after 24 hours (Fig. 4. 7).

The results showed that C/G/GP hydrogel have the potential to be used as sustained-release system for hydrophobic compounds such as FA.

In the degenerated NP, loss of proteoglycans, decrease in type II collagen and increase in type I collagen were observed, however, the changes of collagen are not as significant as those of proteoglycans [1]. Aggrecan is the major component of the proteoglycans, which play an important role to resist external compressive loads. As shown in Fig. 4. 8, the mRNA gene expression of aggrecan was down-regulated in Gel

group compared with control group. There was no significant difference in the expression of aggrecan between the control group and Gel-FA group. Overproduction of ROS can influence several cellular signaling pathways including apoptosis, differentiation and inflammation [45]. The pro-inflammatory mediators such as interlukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) inhibit the synthesis of the desired matrix and promote the MMPs production [4]. FA is a phenolic compound and is known to down-regulate pro-inflammatory gene expression, which may inhibit the proteoglycans degradation [45]. Fig. 4. 8 showed that there was no significant difference between the control group and Gel group in the expression of type II collagen.

The expression of type II collagen was significantly up-regulated in Gel-FA group compared with Gel group. Li et al. [75] reported resveratrol, an antioxidant, can prevent proteoglycans degradation. In our study, we propose that FA might treat H2O2-induced oxidative stress NP cells to normal status by up-regulation Aggrecan; moreover, FA might promote desired ECM synthesis (type II collagen).

High level of MMPs have been found in degenerated NP, which can degrade the proteoglycans and type II collagen of the ECM [2]. It has been reported that overproduction of ROS can up-regulate the MMPs activity by intracellular signaling

Gel group, and then express in normal level in Gel-FA group. As known, FA is a free radical scavenger that may contribute to the anti-inflammatory effect by down-regulation of MMP-3.

TGF-β, BMP-7 and IGF-1 are known as anabolic factors which can promote the synthesis of proteoglycans and type II collagen [21, 69]. However, recent studies demonstrated that the up-regulation of TGF-β might relate to inflammatory reaction in the degenerated NP [9, 22]. The strong expression of TGF-β reflects that the disc cells attempt to repair the degraded ECM. In addition, it has been shown that ROS can stimulate the expression of TGF-β in different types of cells [23, 25-26]. Liu et al. [28]

reported that ROS induced the expression of TGF-β via activation of MMPs. As shown in Fig. 4. 8, the expression of TGF-β was up-regulated in Gel group, and then down-regulated to normal level in Gel-FA group. We could expect the increase of TGF-β with an associated increase in the expression of MMP-3 as shown in Fig. 4. 8.

In the normal NP, proteoglycans and type II collagen play a role to provide the mechanical support to the disc. Proteoglycans consist of core protein with attached GAGs such as chondroitin sulfate and keratin sulfate. In the degenerated NP, a decrease in the content of chondroitin sulfate results in a decrease of water content that affects the capability of NP to absorb the external stress [1]. Overproduction of ROS can

directly damage the cell and degrade the ECM [71-73]. Fig. 4. 9 showed the sulfated GAGs to DNA ratio of Gel group is significantly lower than normal NP cells (control group). The release of FA from C/G/GP hydrogel can terminate the free radical reaction, and then prevent NP cells from the further damage caused by ROS. As shown in Fig. 4.

9, the sulfated GAGs to DNA ratio was reached to the normal level in the Gel-FA group.

The results of alcian blue staining also showed that Gel-FA group was stained positive in sulfated GAGs (Fig. 4. 10(c)). The results indicated that the release of FA from C/G/GP hydrogel might treat H2O2-induced oxidative stress NP cells to produce desired ECM.

Apoptosis via intrinsic pathway can be initiated by oxidative stress which stimulates cytochrome c release from mitochondria. The formation of cytochrome c-Apaf-1 (apoptotic protease activating factor-1) complex can activate the procaspase-9 that results in the activation of caspase-3. Activation of caspase-3 is strongly associated with apoptosis [37-38, 74]. As shown in Fig. 4. 11, the caspase-3 activity was significantly increased in Gel group compared with control group. The caspase-3 activity was decreased to normal level in Gel-FA group. DNA fragmentations can be observed in apoptotic cells [40]. The results of TUNEL staining also showed that there were lots of

less TUNEL-positive cells in Gel-FA group (Fig. 4. 12(c)). Li et al. [75] suggested that resveratrol, an antioxidant, can down-regulate the expression of IL-1 which might response to oxidative stress induced apoptosis. Kim et al. [36] reported that caspase inhibitors can significantly inhibit the apoptosis of disc cells which induced by oxidative stress. In our study, the results demonstrated that FA can inhibit apoptosis in NP cells by inactivation of capase-3 activity.

5.3 The effects of thermosensitive ferulic acid-immobilized chitosan/gelatin/β-glycerol phosphate hydrogel on nucleus pulposus cells under hydrogen peroxide-induced oxidative stress

FA was immobilized on gelatin by EDC/NHS crosslinking method. The carboxyl group of FA can react to amino group of gelatin to form the amide bond. After immobilization, the residual amino group of gelatin was significantly decreased compared with gelatin group (without immobilization of FA) as shown in Fig. 4. 13.

The results indicated that FA was successfully immobilized on C/G/GP hydrogel. The mechanism of antioxidant action of FA is through the hydroxyl group of FA which has the ability to donate a proton, and then to form the phenoxy radical. Phenoxy radical is a stable structure, which has five different resonance structures due to electron delocalization [44]. In this study, FA was immobilized on gelatin through amide bond formation; therefore, we believe that this immobilization process would not affect the antioxidant property of FA.

In the previous study [59], we suggested that the gelation mechanism of the thermosensitive C/G/GP hydrogel included hydrophobic interaction, hydrogen bonding, electrostatic interaction and molecular chain movement. The hydrophobic interactions

chitosan and gelatin at 37C. This type of gelation mechanism has also been reported in other cases [54, 58]. In the study, we believe that the immobilization reaction between the carboxyl group of FA and the amino group of gelatin would not affect the gelation properties of developed hydrogel. The results of rheological analysis indicated that FA-immobilized C/G/GP solution could turn into a gel within 1 minute at 37C and remain liquid for 15 minutes at 25C (Fig. 4. 14). The results suggested that developed hydrogel also had good gelation and handling property for clinical application.

The results of cytotoxicity test showed that FA-immobilized C/G/GP hydrogel was biocompatible (Fig. 4. 15). The concentration of FA in the FA-immobilized C/G/GP hydrogel was 500 μM. In the study, we demonstrated that there was no significant cytotoxicity to NP cells if the concentration of FA was lower than 500 μM. Recent studies indicated that the apoptosis of NP cells could be induced by 100 μM H2O2 and the content of H2O2 was 0.13 ± 0.09 μmole in the human degenerative NP [34, 41]. As shown in Fig. 4. 2, we found that the oxidative stress induced by 100 μM H2O2 can be significantly decreased to normal level by 0.5 μM of FA. As shown in Fig. 4. 16(b), the cumulative concentration of FA released from developed hydrogel was 1.167 ± 0.179 μM within 30 minutes that was enough to stop the free radical reaction induced by 100 μM H2O2. As shown in Fig 4.8, the percentage of cumulative release of FA from

FA-incorporated C/G/GP hydrogel was 16.6 ± 2.1% in the first 2 hours, and it could be significantly decreased to 9.3 ± 0.7% in the FA-immobilized hydrogel (Fig. 4. 16(b)).

The results demonstrated that immobilization of FA on C/G/GP hydrogel could sustain the delivery and prolong the release period of FA compared with the direct incorporation of FA.

Oxidative stress can induce inflammation through activation of nuclear factor-kappa B (NF-κB) and then up-regulate the expression of pro-inflammatory gene such as IL-1β and iNOS [45-76]. As shown in Fig. 4. 17, the mRNA gene expression of iNOS and IL-1β were significantly up-regulated in C/G/GP group compared with control

group. Anti-inflammatory effects of FA have been demonstrated in recent years [44, 77].

FA is a phenolic acid which can inhibit the activation of NF-κB by inhibition of IκB kinase activity and down-regulate the expression of pro-inflammatory genes [45]. Fig. 4.

17 showed that there was no significant difference in the expression of iNOS and IL-1β between the FA-immobilized C/G/GP group and control group. The results indicated that FA release from the developed hydrogel could reduce the level of inflammation caused by oxidative stress.

In the degenerative NP, the imbalance between anabolisms and catabolism of the

collagen [1, 8, 18]. As shown in Fig. 4. 17, the expression of type II collagen and aggrecan were significantly down-regulated in C/G/GP group compared with control

group. There was no significant difference in the expression of aggrecan between the control group and FA-immobilized C/G/GP group. The expression of type II collagen was significantly up-regulated in FA-immobilized C/G/GP group compared with other groups. Recent studies showed that polyphenol has the ability to prevent proteoglycan degradation [75]. In the study, we believe that FA might not only prevent proteoglycan degradation but also promote type II collagen synthesis. Urban et al. [1] indicated that the changes of proteoglycan are more obvious than those of collagen in the degenerative NP. As shown in Fig. 4. 17, there was no significant difference in the expression of type I collagen between the control group and experimental group (C/G/GP and

FA-immobilized C/G/GP group).

In the normal NP, there is a balance between MMPs and TIMPs. MMPs have ability to degrade ECM components including proteoglycans and type II collagen [2].

Recent studies indicated that overproduction of ROS can promote MMPs production through an increase of pro-inflammatory mediators [4, 45]. Activation of MMPs can induce the expression of TGF- which can inhibit the MMPs activity by up-regulation of TIMPs [4, 78]. As shown in Fig. 4. 17, the expression of MMP-3, TIMP-1 and TGF-

were significantly up-regulated in C/G/GP group compared with control group. The results suggested that the up-regulation of TGF- might reflect that H2O2-induced oxidative stress NP cells attempt to balance the rate of synthesis and degradation of matrix by up-regulation of TIMP-1. There was no significant difference in the expression of MMP-3, TIMP-1 and TGF- between the FA-immobilized C/G/GP group and control group. High expression of TGF- has been observed in the degenerative NP that is associated with inflammation [9, 22]. The results showed that FA could reduce cellular inflammation caused by oxidative stress. BMP-7 and IGF-1 are known as anabolic factors which can stimulate the proteoglycans and collagen synthesis [21]. As shown in Fig. 4. 17, there was no significant difference in the expression of BMP-7 and IGF-1 between the experimental group and the control group.

Aggrecan is a major proteoglycan in the NP and consists of core protein with attached glycosaminoglycan (GAG) including chondroitin sulfate and keratin sulfate.

The decrease in the GAG content has been observed in the degenerative NP [1]. Fig. 4.

18 showed that the sulfated GAG production per cell was significantly decreased in C/G/GP group compared with other groups. The sulfated GAG content was reached to the normal level in the FA-immobilized C/G/GP group.

the release of cytochrome c from mitochondria and activates the caspase-9 activity.

Activation of caspase-9 can lead to activation of caspase-3 which can cleave cellular substrate and result in apoptosis [37-39]. As shown in Fig. 4. 19, the caspase-3 activity was significantly increased in C/G/GP group compared with control group. The caspase-3 activity was decreased to normal level in FA-immobilized C/G/GP group.

The results of TUNEL staining showed that there were lots of TUNEL-positive cells in C/G/GP group (Fig. 4. 20(b)), but only a few TUNEL-positive cells in both control group (Fig. 4. 20(a)) and FA-immobilized C/G/GP group (Fig. 4. 20(c)). Recent studies indicated that high level of ROS can directly damage both cells and ECM [71-73].

Polyphenol family has been demonstrated to have ability to inhibit apoptosis by decreasing ROS levels [45, 75]. FA processes an excellent antioxidant property that can inhibit oxidant reaction caused by ROS. In the study, the results showed that FA could inhibit the apoptosis of H2O2-induced oxidative stress NP cells and prevent further cellular damage.