Strontium ranelate-laden near-infrared photothermal-inspired methylcellulose hydrogel for arthritis treatment
用于关节炎治疗的含雷奈酸锶的近红外光热启发甲基纤维素水凝胶

https://doi.org/10.1016/j.msec.2021.111980Get rights and content 获取权利和内容

Highlights 突出

  • •
    Polysaccharide-drug delivering systems have the promise to augment therapeutic outcomes by offering controlled release of bioactive materials, diminishing the required frequency of administration, and preserving therapeutic levels in affected pathological regions.
    多糖药物递送系统有望通过提供生物活性物质的控释、减少所需的给药频率和保持受影响病理区域的治疗水平来增强治疗结果。
  • •
    Herein, an intra-articular photothermal-laden injectable methylcellulose (MC) polymeric hydrogel carrier incorporating strontium ranelate (SrR) and sodium chloride was investigated.
    在此，研究了一种包含雷奈酸锶 （SrR） 和氯化钠的关节内载光热可注射甲基纤维素 （MC） 聚合物水凝胶载体。
  • •
    Characteristics of the MC carrier system were thoroughly evaluated.
    对 MC 载体系统的特性进行了全面评估。
  • •
    The slow release of SrR, enhancement of the material mechanical strength, and the potential of the non-invasive near-infrared photothermal gel to suppress inflammation was examined. Biocompatibility and suppression of intracellular ROS-induced inflammation were observed.
    研究了 SrR 的缓慢释放、材料机械强度的增强以及非侵入性近红外光热凝胶抑制炎症的潜力。观察到生物相容性和细胞内 ROS 诱导的炎症抑制。
  • •
    This multifunctional photothermal MC hydrogel carrier is anticipated to be an alternative approach for future orthopedic disease treatment.
    这种多功能光热 MC 水凝胶载体有望成为未来骨科疾病治疗的替代方法。

Abstract 抽象

Rheumatoid arthritis (RA) is of foremost concern among long-term autoimmune disorders, as it leads to inflammation, exudates, chondral degeneration, and painful joints. Because RA severity often fluctuates over time, a local drug delivery method that titrates release of therapeutics to arthritis bioactivity should represent a promising paradigm of RA therapy. Given the local nature of RA chronic illnesses, polysaccharide-drug delivering systems have the promise to augment therapeutic outcomes by offering controlled release of bioactive materials, diminishing the required frequency of administration, and preserving therapeutic levels in affected pathological regions. Herein, an intra-articular photothermal-laden injectable methylcellulose (MC) polymeric hydrogel carrier incorporating strontium ranelate (SrR) and sodium chloride was investigated to resolve these issues. Physicochemical and cellular characteristics of the MC carrier system were thoroughly evaluated. The slow release of SrR, enhancement of the material mechanical strength, and the potential of the non-invasive near-infrared photothermal gel to improve blood circulation and suppress inflammation in a mini-surgical model of RA were examined. Biocompatibility and suppression of intracellular ROS-induced inflammation were observed. This multifunctional photothermal MC hydrogel carrier is anticipated to be an alternative approach for future orthopedic disease treatment.
类风湿性关节炎 （RA） 是长期自身免疫性疾病中最受关注的问题，因为它会导致炎症、渗出物、软骨变性和关节疼痛。由于 RA 的严重程度通常会随着时间的推移而波动，因此一种将治疗药物释放到关节炎生物活性的局部药物递送方法应该代表 RA 治疗的一个有前途的范式。鉴于 RA 慢性疾病的局部性质，多糖药物递送系统有望通过提供生物活性物质的受控释放、减少所需的给药频率和保持受影响病理区域的治疗水平来增强治疗结果。在此，研究了一种含有雷奈酸锶 （SrR） 和氯化钠的关节内充满光热的可注射甲基纤维素 （MC） 聚合物水凝胶载体来解决这些问题。对 MC 载体系统的物理化学和细胞特性进行了全面评估。在 RA 的小型手术模型中，检查了 SrR 的缓慢释放、材料机械强度的增强以及非侵入性近红外光热凝胶改善血液循环和抑制炎症的潜力。观察到生物相容性和细胞内 ROS 诱导的炎症抑制。这种多功能光热 MC 水凝胶载体有望成为未来骨科疾病治疗的替代方法。

Keywords 关键字

Strontium ranelate
Near-infrared
Photothermal
Functional methylcellulose
Drug delivery
Arthritis

雷奈酸锶
近红外
光热
功能性甲基纤维素
药物输送
关节炎

1. Introduction 1. 引言

Rheumatoid arthritis (RA) [1,2] is an inflammation illness with an unidentified etiology and complicated multifactorial pathogenesis [3]. Disease-modifying antirheumatic drugs (DMARDs) are a category of medications defined by their use in RA to retard the disease progression. Although DMARDs have revealed good disease control over RA, the long-term sequelae of RA and side effects of DMARDs are still major concerns for physicians [4]. There is a rising focus on intra-articular drug/medicine delivery systems. Numerous efforts to maximize local anti-inflammatory effects of administrated formulations have been undertaken by improving cell exposure in articular joints. This has been done by altering the position of the injection. However, researchers have determined that intra-articular drug administration is unsuccessful in producing a therapeutic effect due to rapid drug clearance from the articular joint [5]. Therefore, maintaining a therapeutic level requires a minimal injection frequency to the articular joint [6]. Therapeutic levels in articular joints for sustained periods should be accomplished by administrating injectable depot formulations [6].
类风湿性关节炎（rheumatoid arthritis， RA）[1,2]是一种病因不明、多因素发病机制复杂的炎症性疾病[3]。改善病情的抗风湿药 （DMARDs） 是一类药物，其定义为在 RA 中使用以延缓疾病进展。尽管 DMARD 显示 RA 具有良好的疾病控制，但 RA 的长期后遗症和 DMARD 的副作用仍然是医生的主要担忧 [4]。人们越来越关注关节内药物/药物输送系统。通过改善关节中的细胞暴露，已经采取了许多努力来最大限度地发挥给药制剂的局部抗炎作用。这是通过改变注射位置来完成的。然而，研究人员已经确定，由于关节内药物的快速清除，关节内给药无法成功产生治疗效果 [5]。因此，维持治疗水平需要对关节进行最低的注射频率 [6]。应通过注射长效制剂来达到关节持续治疗水平 [6]。
Strontium ranelate (SrR), used in orthopedic clinics and commercially recognized as a protos [7,8], is utilized as an oral anti-osteoporosis medicine for osteoporotic fracture prevention. SrR displays a unique dual capacity for decreasing bone resorption and enhancing bone formation [9]. Due to its dual purposes, it can avoid bone loss, increase bone strength, reduce risks of fracture [10], and also be utilized in RA treatment. However, traditional administration of SrR by oral ingestion can be challenging due to obstructive gastrointestinal (GI) mucosa with other undesired side effects. All adverse reactions are related to oral treatment and serum levels [11]. A risk of vessel occlusion associated with SrR was noted in oral medical treatment. A harmless and more-useful method of local administration of SrR is undoubtedly needed [12].
雷奈酸锶 （SrR） 用于骨科诊所，在商业上被认为是原型 [7,8]，被用作口服抗骨质疏松症药物，用于预防骨质疏松性骨折。SrR 在减少骨吸收和增强骨形成方面具有独特的双重能力 [9]。由于其双重用途，它可以避免骨质流失、增加骨强度、降低骨折风险 [10]，也可用于 RA 治疗。然而，由于胃肠道 （GI） 粘膜阻塞性以及其他不良副作用，通过口服摄入传统给药 SrR 可能具有挑战性。所有不良反应都与口服治疗和血清水平有关 [11]。在口腔药物治疗中观察到与 SrR 相关的血管闭塞风险。毫无疑问，需要一种无害且更有用的 SrR 局部给药方法 [12]。
Several niches of slow-release drug delivery [[13], [14], [15], [16], [17]] are superior to some conventional dosage forms in terms of improved patient compliance owing to a decrease in fluctuating medication levels. Furthermore, slow-release drug delivery allows for less-frequent drug administration, augments a potent drug's safety margin, and most significantly for drug utilization, shortens the therapeutic period and decreases healthcare costs via improved therapy [18]. Several polymer hydrogels [[19], [20], [21], [22], [23], [24], [25]] were established as delivery vehicles of bioactive substances and even cells in tissue engineering. Indeed, the advantageous benefits of hydrogels for delivering drugs are primarily due to drug pharmacokinetics. Depot formulations are based on which drugs elute slowly and maintain a high local concentration of medicine within the surrounding tissues over a prolonged period [26].
缓释药物递送的几个细分市场 [[13]， [14]， [15]， [16]， [17]] 由于药物水平波动的降低，在提高患者依从性方面优于一些常规剂型。此外，缓释药物递送可以减少给药频率，提高强效药物的安全边际，最重要的是，对于药物利用，缩短了治疗期并通过改进治疗降低了医疗成本 [18]。几种聚合物水凝胶 [[19]， [20]， [21]， [22]， [23]， [24]， [25]] 被确立为组织工程中生物活性物质甚至细胞的递送载体。事实上，水凝胶递送药物的有利优势主要是由于药物药代动力学。长效制剂的依据是药物洗脱缓慢，并在周围组织内长时间保持较高的局部药物浓度[26]。
Research toward developing an injectable in situ-forming polymeric hydrogel carrier has been conducted for biomedical uses in diverse fields over a long period. The sol-to-gel phase transition of these designed hydrogels rely on external stimuli such as a rapid temperature change. Unlike conventional surgical techniques, hydrogels exhibit minimal invasiveness and easy management through simple aqueous-phase administration to target organs. Recently, various polymeric materials were reported to be potential stimulus-triggered in situ-forming hydrogels. In situ-forming hydrogels that employ hydrophobic and electrostatic interactions are tremendously valuable due to their biomedical applications and mechanistic characteristics in regenerative medicine [27].
长期以来，一直在不同领域进行开发可注射原位形成聚合物水凝胶载体的研究，用于生物医学用途。这些设计的水凝胶的溶胶-凝胶相变依赖于外部刺激，例如温度的快速变化。与传统手术技术不同，水凝胶表现出最小的侵入性，并且通过简单的水相给药到目标器官且易于管理。最近，据报道，各种聚合物材料是潜在的刺激触发的原位形成水凝胶。由于其在再生医学中的生物医学应用和机制特性，采用疏水和静电相互作用的原位形成水凝胶非常有价值[27]。
Cellulose-based biomaterials are bio-durable in the body and can adapt to the rigid and mechanical load-bearing environment of bone/cartilage tissues [[28], [29], [30]]. They exhibit durability and hence support cell adhesion, integration, and viability when employed. Methylcellulose (MC) and its related derivatives were proven to be potential biomaterials [[31], [32], [33], [34], [35], [36], [37]], as they possess distinctive biochemical structures which offer a good platform for hydrogel network construction. Thus, hydrogels possess unique properties of swelling capability, bio-durability, and responsiveness toward external stimuli. Hence, they can be used for orthopedic disease treatments [38]. The effects of metal ions and salts on the sol-gel transition of MC hydrogels are of great interest and significance as they are commonly present in many systems [39]. Metal ions can be stably hydrated, revealing sturdy interactions with water molecules. Accordingly, they tend to result in “salting-out” or enhancing a solute hydrophobicity in water. The addition of metal ions to cellulose polymeric solutions dramatically decreases the cellulose gelation time because of the dehydration mechanism [40]. This implies that the addition of ions can enhance the hydrophobic aggregation of cellulose polymers thus enhancing the mechanical strength.
纤维素基生物材料在体内具有生物持久性，可以适应骨骼/软骨组织的刚性和机械承重环境 [[28]， [29]， [30]]。它们具有耐用性，因此在使用时支持细胞粘附、整合和活力。甲基纤维素 （MC） 及其相关衍生物被证明是潜在的生物材料 [[31]， [32]， [33]， [34]， [35]， [36]， [37]]，因为它们具有独特的生化结构，为水凝胶网络构建提供了良好的平台。因此，水凝胶具有独特的溶胀能力、生物耐久性和对外部刺激的响应性。因此，它们可用于骨科疾病治疗 [38]。金属离子和盐对MC水凝胶溶胶-凝胶转变的影响具有重要意义，因为它们通常存在于许多系统中[39]。金属离子可以稳定地水合，揭示与水分子的牢固相互作用。因此，它们往往会导致“盐析”或增强溶质在水中的疏水性。由于脱水机制，在纤维素聚合物溶液中添加金属离子会显著缩短纤维素凝胶化时间[40]。 这意味着离子的添加可以增强纤维素聚合物的疏水聚集，从而提高机械强度。
Clinically, hyperthermia [[41], [42], [43], [44], [45], [46]] is encouraged by altering the perfusion medium temperature in the characteristic range of ca. 38–45 °C, designated as the transition zone. Indeed, healthy cells can withstand a temperature range of 42–45 °C [47], and thus hyperthermia can increase blood flow, thereby improving tissue oxygenation and tissue wound healing [48]. The photothermal hyperthermia-aided function of polypyrrole-polyethylenimine based nanoparticles (PPY-PEI NPs) was developed for biomedical applications utilizing remote controlled near infrared (NIR) [[49], [50], [51], [52]]. However, to the best of our knowledge, MC hydrogel photothermal nanocarrier systems incorporating sustained-release strontium ralenate (SrR) for orthopedic disease treatment have rarely been investigated. It is thought that injectable drug depot-formulated bio-scaffolds with photothermal hyperthermia would increase blood flow, which could alleviate local anti-inflammatory effects of medicines and lead to successful treatment of RA. In addition, hydrogels can treat RA by absorbing extra fluids in swollen joints to relieve pain. Notably, a synergistic effect of RA treatment was hypothesized through a cellulose-based carrier system combining photothermal therapy with locally slow SrR delivery. Obtaining appropriate physicochemical properties, biocompatibility, and the therapeutic delivery facet of cellulose polysaccharides should be considered even though they have rarely been investigated. Therefore, we hypothesized that photothermal-hyperthermia NPs and SrR for loading into MC hydrogel carriers might be an effective RA treatment.
The hypothesized MC hydrogel could be used to load multiple ions such as sodium chloride (NaCl) and SrR to enhance the hydrophobic interaction of cellulose polymers to enhance the mechanical strength and serve as a firm, injectable, sustainable drug-carrier system for RA treatment. It was also hypothesized that the addition of PPY-PEI NPs could interact with MC to produce an in vivo remote photothermally boosted blood flow rate locally. Herein, MC-(PPY PEI NPs) were formulated and characterized physicochemically (by rheology, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), surface charge, and x-ray diffractometry (XRD)). Thus, we investigated whether the existence of NPs would enhance the mechanical strength of the MC hydrogel carriers (MC-NPs). The in vitro biocompatibility and in vivo therapeutic efficacy for cartilage repair and toxicity were also assessed.
假设的 MC 水凝胶可用于负载氯化钠 （NaCl） 和 SrR 等多种离子，以增强纤维素聚合物的疏水相互作用，从而提高机械强度，并作为 RA 治疗的坚固、可注射、可持续的药物载体系统。还假设添加 PPY-PEI NP 可以与 MC 相互作用，从而在局部产生体内远程光热增强血流速率。在此，对 MC-（PPY PEI NPIs） 进行了配制和物理化学表征（通过流变学、核磁共振 （NMR）、傅里叶变换红外 （FTIR）、表面电荷和 X 射线衍射法 （XRD））。因此，我们研究了 NPs 的存在是否会增强 MC 水凝胶载体 （MC-NPs） 的机械强度。还评估了体外生物相容性和体内对软骨修复和毒性的治疗效果。

2. Experimental section 2. 实验部分

2.1. Materials 2.1. 材料

Chemicals were obtained from Sigma-Aldrich (St. Louis, MO). All other cell reagents and analytical-grade chemicals were purchased from Life Technologies (Carlsbad, CA) or local Taiwanese companies.
化学品购自Sigma-Aldrich（密苏里州圣路易斯）。所有其他细胞试剂和分析级化学品均购自 Life Technologies（加利福尼亚州卡尔斯巴德）或台湾当地公司。

2.2. MC-NP preparation 2.2. MC-NP 制备

Preclinically, MC (10% concentration by weight) is generally used for biomedical applications [53]. Incorporation of PPY-PEI NPs adds a photothermal property to MC. PPY-PEI NPs were manufactured through nano-formulation, according to previous studies [49,51,52]. Concisely, the cationic polymeric PEI (600 Da, 0.2 g) was mixed with deionized (DI) water (20 mL) and blended with pyrrole (monomer, 12.5 μL). The solution was then stirred for approximately 0.5 h (at pH 0.8). Subsequently, ferric chloride hexahydrate (12.5 mg/mL, 1 mL) was slowly added to the aqueous solution, with continued stirring for 0.5 h; a black (or dark) color solution was formed, and this mixture solution was dialyzed using a dialysis bag/membrane (3000 Da) against DI water to remove impurities of either ferric ions or free PEI. Liquefied PPY-PEI NPs were then adjusted to the designated concentrations and mixed with an MC polymer solution. NaCl with SrR was added to the MC-based polymer to reduce the gelation time and to enhance mechanical strength.
临床前，MC（按重量计浓度的 10%）通常用于生物医学应用 [53]。PPY-PEI NPs的掺入为MC增加了光热性能。根据以前的研究，PPY-PEI NPs是通过纳米制剂制造的[49,51,52]。简而言之，将阳离子聚合物 PEI （600 Da， 0.2 g） 与去离子 （DI） 水 （20 mL） 混合，并与吡咯 （单体， 12.5 μL） 混合。然后将溶液搅拌约 0.5 小时（pH 值为 0.8）。随后，将六水合氯化铁（12.5 mg/mL，1 mL）缓慢加入水溶液中，继续搅拌 0.5 h;形成黑色（或深色）溶液，并使用透析袋/膜 （3000 Da） 对去离子水透析该混合物溶液，以去除铁离子或游离 PEI 的杂质。然后将液化的 PPY-PEI NP 调节至指定浓度并与 MC 聚合物溶液混合。将含 SrR 的 NaCl 添加到 MC 基聚合物中，以减少凝胶化时间并提高机械强度。

2.3. Surface charge and chemical characterization
2.3. 表面电荷和化学表征

To optimize the MC-NP hydrogel carrier colloid formulation and its characteristics, dynamic light scattering (DLS) and ζ-potential (ζ) measurements were conducted on a ZetaSizer (Malvern Instruments) for polymeric MC hydrogel solutions with different-concentration NP solutions (MC/NPs, 0/30, 100/30, 100/15, 100/0, wt/wt). The MC-NP hydrogel carrier system was chemically characterized through FTIR, nuclear NMR, and XRD.
为了优化 MC-NP 水凝胶载体胶体配方及其特性，在 ZetaSizer （Malvern Instruments） 上对具有不同浓度 NP 溶液（MC/NPs、0/30、100/30、100/15、100/0、wt/wt）的聚合物 MC 水凝胶溶液进行了动态光散射 （DLS） 和ζ电位 （ζ） 测量。通过 FTIR、核 NMR 和 XRD 对 MC-NP 水凝胶载体系统进行化学表征。

2.4. Photothermal properties
2.4. 光热特性

These designed NIR-photo-absorbable hydrogels can be used for repeated photothermal treatments. To confirm the NIR-photothermal switchable behavior, a test polymer solution was placed in an Eppendorf tube, as mentioned above, and then irradiated with an NIR laser (808 nm, for 10 min at 2.5 W/cm2). Thermographs and temperatures were simultaneously recorded using an infrared camera (A-BF, RX300) to evaluate the NIR-photothermal transitional effect.
这些设计的 NIR 光吸收水凝胶可用于重复的光热处理。为了确认 NIR 光热可切换行为，如上所述，将测试聚合物溶液置于 Eppendorf 管中，然后用 NIR 激光（808 nm，以 2.5 W/cm2 照射 10 分钟）。使用红外相机 （A-BF， RX300） 同时记录热像仪和温度，以评估 NIR 光热过渡效应。

2.5. Molecular dynamics (MD) simulation
2.5. 分子动力学 （MD） 模拟

The interaction between MC and PEI-NPs was assessed using a previously described method that acquired images of the biomolecules with atomic details generated by UCSF Chimera software [54].
使用先前描述的方法评估MC和PEI-NPs之间的相互作用，该方法获取生物分子的图像以及UCSF Chimera软件生成的原子细节[54]。

2.6. Mechanical properties of the prepared polymeric samples
2.6. 制备的聚合物样品的机械性能

An inverted Eppendorf tube test was used to examine the polymeric aqueous solutions [55]. An aqueous polymeric solution was placed in a test tube. Photographic images of the gel invert were obtained with a digital camera. The gelation time of the polymer solutions was obtained through the inversion test. A rheology meter was used to further examine rheological features, including viscosity and elasticity, of the formulated polymeric solutions. Lap shear modified test for measuring the adhesion of formulated materials to various gels. Another mechanical property of polymeric matrixes was estimated with a texture analyzer (TA; TA-XT21).
采用倒置 Eppendorf 试管法检测聚合物水溶液 [55]。将聚合物水溶液置于试管中。用数码相机获得凝胶倒置的照片图像。通过倒置试验获得聚合物溶液的凝胶化时间。流变仪用于进一步检查配制的聚合物溶液的流变特性，包括粘度和弹性。Lap 剪切改性测试，用于测量配方材料对各种凝胶的附着力。聚合物基质的另一种机械性能是用质构分析仪 （TA;TA-XT21 的 S

2.7. In vitro release
2.7. 体外放行

It is anticipated that locally administered SrR would have marked benefits for orthopedic applications [12]. MC hydrogel gelation times under different SrR concentrations were estimated to select an optimized formulation for further use. In the in vitro release study, the suspended SrR, MC-NP-NaCl (N)-SrR (with or without NIR) was incubated on a 37 °C shaker. Drug-release studies were conducted with dialysis bags/membranes (3000 Da) for 2 weeks. Dialysis technology appears to be an appealing option for studying drug release from polymer formulations or in situ depot-forming systems. Characteristics of the test of in vitro drug release for drug formulations include, but are not limited to, mimicking drug release in in vivo environments [56]. Absorption was measured at approximately 360 nm [57], and the optical density value was used to determine the concentration of the drug (SrR) released from the formulated polymer matrix. The remaining drug formulation was determined after 5 min of sonication and centrifugation at 1600g for 10 min. The presented values refer to the percentages of drug released and absorbed.
预计本地施用的 SrR 将对骨科应用产生显著的好处 [12]。估计不同 SrR 浓度下的 MC 水凝胶凝胶凝胶时间，以选择优化配方以供进一步使用。在体外释放研究中，将悬浮的 SrR、MC-NP-NaCl （N）-SrR（带或不带 NIR）在 37 °C 摇床上孵育。使用透析袋/膜 （3000 Da） 进行药物释放研究 2 周。透析技术似乎是研究聚合物制剂或原位仓库形成系统中的药物释放的一种有吸引力的选择。药物制剂的体外药物释放测试的特点包括但不限于模拟体内环境中的药物释放[56]。在大约 360 nm 处测量吸收 [57]，并使用光密度值来确定从配制的聚合物基质中释放的药物浓度 （SrR）。超声处理 5 分钟并以 1600 g 离心 10 分钟后测定剩余的药物制剂。所示值是指药物释放和吸收的百分比。

2.8. Cell viability 2.8. 细胞活力

L929 mouse fibroblasts (ATCC® CCL-1™, American Type Culture Collection, Manassas, VA) are routinely used for the in vitro assessments of the cytotoxic properties of dental materials due to their reproducible growth rates and bio-responses [58]. In this study, the SW1353 (ATCC® HTB-94™) and MG-63 (ATCC® CRL-1427™) cell lines were used as in vitro models to examine chondrocyte and osteoblast-like cell functions. Cells were individually maintained in recommended cell growth culture medium. A cell viability test was carried out after cell seeding onto a 96-well plate (10,000 cells/well) or confocal dishes (1.0 × 106 cells/dish) and co-cultured with test samples. Untreated cells (only cell medium) served as the control group. The calcein/AM assay was selected for the quantitative and qualitative examinations of cell viability. Calcein/AM is a cell-permeable dye that can be applied to determine cell viability in living cells after they interact with test materials. The non-fluorescent calcein/AM in living cells is transformed to green-fluorescent calcein after acetoxymethyl ester hydrolysis occurs with intracellular esterases. For the study of effects of NIR irradiation on cell biocompatibility, L929 cells were seeded on a 96-well plate (10,000 cells/well). After being incubated for 24 h, cells were exposed to 808-nm NIR laser irradiation for 10 min. The NIR laser power densities were set in the range of 0– 2.5 W cm−2. Once cells were exposed to NIR laser irradiation, an MTT assay was carried out to determine cell viability.
L929小鼠成纤维细胞（ATCC® CCL-1，American™ Type Culture Collection，Manassas，VA）由于其可重复的生长速率和生物反应，通常用于体外评估牙科材料的细胞毒性特性[58]。在本研究中，使用 SW1353 （ATCC® HTB-94™） 和 MG-63 （ATCC® CRL-1427™） 细胞系作为体外模型来检查软骨细胞和成骨细胞样细胞功能。将细胞单独保存在推荐的细胞生长培养基中。将细胞接种到 96 孔板 （10,000 个细胞/孔） 或共聚焦培养皿 （1.0 × 106 个细胞/皿） 并与测试样品共培养后进行细胞活力测试。未处理的细胞（仅细胞培养基）作为对照组。选择钙黄绿素/AM 测定用于细胞活力的定量和定性检查。钙黄绿素/AM 是一种细胞渗透性染料，可用于测定活细胞与测试材料相互作用后的细胞活力。在乙酰氧基甲酯与细胞内酯酶发生水解后，活细胞中的非荧光钙黄绿素/AM 转化为绿色荧光钙黄绿素。为了研究 NIR 照射对细胞生物相容性的影响，将 L929 细胞接种在 96 孔板（10,000 个细胞/孔）上。孵育 24 小时后，细胞暴露于 808 nm NIR 激光照射 10 分钟。NIR 激光功率密度设置在 0-2.5 W cm-2 的范围内。一旦细胞暴露于 NIR 激光照射下，进行 MTT 测定以确定细胞活力。

2.9. Cell interactions (live cells and reactive oxygen species (ROS))
2.9. 细胞相互作用（活细胞和活性氧 （ROS））

Lipopolysaccharide (LPS) is typically used to induce chondrocyte inflammatory injury in vitro. Cells were exposed to LPS (1 μg/mL) to simulate inflammatory lesions. Cells without LPS induction (0 μg/mL) were used as controls. The cell medium was changed every 24 h. Chondrocytes with LPS-mediated inflammatory lesions (mimicking RA) were considered in vitro cell models. LPS induces signaling by binding to specific cell surface receptors, such as Toll-like receptor 4, inducing intracellular signaling cascades that result in the activation of numerous proinflammatory signaling pathways [59]. The experimental groups included LPS-induced cells treated with SrR samples and LPS-induced cells indirectly treated with an SrR hydrogel-extracted sample (same amount of SrR). Calcein/AM was used to determine cell viability.
脂多糖 （LPS） 通常用于在体外诱导软骨细胞炎症损伤。将细胞暴露于 LPS （1 μg/mL） 以模拟炎症病变。使用未诱导 LPS 的细胞 （0 μg/mL） 作为对照。每 24 小时更换一次细胞培养基。具有 LPS 介导的炎症病变 （模拟 RA） 的软骨细胞被认为是体外细胞模型。LPS 通过与特定的细胞表面受体（如 Toll 样受体 4）结合来诱导信号传导，诱导细胞内信号级联反应，从而激活许多促炎信号通路 [59]。实验组包括用 SrR 样品处理的 LPS 诱导的细胞和用 SrR 水凝胶提取的样品 （等量的 SrR） 间接处理的 LPS 诱导的细胞。钙黄绿素/AM 用于测定细胞活力。
ROS produced during inflammation are crucial effectors connecting inflammation and the increased incidence of various chronic diseases [60]. ROS are believed to play essential biological roles in illness pathogeneses through various mechanisms by instigating injury to cell components. To determine intracellular ROS levels, cell-permeable 2′,7′-dichlorodihydrofluorescein (DCFH) diacetate (DCFH-DA, Thermo Fisher Scientific) was co-cultured with cells and hydrolyzed with DCFH cell esterases. After oxidization by ROS, DCFH is converted into the green fluorescent dichlorofluorescein (DCF). Oxidative stress (inflammatory response or severity) was measured using a DCFH-DA assay. For DCF staining, DCFH was used at a concentration of 20 μM and incubated at 37 °C for 30 min. After staining, tested cells were rinsed with phosphate-buffered saline (PBS) and maintained in cell medium. The fluorescent signal of cells was recorded using fluorescence microscopy.
炎症过程中产生的 ROS 是连接炎症和各种慢性疾病发病率增加的关键效应器 [60]。ROS 被认为通过各种机制通过刺激细胞成分损伤在疾病发病机制中发挥重要的生物学作用。为了确定细胞内 ROS 水平，将细胞渗透性 2′，7′-二氯二氢荧光素 （DCFH） 二乙酸酯（DCFH-DA，Thermo Fisher Scientific）与细胞共培养并与 DCFH 细胞酯酶水解。经 ROS 氧化后，DCFH 转化为绿色荧光二氯荧光素 （DCF）。使用 DCFH-DA 测定法测量氧化应激 （炎症反应或严重程度）。对于 DCF 染色，使用 20 μM 浓度的 DCFH 并在 37 °C 下孵育 30 分钟。染色后，用磷酸盐缓冲盐水 （PBS） 冲洗测试细胞，并维持在细胞培养基中。使用荧光显微镜记录细胞的荧光信号。

2.10. In vivo studies
2.10. 体内研究

2.10.1. Animals 2.10.1. 动物

Male Wistar rats were acquired from the BioLASCO Animal Center (Taiwan BioLASCO, Taipei, Taiwan). All in vivo studies were performed following guidelines of the Guide for the Care and Use of Laboratory Animals and the Taipei Medical University-stipulated study protocol.
雄性 Wistar 大鼠购自 BioLASCO 动物中心 （Taiwan BioLASCO， Taipei， Taiwan）。所有体内研究均按照实验动物护理和使用指南和台北医学大学规定的研究方案的指南进行。

2.10.2. RA model and formulation therapeutic effects
2.10.2. RA 模型和制剂治疗效果

RA is a chronic, systemic, inflammatory disease of unknown etiology that affects connective tissues. A rat model of RA is created [61] by a zymosan injection (15 mg/mL, 20 m microliter, at 0, 1, 5, and 7 days) administered intra-articularly with 1% ~ 4% isoflurane-anesthetized rats. After induction for 1 h, the different formulations were also administered intra-articularly. At the aforementioned time points of multiple zymosan inductions, rat knee tissues of designated groups received NIR (808 nm, 2.5 W/cm2). Tissues were sampled to analyze their inflammatory responses (interleukin (IL)-1) before sacrificing the test animals. Superficial tissues were dissected for further histological examination after sacrificing the animals. Moreover, an In Vivo Imaging System (IVIS) was used to test in vivo drug release. In this in vivo biodistribution study, Cy5 or cyanine dye [62] was selected as a model drug (represented as embedded SrR) and was blended with the prepared hydrogel. Rats received the Cy5-hydrogel carrier through the same administration route (intra-articularly) and were sacrificed for the fluorescent IVIS study to detect the released Cy5 signal 1 week after administration. A thermal camera was used to detect the photothermally responsive hydrogel carrier to validate the in situ NIR-photothermal switching effect.
RA 是一种病因不明的慢性全身性炎症性疾病，影响结缔组织。通过关节内注射酵母聚糖（15 mg/mL，20 m微升，在0、1、5和7天）与1%~4%异氟醚麻醉的大鼠进行关节内注射，建立RA大鼠模型[61]。诱导 1 小时后，也关节内施用不同的制剂。在上述多次酵母聚糖诱导的时间点，指定组的大鼠膝关节组织接受 NIR （808 nm，2.5 W/cm2）。在处死试验动物之前，对组织进行取样以分析其炎症反应 （白细胞介素 （IL）-1）。在杀死动物后，解剖浅表组织以进行进一步的组织学检查。此外，体内成像系统 （IVIS） 用于测试体内药物释放。在这项体内生物分布研究中，Cy5 或花青染料 [62] 被选为模型药物（表示为包埋的 SrR），并与制备的水凝胶混合。大鼠通过相同的给药途径 （关节内） 接受 Cy5-水凝胶载体，并在给药 1 周后处死用于荧光 IVIS 研究以检测释放的 Cy5 信号。使用热像仪检测光热响应式水凝胶载体，以验证原位 NIR 光热切换效应。

2.10.3. ROS – in vivo analysis
2.10.3. ROS – 体内分析

In light of the essential roles ROS play in tissue healing and the continued quest for therapeutic strategies to treat wounds, ROS represent a promising avenue for improving wound-healing responses. Therefore, in vivo ROS levels were studied through DCF staining [63]. The non-fluorescent DCFH-DA is transformed into a highly fluorescent DCF when the acetate groups are removed from the intracellular esterases and oxidized. DCF staining is widely used for direct ROS measurements. One hour before sacrifice, test animals received 10 μM DCFH-DA (intravenously (IV)) to facilitate the accumulation of the visible green fluorescent DCF in desired tissues. Immediately after animals were sacrificed, harvested tissues were imaged using a fluorescent microscope.
鉴于 ROS 在组织愈合中发挥的重要作用以及对治疗伤口的治疗策略的持续追求，ROS 代表了改善伤口愈合反应的有前途的途径。因此，通过 DCF 染色研究体内 ROS 水平 [63]。当乙酸盐基团从细胞内酯酶中去除并氧化时，非荧光 DCFH-DA 转化为高荧光 DCF。DCF 染色广泛用于直接 ROS 测量。处死前一小时，测试动物接受 10 μM DCFH-DA （静脉内 （IV）） 以促进可见绿色荧光 DCF 在所需组织中积累。处死动物后，立即使用荧光显微镜对收获的组织进行成像。
Before the pathological examination, treated animals were sacrificed under CO2. Tissues were harvested, fixed in 10% formalin, and dehydrated in consecutive gradients of alcohol (dehydration process) before paraffin embedding. Paraffin-embedded tissues were sectioned into slices using a microtome and stained with hematoxylin and eosin (H&E) for subsequent observations. Before sacrificing the tested animals, magnetic resonance imaging (MRI) was performed under anesthesia to gain in vivo information on the pathogenic (inflammatory) changes in the rat knee cartilage tissue. This approach served as a supplement to histology as a helpful and practical longitudinal follow-up of the in vivo cartilage defect model. A three-dimensional MRI dataset was acquired using a three-dimensional spoiled gradient-echo sequence. Gray-level MRI images were used to recognize inflammatory patterns in joints [64], which were measured by ImageJ software for further comparison.
在病理检查之前，在 CO2 下处死治疗过的动物。收获组织，用 10% 福尔马林固定，并在石蜡包埋前在连续的酒精梯度中脱水（脱水过程）。使用切片机将石蜡包埋的组织切成薄片，并用苏木精和伊红 （H&E） 染色以进行后续观察。在牺牲受试动物之前，在麻醉下进行磁共振成像 （MRI），以获得大鼠膝关节软骨组织中致病（炎症）变化的体内信息。这种方法作为组织学的补充，作为体内软骨缺损模型的有用且实用的纵向随访。使用三维变质梯度回波序列获取三维 MRI 数据集。使用灰度 MRI 图像识别关节中的炎症模式 [64]，并通过 ImageJ 软件进行测量以进一步比较。

2.11. Statistical analysis
2.11. 统计分析

Data are presented as the mean ± standard deviation (SD). Statistical evaluation of the obtained outcomes was carried out using Student's t-test. The calculated value was considered statistically significant at p < 0.05
数据以平均值±标准差 （SD） 表示。使用 Student 的 t 检验对获得的结果进行统计评估。计算值在 p < 0.05 时被认为具有统计学意义

3. Results and discussions
3. 结果和讨论

3.1. Optimal formulation of the MC-NP hydrogel carrier and its characteristics
3.1. MC-NP 水凝胶载体的最佳配方及其特性

In this study, PEI-polypyrrole-based nanomaterials were first used to formulate MC hydrogels. Their photothermal properties could induce local hyperthermia after being subjected to in vivo non-invasive NIR. The molecular structures of biocompatible, neutrally charged components applied in tissue engineering have been extensively verified [65]. Generally, neutrally charged biomaterials exhibit low cytotoxicity to cells [66] and are suitable for cartilage cell tissue-healing applications. Accordingly, zeta potentials of the formulations containing cellulose with positively charged PPY NPs with NaCl were verified. Pure MC exhibited a slightly negative charge (−2.5 mV). The NP-alone group displayed a strong positive charge (+20.1 mV). MC/NPs at 100/30 (w/w) showed a +2.0 mV charge, suggesting that the reduced amount of positively charged NPs might shield a part of the cationic property by the presence of NaCl with MC. Interestingly, the cellulose + PPY NP group at a dose of MC/NPs of 100/15 (w/w) showed a slightly cationic characteristic (+0.9 mV) close to neutrality (Fig. 1a, with NaCl). As is known, a zeta potential of +10 to −10 mV is considered nearly neutral [67], and a neutral charge should be less cytotoxic [68]. Considering slightly cationic nano-formulation systems able to bear sufficient drugs [69] through electrostatic interactions while preserving biocompatibility, the desired formulation (MC/NP, 100/15 (w/w)) was thus selected for further testing. Since PPY-based NPs were present in the hydrogel carrier, this composed hydrogel carrier could receive NIR and induce a photothermal-hyperthermic effect (Fig. 1b, imaged with a thermal camera), compared to the low temperature before NIR irradiation. Photothermal agents such as PPY can competently generate hyperthermia by converting NIR light irradiation energy to heat. The MD simulation study results showed that hydrogen bonding occurred within the MC and PEI of NPs, suggesting that stronger bonding occurred (Fig. 1c binding energy: −1092.8 ± 29.8 kcal/mol). This observed behavior could be attributed to complex formation between PEI and MC through hydrogen bonding, as reported previously [70].
在这项研究中，基于 PEI-聚吡咯的纳米材料首次用于配制 MC 水凝胶。它们的光热特性在经受体内非侵入性 NIR 后可诱导局部体温过高。组织工程中应用的生物相容性、中性电荷组分的分子结构已得到广泛验证[65]。通常，带中性电荷的生物材料对细胞的细胞毒性较低[66]，适用于软骨细胞组织愈合应用。因此，验证了含有带正电荷的 PPY NPs 和 NaCl 的纤维素的制剂的 zeta 电位。纯 MC 表现出轻微的负电荷 （−2.5 mV）。单独 NP 组显示出强正电荷 （+20.1 mV）。100/30 （w/w） 的 MC/NPs 显示出 +2.0 mV 电荷，这表明带正电荷的 NPs 量的减少可能会通过 MC 中存在 NaCl 来屏蔽部分阳离子特性。有趣的是，在 MC/NPs 剂量为 100/15 （w/w） 时，纤维素 + PPY NP 组显示出接近中性的轻微阳离子特性 （+0.9 mV）（图 1a， 与 NaCl）。众所周知，+10 至 -10 mV 的 zeta 电位被认为是近乎中性的 [67]，中性电荷的细胞毒性应该较小 [68]。考虑到轻微阳离子的纳米制剂系统能够通过静电相互作用承受足够的药物[69]，同时保持生物相容性，因此选择了所需的制剂（MC/NP，100/15 （w/w））进行进一步测试。 由于基于 PPY 的 NP 存在于水凝胶载体中，因此与 NIR 照射前的低温相比，这种组合的水凝胶载体可以接收 NIR 并诱导光热热疗效应（图 1b，用热像仪成像）。PPY 等光热剂可以通过将 NIR 光照射能量转化为热量来产生热疗。MD 模拟研究结果表明，NPs 的 MC 和 PEI 内发生氢键，表明发生了更强的键合（图 1c 结合能：−1092.8 ± 29.8 kcal/mol）。如前所述，观察到的这种行为可归因于 PEI 和 MC 之间通过氢键形成复合物 [70]。
Fig. 1
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Fig. 1. Characterization of test samples: (a) zeta potential, (b) thermal image (under near-infrared irradiation), (c) molecular dynamic simulation, (d) NMR, (e) FTIR, and (f) XRD.
图 1.测试样品的表征：（a） zeta 电位，（b） 热图像（在近红外照射下），（c） 分子动力学模拟，（d） NMR，（e） FTIR 和 （f） XRD。

As shown in Fig. 1d, the NMR data suggested that the MC anomeric Csingle bondH resonance (ca. 4.5 ppm, [71]) and PEI proton resonance of NPs (ca. 2.6–2.8 ppm, [72]) could also be observed in MC-NPs, suggesting that complexes had successfully formed. As shown in Fig. 1e, FTIR data showed that MC C-O-C (ca. 1250–1050 cm−1) and NP Cdouble bondC (ca. 1600–1400 cm−1) were also observed in MC-NPs, suggesting that complexes had successfully formed. As a result, XRD data indicated that characteristic XRD angles of an individual component (MC/NPs) also existed in the combined MC-NP complex group, implying that polymeric complex formation was achieved. XRD data of control MC polymers and MC-NPs showed the crystalline nature with a peak at 2θ of around 20 (Fig. 1f). This indicates no apparent change in the XRD intensity peak of treated MC-NPs. Clean NPs and MC-NPs showed minor border diffraction peaks at 2θ of around 30° suggesting the NP contribution.
如图 1d 所示，NMR 数据表明，在 MC-NP 中也可以观察到 MC 异头 C single bond H 共振（约 4.5 ppm，[71]）和 NP 的 PEI 质子共振（约 2.6-2.8 ppm，[72]），表明复合物已成功形成。如图 1e 所示，FTIR 数据显示，在 MC-NP 中还观察到 MC C-O-C（约 1250-1050 cm-1）和 NP C double bond C（约 1600-1400 cm-1），表明复合物已成功形成。结果，XRD 数据表明，组合的 MC-NP 复合物组中也存在单个组分 （MC/NPs） 的特征 XRD 角，这意味着实现了聚合物复合物的形成。对照 MC 聚合物和 MC-NP 的 XRD 数据显示结晶性质，在 2θ 处的峰约为 20（图 1f）。这表明处理后的 MC-NPs 的 XRD 强度峰没有明显变化。干净的 NPs 和 MC-NPs 在 2θ 处显示出 30° 左右的微小边界衍射峰，表明 NP 的贡献。

3.2. Ion effects on the cellulose hydrogel carrier and SrR release
3.2. 离子对纤维素水凝胶载体和 SrR 释放的影响

Dynamic metal-ion interactions with cellulose-constructed hydrogels can improve the mechanical properties [75]. However, to the best of our knowledge, there are limited or no research studies on the effects of multiple salts such as NaCl and SrR on the photothermal (PEI-PPY) MC sol-gel transition. A rapid sol-gel transition rate at body temperature facilitates the use of injectable gels [76]. Therefore, polymeric materials with high potential should be considered as quick-gelling injectable hydrogels
与纤维素构建的水凝胶的动态金属离子相互作用可以改善机械性能[75]。然而，据我们所知，关于 NaCl 和 SrR 等多种盐对光热 （PEI-PPY） MC 溶胶-凝胶转变影响的研究有限或没有研究。在体温下快速的溶胶-凝胶转变速率有助于使用可注射凝胶[76]。因此，应将具有高电位的聚合物材料视为快速胶凝注射水凝胶
The mechanisms underlying the transitional phase are related to salt-assisted and salt-suppressed sol-gel shifts. First, NaCl [77] was used to evaluate the MC-based polymer mixture gelation temperature due to the salting-out phenomena, and it enhanced cellulose hydrogel carriers for biomedical applications [78].
过渡期的潜在机制与盐辅助和盐抑制的溶胶-凝胶位移有关。首先，NaCl [77] 被用于评估由于盐析现象引起的基于 MC 的聚合物混合物凝胶化温度，并增强了用于生物医学应用的纤维素水凝胶载体 [78]。
In practice, rheology is principally concerned with extending continuum mechanics to characterize the flow of matter, presenting combined viscous and elastic behaviors by properly relating fluid and elasticity mechanics. We thus performed experiments on material mechanical properties to systematically examine the rheological features of our polymeric materials. As shown in Fig. 2a, rheological results revealed that mechanical properties (G′ elastic (storage) modulus and G″ viscous (loss) modulus) of the MC-NP with NaCl groups were stronger than those of the MC-NP and MC groups. Crossover transition temperatures (gel temperature and solution temperature) were obtained where G″ = G′, a temperature often termed initiation of the gelation point determined during heating. With supplementation of material additives, a lower gelation point temperature can be attained. For instance, the addition of NaCl into the MC-MP matrix reduced the gelation point of MC-NPs (Fig. 2a). Rheological data of MC-NPs suggested a lower gelation point than MC, possibly due to the addition of an NP-interacting MC intermolecular structure. Thus, for certain biomedical applications, the gelation temperature of a polymeric solution can be modified by supplementing additives.
在实践中，流变学主要关注扩展连续介质力学以表征物质的流动，通过适当地关联流体和弹性力学来呈现粘性和弹性的组合行为。因此，我们对材料机械性能进行了实验，以系统地检查我们的聚合物材料的流变特性。如图 2a 所示，流变学结果表明，含 NaCl 基团的 MC-NP 的机械性能（G′ 弹性（存储）模量和 G“ 粘性（损耗）模量）比 MC-NP 和 MC 基团强。在 G“ = G′ 中获得交叉转变温度（凝胶温度和溶液温度），该温度通常称为加热过程中确定的凝胶化点的起始温度。通过添加材料添加剂，可以获得较低的凝胶点温度。例如，在 MC-MP 基质中添加 NaCl 会降低 MC-NP 的凝胶化点（图 2a）。MC-NPs 的流变学数据表明凝胶化点低于 MC，这可能是由于添加了 NP 相互作用的 MC 分子间结构。因此，对于某些生物医学应用，可以通过补充添加剂来改变聚合物溶液的凝胶温度。
Fig. 2
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Fig. 2. Rheological properties of methylcellulose nanoparticles (MC-NPs) with NaCl (N) tested by (a) a rheological meter. (b) Gelation time of MC (with NaCl) under different strontium ralenate (SrR) concentrations were determined. (c) Quantitative and qualitative photo gelation time of test materials (from left to right: MC, MC-NPs, MC-NP-N, and MC-NP-N-SrR). (d) Texture analyzer (TA) used to estimate the test polymeric material resistance. (e) Adhesiveness experimental data. (f) In vitro drug release (SrR) of different formulations (SrR, MC-NPs-N-SrR, and MC-NPs-N-SrR + near-infrared (NIR)).
图 2.用 （a） 流变仪测试甲基纤维素纳米颗粒 （MC-NPs） 与 NaCl （N） 的流变特性 。（b） 测定不同浓度雷锶雷烯酸盐 （SrR） 下 MC （含 NaCl） 的凝胶时间。（c） 测试材料的定量和定性光凝胶时间 （从左到右：MC、MC-NPs、MC-NP-N 和 MC-NP-N-SrR）。（d） 用于估计测试聚合物材料阻力的质构仪 （TA）。（e） 粘附性实验数据。（f） 不同制剂 （SrR、MC-NPs-N-SrR 和 MC-NPs-N-SrR + 近红外 （NIR）） 的体外药物释放 （SrR）。

This polymeric MC-NP hydrogel carrier system was fabricated for potential implantation in open surgical procedures or injectable mini-surgery that requires tissue engineering. Its sol-gel properties are triggered by a temperature change. Thus it should be tailored to fit into complex tissue defects. Soft, hydrophilic, nontoxic polymeric biomaterials have lately received considerable attention for delivering active substances into defect sites in a non-invasive manner. After injection with a syringe, they can be physically crosslinked by salting-out and induced to generate drug-containing hydrogels in situ.
这种聚合物 MC-NP 水凝胶载体系统被制造用于可能植入需要组织工程的开放外科手术或可注射小型手术。它的溶胶-凝胶特性是由温度变化触发的。因此，它应该被定制以适应复杂的组织缺损。柔软、亲水、无毒的聚合物生物材料最近因以非侵入性方式将活性物质输送到缺陷部位而受到广泛关注。用注射器注射后，它们可以通过盐析进行物理交联，并诱导原位生成含药物的水凝胶。
In addition, the hydrogel carrier (MC-only) containing NaCl (N) with various amounts of SrR had a merged effect from the salting-out and salting-in interactions (Fig. 2b), which depend on the hydration properties of the constituent ions and the ion strength [39]. The gelation time was reduced with an increasing SrR concentration (Fig. 2b). Previous cell studies that reported the use of strontium (related to ranelate or chloride) at a concentration range of 0.01– 10 mM revealed positive effects on differentiation, proliferation, and generation of mineralized cellular matrix for both mesenchymal stem cells and osteoblastic lineages [79]. Considering the effective, biocompatible, and rapid gelation properties, 10 mM of SrR was chosen for this study (Fig. 2b).
此外，含有不同量SrR的NaCl（N）的水凝胶载体（仅MC）在盐析和盐析相互作用中具有合并效应（图2b），这取决于组成离子的水合特性和离子强度[39]。凝胶化时间随着 SrR 浓度的增加而缩短（图 2b）。先前的细胞研究报告了使用浓度范围为 0.01-10 mM 的锶（与雷奈酸盐或氯化物有关）揭示了对间充质干细胞和成骨细胞谱系的分化、增殖和矿化细胞基质生成的积极影响 [79]。考虑到有效、生物相容性和快速凝胶化特性，本研究选择了 10 mM 的 SrR（图 2b）。
As depicted in Fig. 2c, when SrR was added to MC-NPs-N, it changed the interaction between MC-NP-N chains with water molecules, possbly altering the sol-gel rate and transition temperature. The existence of SrR, with a high effect of salting-in, possibly resulted in a decrease in the gelation time of MC-NPs-N (Fig. 2c). Hence, MC hydrogel carrier gelation can be controlled using a simple salt mixture of NaCl with SrR. As revealed in Fig. 2d (texture analysis, TA data), MC-NPs-N-SrR and MC-NPs-N should be viscoelastic materials while MC and MC-NPs should be fluid polymeric materials. MC-NPs-N-SrR had a higher gel breaking force (higher mechanical strength) than MC-NPs-N (Fig. 2d), suggesting more-substantial mechanical properties in the MC-NPs-N-SrR. As shown in the adhesive results, the MC-NP-NaCl-SrR group had a higher adhesiveness area than that of the MC-NP-NaCl group (Fig. 2e). This suggests that the gel adhesiveness of MC-NPs-NaCl-SrR can be an applicable indicator of the retention time in a wound lesion site [80].
如图 2c 所示，当 SrR 添加到 MC-NPs-N 中时，它改变了 MC-NP-N 链与水分子之间的相互作用，可能改变了溶胶-凝胶速率和转变温度。SrR 的存在，具有高盐渍效应，可能导致 MC-NPs-N 凝胶化时间缩短（图 2c）。因此，可以使用 NaCl 与 SrR 的简单盐混合物来控制 MC 水凝胶载体凝胶化。如图 2d（织构分析、TA 数据）所示，MC-NPs-N-SrR 和 MC-NPs-N 应为粘弹性材料，而 MC 和 MC-NPs 应为流体聚合物材料。MC-NPs-N-SrR 比 MC-NPs-N 具有更高的凝胶断裂力（更高的机械强度）（图 2d），表明 MC-NPs-N-SrR 具有更丰富的机械性能。如胶粘剂结果所示，MC-NP-NaCl-SrR 组的粘附面积高于 MC-NP-NaCl 组（图 2e）。这表明MC-NPs-NaCl-SrR的凝胶粘附性可以作为伤口病变部位保留时间的适用指标[80]。
In situ gelling has been extensively examined as a vehicle for prolonged drug release. It has garnered attention because of its advantages for drug delivery, such as decreased administration frequency, enhanced patient comfort, and an ability to maintain a long-term local concentration of active compounds (such as SrR). In situ gel formation occurs because of body thermal alterations and exchange of solvents. As shown in Fig. 2f, the free form of SrR was quickly released from the dialysis bag. SrR release behaviors from the hydrogel carrier with or without NIR irradiation were similar, indicating that the NIR-driven photothermal hyperthermic effects did not affect drug release (Fig. 2f, both with and without NIR treatment). A possible mechanism is that the body temperature (37 °C) or photothermal-hyperthermia (38– 45 °C) is higher than the gelation point of the carrier (MC-NPs-NaCl, Fig. 2a). The gelation state of MC-NPs-NaCl is thus advantageous for sustained SrR release. A similar theory of research findings supports our release results [81]. Photothermal MC-based gels can be applied as a slow drug-release carrier system.
原位胶凝作为延长药物释放的载体已被广泛研究。它因其在药物递送方面的优势而受到关注，例如降低给药频率、提高患者舒适度以及能够长期保持活性化合物（如 SrR）的局部浓度。原位凝胶形成是由于体热变化和溶剂交换而发生的。如图 2f 所示，游离形式的 SrR 迅速从透析袋中释放出来。有或没有 NIR 照射的水凝胶载体的 SrR 释放行为相似，表明 NIR 驱动的光热热疗效应不会影响药物释放（图 2f，有和没有 NIR 处理）。一种可能的机制是体温 （37 °C） 或光热热疗 （38-45 °C） 高于载体的凝胶化点（MC-NPs-NaCl，图 2a）。因此，MC-NPs-NaCl 的凝胶状态有利于持续释放 SrR。类似的研究结果理论支持我们的发布结果 [81]。基于光热 MC 的凝胶可以作为缓慢释放的药物载体系统使用。

3.3. Cell viability and ROS analysis
3.3. 细胞活力和 ROS 分析

L929 cells were next tested for viability by a calcein/AM assay (Fig. 3a). In vitro results showed that viabilities of cells treated with SrR and NPs were similar to that of the untreated control group. Groups given MC(-N) or MC-NP(-N)-SrR had slightly higher cell numbers compared to the untreated cell group. LPS seemed to decrease both test cell lines (SW1353 and MG63, Fig. 3b), compared to the groups given SrR or MC-NP(-N)-SrR. NIR light external to the high absorption band of an organ's tissue water content can safely penetrate relatively deeply into living tissues [82]. Several findings indicate the biosafety of NIR technology of around 2 W/cm2 for precision treatment in numerous translational bioengineering applications [[83], [84], [85]]. L929 cell viability results showed that cells irradiated with an 808-nm laser at power densities of 0.3, 1, and 2.5 W/cm2 for 10 min of treatment revealed no significant cell death (Fig. 3c).
接下来通过钙黄绿素/AM 测定检测 L929 细胞的活力（图 3a）。体外结果表明，用 SrR 和 NPs 处理的细胞的活力与未处理的对照组相似。与未处理的细胞组相比，给予 MC（-N） 或 MC-NP（-N）-SrR 的组细胞数量略高。与给予 SrR 或 MC-NP（-N）-SrR 的组相比，LPS 似乎减少了两种测试细胞系（SW1353 和 MG63，图 3b）。器官组织含水量高吸收带外的近红外光可以安全地相对较深地渗透到活组织中 [82]。一些发现表明，在众多转化生物工程应用中，约 2 W/cm2 的 NIR 技术对精确治疗的生物安全性 [[83]， [84]， [85]]。L929 细胞活力结果表明，用 808 nm 激光以 0.3、1 和 2.5 W/cm2 的功率密度照射 10 分钟的细胞处理后，没有发现明显的细胞死亡（图 3c）。
Fig. 3
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Fig. 3. Cell viability of different formulations estimated by calcein/AM of (a) L929, (b) SW1353, and MG63 cells. (c) MTT data of L929 cells treated with different power density of NIR. Inhibition of lipopolysaccharide (LPS)-induced cellular inflammation by different indirect treatments (LPS alone, LPS‑strontium ralenate (SrR), (d) LPS-methyl cellulase nanoparticle (MC-NP)-NaCl (N)-SrR) qualitatively and (e) quantitatively.
图 3.通过钙黄绿素/AM 估计 （a） L929、（b） SW1353 和 MG63 细胞的不同制剂的细胞活力。（c） 用不同功率密度的 NIR 处理的 L929 细胞的 MTT 数据。 通过不同的间接处理 （单独 LPS、LPS-锶镍酸酯 （SrR）、（d） LPS-甲基纤维素酶纳米颗粒 （MC-NP）-NaCl （N）-SrR） 定性和 （e） 定量抑制脂多糖 （LPS） 诱导的细胞炎症。

Furthermore, LPS-stimulated intracellular ROS formation was analyzed (Fig. 3d,e). Recent investigations reported that SrR exhibits a protective biological effect by suppressing inflammation [86,87] against experimental osteoarthritis and stimulates analgesia in arthritic rodents, which was linked to suppression of proinflammatory cytokine release to joints [88]. LPS-treated cells that received SrR alone exhibited slightly inhibited intracellular ROS generation with an increment in biocompatibility (Fig. 3b,d,e), which implies that the SrR might block the release of inflammatory cytokines to bone cells or chondrocytes. When LPS-treated cells received MC-NPs(-N)-SrR, intracellular ROS formation was significantly inhibited (Fig. 3d, e). In addition to the potential existence of slowly released SrR, another possible factor that would affect the consequence is the fate of absorbed cellular inflammatory cytokines into the polymeric material or gel [89]. The presence of MC could also be related to higher cell populations, cell viability, and proliferation rates [90]. Therefore, this therapeutic strategy may reduce the number and frequency of drug doses, and even eliminate night-time administration of drugs, minimizing patient discomfort and increasing compliance.
此外，还分析了 LPS 刺激的细胞内 ROS 形成 （图 3d，e）。最近的研究报告称，SrR通过抑制针对实验性骨关节炎的炎症[86,87]并刺激关节炎啮齿动物的镇痛作用而表现出保护性生物学效应，这与抑制促炎细胞因子释放到关节有关[88].单独接受 SrR 的 LPS 处理的细胞表现出细胞内 ROS 生成的轻微抑制，生物相容性增加（图 3b、d、e），这意味着 SrR 可能阻止炎性细胞因子释放到骨细胞或软骨细胞。当 LPS 处理的细胞接受 MC-NPs（-N）-SrR 时，细胞内 ROS 的形成受到显着抑制（图 3d、e）。除了可能存在缓慢释放的 SrR 之外，另一个可能影响后果的因素是吸收的细胞炎性细胞因子进入聚合物材料或凝胶的命运 [89]。MC 的存在也可能与较高的细胞群、细胞活力和增殖率有关 [90]。因此，这种治疗策略可能会减少药物剂量的数量和频率，甚至消除夜间给药，最大限度地减少患者的不适并提高依从性。

3.4. Hydrogel carrier treatment of RA for cartilage regeneration
3.4. RA 的水凝胶载体治疗用于软骨再生

In a zymosan-induced RA rat model, animals were intra-articularly administered SrR-MC-NP(-N) hydrogels and received NIR in vivo (Fig. 4a). An IVIS was used to analyze Cy5, a fluorescent cyanine dye, representing a model drug of the hydrophilic SrR that was encapsulated into an MC carrier (MC-NP(-N)), as mentioned below. The Cy5-MC carrier was individually injected into tissue defects caused by RA. After 1 week, the hydrogel form presented a higher-intensity Cy5 signal than did the liquid form of Cy5 to the MC-based polymeric transitional sol-gel, increasing the mechanical properties while retaining the surrounding knee joint effect (Fig. 4b). Our data showed that in rats with zymosan-induced RA treated with the free-form hydrophilic model drug Cy5 alone, the compound was rapidly eliminated, with minimal accumulation at the knee. However, the Cy5 signal of Cy5-loaded MC hydrogel formulations could be traced in vivo for a longer time at the RA knee, prolonging drug release compared to the group receiving free-from Cy5 (Fig. 4b).
在酵母聚糖诱导的 RA 大鼠模型中，动物关节内施用 SrR-MC-NP（-N） 水凝胶并在体内接受 NIR（图 4a）。IVIS 用于分析 Cy5，一种荧光花青染料，代表封装在 MC 载体 （MC-NP（-N）） 中的亲水性 SrR 的模型药物，如下所述。将 Cy5-MC 载体单独注射到 RA 引起的组织缺损中。1 周后，水凝胶形式对基于 MC 的聚合物过渡溶胶凝胶表现出比液体形式的 Cy5 更高强度的 Cy5 信号，在增加机械性能的同时保留周围的膝关节效应（图 4b）。我们的数据显示，在单独使用游离型亲水模型药物 Cy5 治疗的酶聚糖诱导的 RA 大鼠中，该化合物被迅速消除，在膝关节的积累最小。然而，负载 Cy5 的 MC 水凝胶制剂的 Cy5 信号可以在 RA 膝关节的体内追踪更长的时间，与接受不含 Cy5 的组相比，延长了药物释放时间（图 4b）。
Fig. 4
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Fig. 4. (a) Surgical image of the zymosan-induced rat rheumatoid arthritis (RA) model, intra-articular administration, and near infrared (NIR) treatment. (b) IVIS image showing in vivo drug release profiles. (c) Thermal camera image showing in vivo photothermal effects in the rat knee after injection of formulations. (d) Qualitative and quantitative MRI knee joint data representing the anti-inflammatory efficacy of treatment of RA with the strontium ralenate methylcellulose (SrR MC-NP(-N)) hydrogel carrier.
图 4.（a） 酵母聚糖诱导的大鼠类风湿性关节炎 （RA） 模型、关节内给药和近红外 （NIR） 治疗的手术图像。（b） 显示体内药物释放曲线的 IVIS 图像。（c） 热像仪图像显示注射制剂后大鼠膝关节的体内光热效应。（d） 定性和定量 MRI 膝关节数据，代表用锶镂烯酸酯甲基纤维素 （SrR MC-NP（-N）） 水凝胶载体治疗 RA 的抗炎效果。

Furthermore, NIR photothermal-driven hyperthermia of the MC hydrogel carrier was assessed and compared to MC only in vivo (Fig. 4c). Also, MRI data (Fig. 4d) might support this hypothesis. Qualitative and quantitative MRI knee joint MRI results (Fig. 4d) implied that the region of articular cartilage treated with SrR MC-hydrogel carrier (MC-NP(-N))-NIR healed better and potentially suppressed inflammation (patterns of MRI hyperintense punctiform images representing and correlating with the severity of inflammatory disease) compared to the untreated group and the group treated with hydrogel only. As noted, SrR MC hydrogel carriers are a class of biomaterials with mechanical properties that provide a supportive environment for cartilage formation. For instance, thermosensitive hydrogel formulations were demonstrated to be effective in preclinical cartilage repair studies [91]. The main obstacle to hydrogels, or any biomaterial, in cartilage regeneration is the correct integration with surrounding tissues. This occurs as a result of cartilage properties hampering attachment with a graft. Polymer-based biomaterials are generally only embedded into a chondral defect, which integrates with surrounding tissues vital for hydrogel retention and new tissue development. Some techniques rely on blood clotting or clot components such as fibrinogen for hydrogel integration with the adjacent cartilage and bone. Unfortunately, blood clotting and associated responses produce fibrous repair tissue which has weaker mechanical properties. For example, microfracture surgery induces challenging fibrocartilage formation and osseous overgrowth.
此外，评估了 MC 水凝胶载体的 NIR 光热驱动热疗，并仅在体内与 MC 进行了比较（图 4c）。此外，MRI 数据（图 4d）可能支持这一假设。定性和定量 MRI 膝关节 MRI 结果（图 4d）表明，与未治疗组和仅接受水凝胶治疗组相比，用 SrR MC-水凝胶载体 （MC-NP（-N））-NIR 治疗的关节软骨区域愈合得更好，并可能抑制炎症（MRI 高信号点状图像模式代表炎症性疾病的严重程度并与之相关）。如前所述，SrR MC 水凝胶载体是一类具有机械性能的生物材料，可为软骨形成提供支持环境。例如，热敏性水凝胶制剂在临床前软骨修复研究中被证明是有效的[91]。水凝胶或任何生物材料在软骨再生中的主要障碍是与周围组织的正确整合。这是由于软骨特性阻碍了移植物的附着而发生的。基于聚合物的生物材料通常仅嵌入软骨缺损中，软骨缺损与对水凝胶保留和新组织发育至关重要的周围组织整合。一些技术依靠血液凝固或凝块成分（如纤维蛋白原）与相邻软骨和骨骼进行水凝胶整合。不幸的是，血液凝固和相关反应会产生机械性能较弱的纤维修复组织。 例如，微骨折手术会诱发具有挑战性的纤维软骨形成和骨质过度生长。
Once biological adhesion bonds a hydrogel into a cartilage explant, the mechanical bio-integrity of the irregular organ-hydrogel interface is greater than that of the bulk-hydrogel, and the in vivo growth of cartilage tissue is facilitated. Finally, the concept of tissue cartilage priming can be used for any hydrogel system to improve bio-integration of cartilage tissues. MC is commonly thought to be a bio-safe and bio-durable material. Nevertheless, despite its frequent use as an in vitro cell culture system for cartilage organs, it was rarely evaluated in a photothermal-hyperthermic preclinical setting until this investigation. Interestingly, MC-based hydrogels with NIR photothermal effects are essential for initiating blood circulation and pain relief [92]. A recent study reported the effects of heat treatment on the metabolism of cartilage matrix components, such as collagen and proteoglycans [92].
一旦生物粘附将水凝胶结合到软骨外植体中，不规则器官-水凝胶界面的机械生物完整性大于本体水凝胶的机械生物完整性，并促进软骨组织的体内生长。最后，组织软骨启动的概念可用于任何水凝胶系统，以改善软骨组织的生物整合。MC 通常被认为是一种生物安全和生物耐用材料。然而，尽管它经常用作软骨器官的体外细胞培养系统，但在这项研究之前，它很少在光热热疗临床前环境中进行评估。有趣的是，具有 NIR 光热效应的基于 MC 的水凝胶对于启动血液循环和缓解疼痛至关重要 [92]。最近的一项研究报道了热处理对软骨基质成分（如胶原蛋白和蛋白多糖）代谢的影响[92]。
As mentioned above, unique hydrogel physical properties have emerged as powerful tools in drug-delivery systems. Their porosity also enables the loading of drugs into the hydrogel matrix and their subsequently release at a rate dependent on a specific diffusing coefficient of the biomolecule or biomacromolecule via the hydrogel network. Indeed, the advantages of hydrogels for drug delivery are largely pharmacokinetic. The depot formulation is made from active agent elutes that preserve the local therapeutic concentration of the active drug in the surrounding tissues over a prolonged time, overcoming risks of side effects produced by systemic administration.
如上所述，独特的水凝胶物理特性已成为药物递送系统中的强大工具。它们的孔隙率还使药物能够加载到水凝胶基质中，然后通过水凝胶网络以取决于生物分子或生物大分子的特定扩散系数的速率释放。事实上，水凝胶用于药物递送的优势主要是药代动力学。长效制剂由活性剂洗脱液制成，可在较长时间内保持活性药物在周围组织中的局部治疗浓度，从而克服 全身给药产生的副作用风险。
Inflammation is a general response of the body to trauma. The body may respond with non-infectious inflammation when biomaterials, such as hydrogels, are implanted to promote cartilage repair. ROS formation was used to estimate the degree of healing after RA induction to evaluate cartilage repair. The damage to cartilage by zymosan is described as an inflammatory process (production of ROS). The zymosan-induced RA animals plus NIR exhibited prominent ROS generation, which indicated that the healing process was still ongoing (Fig. 5a). The RA group that received NIR showed rather high levels of intracellular ROS compared to the NIR-SrR treated group. Minimal ROS signals were detected in animals that received the SrR-hydrogel carrier (MC-NPs-N-SrR) with NIR (Fig. 5a), likely due to the locally slow release of SrR with photothermal-hyperthermic effects which accelerated the healing process (Fig. 5a). As mentioned above, ROS generated during inflammation are important effectors linking inflammation and increased incidences of various chronic diseases such as RA. In inflammatory conditions such as RA, large numbers of polymorphonuclear leukocytes and macrophages might infiltrate into the joint space. Once activated, these proinflammatory cells produce respiratory bursts that lead to high amounts of ROS.
炎症是身体对创伤的一般反应。当植入生物材料（如水凝胶）以促进软骨修复时，身体可能会对非感染性炎症做出反应。ROS 形成用于估计 RA 诱导后的愈合程度，以评估软骨修复。酵母聚糖对软骨的损害被描述为炎症过程（产生 ROS）。酵母聚糖诱导的 RA 动物加上 NIR 表现出明显的 ROS 生成，这表明愈合过程仍在进行中（图 5a）。与 NIR-SrR 处理组相比，接受 NIR 的 RA 组显示出相当高水平的细胞内 ROS。在接受 NIR 的 SrR 水凝胶载体 （MC-NPs-N-SrR） 的动物中检测到最小的 ROS 信号（图 5a），这可能是由于 SrR 的局部缓慢释放和光热热疗效应加速了愈合过程（图 5a）。如上所述，炎症过程中产生的 ROS 是将炎症与各种慢性疾病（如 RA）发病率增加联系起来的重要效应器。在 RA 等炎症性疾病中，大量多形核白细胞和巨噬细胞可能会浸润到关节腔。一旦被激活，这些促炎细胞会产生呼吸爆发，导致大量的 ROS。
Fig. 5
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Fig. 5. (a) Fluorescence reactive oxygen species (ROS) and H&E image of zymosan-induced cartilage representing anti-inflammation ability of rheumatoid arthritis (RA) after treatment (RA-near infrared (NIR), RA strontium ralenate (SrR)-NIR, RA SrR-hydrogel carrier-NIR, and cell nuclei staining by DAPI to obtain fluorescent images). (b) Interleukin (IL)-1 was used to confirm the anti-inflammation of RA after treatment, and (c) histological microscopic observations (soft organs) suggested no apparent toxic effects were found in the RA knee group treated with SrR-hydrogel carrier-NIR.
图 5.（a） 酵母聚糖诱导的软骨荧光活性氧 （ROS） 和 H&E 图像，代表类风湿关节炎 （RA） 治疗后的抗炎能力（RA-近红外 （NIR）、RA 锶雷烯酸酯 （SrR）-NIR、RA SrR-水凝胶载体-NIR，以及 DAPI 细胞核染色以获得荧光图像）。（b） 白细胞介素 （IL）-1 用于确认治疗后 RA 的抗炎作用，（c） 组织学显微镜观察 （软器官） 表明在接受 SrR-水凝胶载体-NIR 治疗的 RA 膝关节组中未发现明显的毒性作用。

Nevertheless, it is imperative to note that ROS can enhance and influence wound healing; excessive ROS production causes oxidative stress that can negatively affect wound healing. Sustained and elevated ROS have been detected in vivo and were related to impaired repair of chronic wounds. The proposed low concentrations of cellular ROS, produced in wound healing, are vital for repair [93] and represent therapeutic effects of administered formulations.
尽管如此，必须注意 ROS 可以增强和影响伤口愈合;过量的 ROS 产生会导致氧化应激，从而对伤口愈合产生负面影响。已在体内检测到持续和升高的 ROS，并且与慢性伤口修复受损有关。在伤口愈合过程中产生的低浓度细胞 ROS 对修复至关重要 [93]，并且代表了给药制剂的治疗效果。
According to the therapeutic efficacy, the group that received SrR-NIR showed middle suppression of the inflammation marker, IL-1, compared to the group treated with MC-NPs-N-SrR and NIR irradiation (Fig. 5b). Interestedly, RA rats that received MC-NP-N-SrR and NIR irradiation presented the lowest inflammation levels. A possible mechanism might have been the slow release of SrR with increased blood flow because of the photothermal effects, which further facilitates the transport of waste products, ROS, and nutrients, and contributes to reducing inflammation in vivo (Fig. 5a,b). In the H&E histological results, inflammation indications seems present in the zymosan-induced RA rat knee with NIR (Fig. 5a). As a result, RA rats that individually received SrR-NIR showed some inflammation suppression. The group treated with the SrR-hydrogel carrier plus NIR showed greater suppression of inflammation than did the other groups (Fig. 5a).
根据治疗效果，与接受 MC-NPs-N-SrR 和 NIR 照射的组相比，接受 SrR-NIR 的组显示出炎症标志物 IL-1 的中等抑制（图 5b）。有趣的是，接受 MC-NP-N-SrR 和 NIR 照射的 RA 大鼠表现出最低的炎症水平。一种可能的机制可能是由于光热效应，随着血流量的增加，SrR 的缓慢释放，这进一步促进了废物、ROS 和营养物质的运输，并有助于减少体内炎症（图 5a、b）。在 H&E 组织学结果中，酶聚糖诱导的 RA 大鼠膝关节中似乎存在炎症迹象 NIR （图 5a）。结果，单独接受 SrR-NIR 的 RA 大鼠表现出一些炎症抑制。与其他组相比，接受 SrR 水凝胶载体加 NIR 治疗的组显示出更强的炎症抑制作用（图 5a）。
As described in the published literature, treatment with SrR is associated with significantly lower osteoarthritis progression [94]. However, researchers have reported that intra-articular or systemic administration of free-form SrR failed to produce significant effects due to rapid drug clearance from articular joints [6]. Hence, to maintain therapeutic levels of therapeutics for an extended duration, a reduced injection frequency to the articular joint is indeed required. Prolonged active drug concentrations in the articular joint can be attained by administering an injectable depot formulation (Fig. 4b). Therefore, it is conceivable that injectable drug depot formulations with NIR-mediated in vivo hyperthermia developed by our group could facilitate local anti-inflammatory therapeutic effects of SrR and encourage repair of RA damage.
如已发表的文献所述，SrR 治疗与显著降低骨关节炎进展相关 [94]。然而，研究人员报告说，由于关节中的药物快速清除，关节内或全身施用游离型 SrR 未能产生显着效果 [6]。因此，为了延长治疗水平，确实需要降低关节注射频率。通过施用可注射长效制剂，可以延长关节中的活性药物浓度（图 4b）。因此，可以想象，我们小组开发的具有 NIR 介导的体内热疗的注射药物长效制剂可以促进 SrR 的局部抗炎治疗效果并促进 RA 损伤的修复。
Early hyperthermia bioactivates macrophages with cytokines found to release an adaptive immune response. A potential immunogenic role of exosomes released from hyperthermia-treated abnormal cells/lesions was previously reported [95]. Thus, cellulose-based materials are likely to be cleared with mechanical movements of activated macrophages out of the lesion with eventual excretion [96]. Cellulose-based materials employed as a stable implantable scaffold matrix for tissue engineering can be considered bio-durable and biodegradable [97]. Further, an increase in lesion oxygenation of cells appeared to be caused by an increase in local blood flow due to mild hyperthermia [98], which would presumably enhance the biodegradation of cellulose materials [97]. As shown by the histological data (Fig. 5c), no apparent toxic effects were found in the RA knee SrR-hydrogel carrier with NIR-treated group. Our findings on the biocompatibility and non-toxicity make polysaccharide-based materials great candidates for injectable biomaterials to repair cartilage [99].
早期热疗通过发现释放适应性免疫反应的细胞因子对巨噬细胞进行生物激活。既往报道过热疗处理的异常细胞/病变释放的外泌体具有潜在的免疫原性作用 [95]。因此，纤维素基材料很可能通过活化的巨噬细胞的机械运动从病灶中清除，并最终排泄[96]。用作组织工程的稳定植入式支架基质的纤维素基材料可以被认为是生物耐用和可生物降解的[97]。此外，病变细胞氧合的增加似乎是由轻度高热引起的局部血流增加引起的[98]，这可能有助于增强纤维素材料的生物降解[97]。如组织学数据所示（图 5c），在 IRI 处理组的 RA 膝关节 SrR-水凝胶载体中没有发现明显的毒性作用。我们在生物相容性和无毒方面的发现使多糖基材料成为可注射生物材料修复软骨的绝佳候选者 [99]。

4. Conclusions 4. 结论

The primary goal of this study was to investigate the impact of photothermal SrR-incorporated MC based polymeric hydrogels in inducing inflammation suppression in a rat RA model. Notably, a synergistic effect of RA treatment was expected through the cellulose-based carrier system that combined photothermal therapy with locally SrR delivery with slow release aspect. Sol-gel properties were modulated through two ions (NaCl and SrR) that produced hydrophobic effects to enhance the hydrogel mechanical strength. The sustained long-term release of active SrR of this regulated gel system was proposed in this study to inhibit RA symptoms. The drug carrier system preserved cell viability and functioned to suppress inflammation. Our hydrogel system containing SrR with sustained drug release was used to increase the therapeutic effect in a well-established zymosan-induced rat RA model. The hydrogel carrier system was physically and chemically characterized. This novel and functional photothermal MC based hydrogel drug-delivery system possesses multiple modalities for mini-surgical treatment of RA (Fig. 6). It could be one of the important drug delivery administrative approaches for advanced orthopedic technologies in the near future.
本研究的主要目标是研究光热 SrR 掺入的基于 MC 的聚合物水凝胶在大鼠 RA 模型中诱导炎症抑制的影响。值得注意的是，通过基于纤维素的载体系统，预期 RA 治疗的协同效应，该系统将光热疗法与局部 SrR 递送相结合，具有缓释方面。溶胶-凝胶性能通过两个离子 （NaCl 和 SrR） 进行调节，产生疏水效应以增强水凝胶的机械强度。本研究提出这种受调节的凝胶系统持续长期释放活性 SrR 以抑制 RA 症状。药物载体系统保留了细胞活力并起到抑制炎症的作用。我们含有 SrR 且药物持续释放的水凝胶系统用于增加已建立的酵母聚糖诱导的大鼠 RA 模型的治疗效果。对水凝胶载体系统进行了物理和化学表征。这种基于光热 MC 的新型功能性水凝胶药物递送系统具有多种 RA 小型手术治疗模式（图 6）。在不久的将来，它可能是先进骨科技术的重要药物输送管理方法之一。
Fig. 6
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Fig. 6. Methyl cellulose nanoparticle (MC-NP) carrier system possesses a near infrared (NIR) photothermal effect with the local slow-release of strontium ralenate (SrR), potentially enabling suppression of arthritis-related inflammation.
图 6.甲基纤维素纳米颗粒 （MC-NP） 载体系统具有近红外 （NIR） 光热效应，局部缓慢释放罗烯酸锶 （SrR），可能能够抑制关节炎相关的炎症。

Author information 作者信息

The first three co-authors equally contributed to this work.
前三位合著者对这项工作做出了同样的贡献。

Declaration of competing interest
利益争夺声明

The authors declare no competing financial interests and no conflicts of interest.
作者声明没有竞争性的经济利益，也没有利益冲突。

Acknowledgments 确认

This research work was financially supported by the Taiwan Ministry of Science and Technology (grant nos.: MOST 108-2320-B-038-061-MY3 and 108-2221-E-038-017-MY3).
这项研究工作得到了台湾科学技术部的财政支持（批准号：MOST 108-2320-B-038-061-MY3 和 108-2221-E-038-017-MY3）。

Conflict of interest 利益冲突

The authors (Chih-Wei Chiang, Yu-Cheng Hsiao, Pei-Ru Jheng, Chih-Hwa Chen, Yankuba B. Manga, Lekha R, Kun-Mao Chao, Yi-Cheng Ho and Er-Yuan Chuang) declare Non-financial interest and No conflicts of interest in this research work.
作者（Chih-Wei Chiang， Yu-Cheng Hsiao， Pei-Ru Jheng， Chih-Hwa Chen， Yankuba B. Manga， Lekha R， Kun-毛 Chao， Yi-Cheng Ho 和 Er-Yuan Chuang）声明在本研究工作中无经济利益和无利益冲突。

Author statement 作者陈述

This original research manuscript and our interesting/novel findings was not considered by any scientific journal yet and should attract major interest of the broad audience of Materials Science and Engineering: C. Further, we believe that this article will be appealing to a global audience of photothermal methylcellulose or related biomacromolecular hydrogel, arthritis treatment, advanced polymeric and polysaccharide functional materials, local administration of formulation of slowly released Strontium Ranelate at the same time.
这份原始研究手稿和我们有趣/新颖的发现尚未被任何科学期刊考虑，应该会引起材料科学与工程的广泛读者的重大兴趣：C. 此外，我们相信这篇文章将吸引光热甲基纤维素或相关生物大分子水凝胶、关节炎治疗、先进聚合物和多糖功能材料的全球读者。 同时局部施用缓慢释放的雷亚酸锶制剂。

References

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