负载银和重组人碱性成纤维细胞生长因子的甲基丙烯酸酐化明胶水凝胶对兔深Ⅱ度烧伤创面的影响

Effects of methacrylic anhydride gelatin hydrogel loaded with silver and recombinant human basic fibroblast growth factor on deep partial-thickness burn wounds in rabbits

目的探讨负载银和重组人碱性成纤维细胞生长因子(rh-bFGF)的甲基丙烯酸酐化明胶(GelMA)水凝胶对兔深Ⅱ度烧伤创面的影响。方法采用实验研究方法。制备含不同浓度甲基丙烯酸酐(MA)的低浓度MA明胶(GelMA)材料、中浓度GelMA材料和高浓度GelMA材料,加入光引发剂后分别制得低浓度GelMA水凝胶、中浓度GelMA水凝胶和高浓度GelMA水凝胶。采用核磁共振波谱仪检测前述3种浓度GelMA材料的氢核磁共振谱并根据波谱图计算其取代度,采用场发射扫描电子显微镜(FESEM)检测前述3种浓度GelMA水凝胶的三维微观结构及孔径,样本数均为9。根据前述筛选出的MA浓度合成含10种浓度银的GelMA(含银GelMA)溶液,将每种浓度的含银GelMA溶液均分为3份,加入光引发剂后分别暴露于紫外光下持续20、25、35 s,制得相应的含银GelMA水凝胶。采用胶原酶降解法测定不同光交联时间含银GelMA水凝胶降解12、24、36、48 h的降解剩余率及彻底降解所需时长,样本数为5。测定前述筛选出光交联时间下含10种浓度银GelMA水凝胶对金黄色葡萄球菌的抑菌圈直径反映其抑菌能力,样本数均为5。以与含最低浓度银(即不含银)GelMA水凝胶抑菌圈直径相比有统计学意义的含银GelMA水凝胶为有抑菌活性。选取具有抑菌活性的且载药浓度最低的含银GelMA水凝胶,采用FESEM检测其三维微观结构及孔径,采用能谱仪检测其内部银元素的存在情况,样本数均为9。将冻干单纯GelMA水凝胶和冻干含银GelMA水凝胶分别浸没于磷酸盐缓冲液中24 h,通过称重法计算并比较2种水凝胶的溶胀率,样本数为5。根据预实验及前述实验结果,制备含银和rh-bFGF的GelMA水凝胶(简称复合水凝胶)。大体观察复合水凝胶的外观,并采用FESEM检测其三维微观结构与孔径。取30只4~6个月龄、雌雄各半日本大耳兔,在其背部制作深Ⅱ度烧伤创面。以兔头侧为基准,将脊柱左侧创面作为复合水凝胶治疗组,右侧作为纱布对照组,2组创面分别作相应处理。观察伤后3、7、14、21、28 d创面愈合情况;记录伤后7、14、21、28 d创面愈合面积并计算其愈合率,样本数为30。对数据行重复测量方差分析、单因素方差分析、独立样本t检验。结果低浓度GelMA材料、中浓度GelMA材料及高浓度GelMA材料的取代度,差异明显(F=1628.00,P<0.01)。低浓度GelMA水凝胶存在疏松、不规则三维空间网状结构,孔径为(60±17)μm;中浓度GelMA水凝胶的三维空间网络、孔径大小均较均匀规则,孔径为(45±13)μm;高浓度GelMA水凝胶的三维空间网状结构致密、层次混乱,孔径为(25±15)μm。3种GelMA水凝胶孔径大小差异有统计学意义(F=12.20,P<0.01),选取(MA)中浓度为后续材料制作浓度。相同光交联时间下的含不同浓度银GelMA水凝胶的降解性基本一致;20、25、35 s光交联时间下含银GelMA水凝胶降解12 h的降解剩余率分别为(74.2±1.7)%、(85.3±0.9)%、(93.2±1.2)%,降解24 h的降解剩余率分别为(58.3±2.1)%、(65.2±1.8)%、(81.4±2.6)%,降解36 h的降解剩余率分别为(22.4±1.9)%、(45.2±1.7)%、(68.1±1.4)%,降解48 h的降解剩余率分别为(8.2±1.7)%、(32.4±1.3)%、(54.3±2.2)%;20、25、30 s光交联时间下含银GelMA水凝胶彻底降解所需时间分别为(50.2±2.4)、(62.4±1.4)、(72.2±3.2)h,差异有统计学意义(F=182.40,P<0.01),选取25 s作为后续光交联时间。低浓度至高浓度的10种含银GelMA水凝胶对金黄色葡萄球菌的抑菌圈直径依次为(2.6±0.4)、(2.5±0.4)、(3.2±0.4)、(12.1±0.7)、(14.8±0.7)、(15.1±0.5)、(16.2±0.6)、(16.7±0.5)、(16.7±0.4)、(16.7±0.6)mm,基本呈浓度依赖性升高趋势,总体比较差异有统计学意义(F=428.70,P<0.01),与含最低浓度银GelMA水凝胶相比,其他有抑菌活性的含低浓度至高浓度银GelMA水凝胶的抑菌圈直径均明显增大(t值分别为26.35、33.84、43.65、42.17、49.24、55.74、43.72,P<0.01)。对金黄色葡萄球菌抑菌圈直径为(12.1±0.7)mm的含银GelMA水凝胶具有抑菌活性且载药浓度最低,选取该含银浓度为后续材料制作浓度。含银GelMA水凝胶的微观形貌为规律的趋于平行线性的条索状结构,孔径为(45±13)μm,且含有银元素。浸没24 h,含银GelMA水凝胶的溶胀率与单纯GelMA水凝胶相近(P>0.05)。复合水凝胶呈无色清亮透明状;其三维结构为规则、均匀的网格状,内部存在细丝网状结构,孔径为(40±21)μm。伤后3 d,复合水凝胶组兔创面可见大量坏死组织及渗出物;纱布对照组兔创面可见散在结痂,亦可见少量坏死组织及渗出物。伤后7 d,复合水凝胶组兔创面已明显缩小,纱布对照组兔出现创面存在与纱布粘连情况。伤后14 d,复合水凝胶组兔创面红润、可见肉芽组织生长;纱布对照组兔创面基底呈苍白色、血运差。伤后21 d,复合水凝胶组兔创面完全愈合,纱布对照组兔创面出现愈合趋势。伤后28 d,复合水凝胶组兔创面部位可见新生毛发,纱布对照组兔仍残存椭圆形创面。伤后7、14、21、28 d,复合水凝胶组兔创面愈合率均明显大于纱布对照组(t值分别为2.24、4.43、7.67、7.69,P<0.05或P<0.01)。结论中浓度GelMA水凝胶在溶胀性、可降解性方面具有良好的理化特性,筛选出的含银GelMA水凝胶具有抑菌活性且载药浓度最低,制得的复合水凝胶可明显缩短兔深Ⅱ度烧伤创面愈合时间。

Objective To investigate the effects of methacrylic anhydride gelatin(GelMA)hydrogel loaded with silver and recombinant human basic fibroblast growth factor(rh-bFGF)on deep partial-thickness burn wounds in rabbits.Methods The experimental research method was adopted.Low-concentration GelMA materials,medium-concentration GelMA materials and high-concentration GelMA materials containing different concentrations of methacrylic anhydride(MA)were prepared,after adding photoinitiator,low-concentration GelMA hydrogels,medium-concentration GelMA hydrogels,and high-concentration GelMA hydrogels were obtained,respectively.The nuclear magnetic resonance spectroscopy was performed to detect the hydrogen nuclear magnetic resonance spectra of the above-mentioned three concentrations of GelMA materials,and to calculate the degree of substitution according to the spectrum diagram.The three-dimensional microstructure and pore size of 3 types of above-mentioned GelMA hydrogels were detected by field emission scanning electron microscopy(FESEM),with 9 samples measured.According to the selected concentration of MA,ten kinds of solutions of GelMA with different concentration of silver(silver-containing GelMA)were synthesized,and the silver-containing GelMA solution of each concentration was divided into three parts,and then exposed to ultraviolet light lasting for 20,25,and 35 s,respectively.After adding photoinitiator,the corresponding silver-containing GelMA hydrogels were obtained.The residual degradation rate of silver-containing GelMA hydrogel with different photocrosslinking times was detected by collagenase degradation method at degradation of 12,24,36,and 48 h;and the time required for complete degradation was detected,and the sample number was 5.The inhibition zone diameter of GelMA hydrogel under above screened photocrosslinking times containing 10 concentrations of silver against Staphylococcus aureus was measured to reflect its antibacterial ability,and the sample numbers were all 5.The silver-containing GelMA hydrogel with statistical significance compared with the antibacterial circle diameter of the silver-containing GelMA hydrogel containing the lowest concentration(no silver)was considered as having antibacterial activity.The three-dimensional microstructure and pore size of the silver-containing GelMA hydrogels with antibacterial activity and the lowest drug concentration selected were detected by FESEM,and the sample numbers were all 9.The freeze-dried alone GelMA hydrogel and the freeze-dried silver-containing GelMA hydrogel were soaked in phosphate buffer solution for 24 h,respectively,then the swelling rate of the two GelMA hydrogel were calculated and compared by weighing method,and the sample number was 5.GelMA hydrogel containing silver and rh-bFGF,namely compound hydrogel for short,was prepared according to the preliminary experiment and the above experimental results.The appearance of the composite hydrogel was observed in general,and its three-dimensional microstructure and pore size were detected by FESEM.The deep partial-thickness burn wound was made on the back of 30 rabbits(aged 4-6 months,female half and half).Meanwhile,with the rabbit head as the benchmark,the wounds on the left side of the spine were treated as composite hydrogel treatment group,and the wounds on the right side were treated as gauze control group,and which were treated accordingly.On post injury day(PID)3,7,14,21,and 28,the healing of wounds in the two groups was observed.On PID 7,14,21,and 28,the wound healing area was recorded and the healing rate was calculated,with a sample number of 30.Data were statistically analyzed with analysis of variance for repeated measurement,one-way analysis of variance,and independent sample t test.Results The substitution degree among low-concentration GelMA materials,medium-concentration GelMA materials,and high-concentration GelMA materials was significantly different(F=1628.00,P<0.01).The low-concentration GelMA hydrogel had a loose and irregular three-dimensional spatial network structure with a pore size of(60±17)μm;the medium-concentration GelMA hydrogel had a relatively uniform three-dimensional spatial network and pore size with a pore size of(45±13)μm;the high-concentration GelMA hydrogel had the dense and disordered three-dimensional spatial network with a pore size of(25±15)μm,the pore sizes of 3 types of GelMA hydrogels were significantly differences(F=12.20,P<0.01),and medium concentration of MA was selected for the concentration of subsequent materials.The degradability of silver-containing GelMA hydrogels with different concentrations of the same photocrosslinking time was basically same.The degradation residual rates of silver-containing GelMA hydrogels with 20,25,and 35 s crosslinking time at 12 h were(74.2±1.7)%,(85.3±0.9)%,and(93.2±1.2)%,respectively;the residual rates of degradation at 24 h were(58.3±2.1)%,(65.2±1.8)%,and(81.4±2.6)%,respectively;the residual rates of degradation at 36 h were(22.4±1.9)%,(45.2±1.7)%,and(68.1±1.4)%,respectively;the residual rates of degradation at 48 h were(8.2±1.7)%,(32.4±1.3)%,and(54.3±2.2)%,respectively,and 20,25,and 30 s photocrosslinking time required for complete degradation of silver-containing GelMA hydrogels were(50.2±2.4),(62.4±1.4),and(72.2±3.2)h,and the difference was statistically significant(F=182.40,P<0.01),25 s were selected as the subsequent photocrosslinking time.The antibacterial diameters of 10 types of silver-containing GelMA hydrogels against Staphylococcus aureus from low to high concentrations were(2.6±0.4),(2.5±0.4),(3.2±0.4),(12.1±0.7),(14.8±0.7),(15.1±0.5),(16.2±0.6),(16.7±0.5),(16.7±0.4),and(16.7±0.6)mm,respectively,and which basically showed a concentration-dependent increasing trend,and the overall difference was statistically significant(F=428.70,P<0.01).Compared with the silver-containing GelMA hydrogel with the lowest concentration,the antibacterial circle diameters of other silver-containing GelMA hydrogels with antibacterial ability from low to high concentration were significantly increased(with t values of 26.35,33.84,43.65,42.17,49.24,55.74,and 43.72,respectively,P<0.01).The silver-containing GelMA hydrogel with the antibacterial diameter of(12.1±0.7)mm had the lowest antibacterial activity against Staphylococcus aureus and the lowest drug loading concentration,and the concentration of silver was selected for the concentration of subsequent materials.The microscopic morphology of the silver-containing GelMA hydrogel containing silver element with a pore size of(45±13)μm had a regular and linear strip-like structure.After soaking for 24 h,the swelling ratio of silver-containing GelMA hydrogel was similar to that of alone GelMA hydrogel.The composite hydrogel was colorless,clear and transparent,and its three-dimensional microstructure was a regular and uniform grid,with a filament network structure inside,and the pore size of(40±21)μm.On PID 3,a large amount of necrotic tissue and exudate of rabbit wound in composite hydrogel group were observed,and scattered scabs,a small amount of necrotic tissue and exudate of rabbit wound in gauze control group were observed.On PID 7,the area of rabbit wound in composite hydrogel group was significantly reduced,and adhesion of rabbit wound and gauze in gauze control group was observed.On PID 14,In composite hydrogel group,the rabbit wound surface was ruddy,and the growth of granulation tissue was observed,and in gauze control group,the rabbit wound base was pale,and the blood supply was poor.On PID 21,the rabbit wounds in composite hydrogel group healed completely,and rabbit wound in gauze control group had healing trend.On PID 28,new hair could be seen on rabbit wound surface in composite hydrogel group;oval wound of rabbit in gauze control group still remained.On PID 7,14,21,and 28,the wound healing areas of rabbit in composite hydrogel group were significantly larger than those in gauze control group(with t values of 2.24,4.43,7.67,and 7.69,respectively,P<0.05 or P<0.01).Conclusions The medium-concentration GelMA hydrogel has good physical and chemical properties in terms of swelling and degradability.The screened silver-containing GelMA hydrogels had the lowest antibacterial activity and the lowest drug loading concentration.Composite hydrogel can significantly shorten the healing time of deep partial-thickness burn wounds in rabbits.