兽研摘要
本病例报告描述了一只3岁混种犬右前肢严重创伤后发生复杂性体积性肌肉丢失（VML）损伤的处理过程，该损伤导致广泛的多肌肉缺损，不适合一期闭合。在初始稳定和手术清创后，采用转位皮瓣完成重建，同时植入金纳米颗粒-生物活性玻璃-海藻酸盐-纤维素（BGAuSP-Alg-MCC）复合材料以填充死腔并支持组织再生。通过临床随访、术后即刻及术后1个月和3个月的计算机断层扫描，以及组织学和免疫组织化学分析评估术后愈合情况。影像学显示生物材料逐步整合，组织密度恢复至与邻近肌肉相当，且无重大并发症。组织学评估显示中度炎症和肉芽组织形成，其中含有大量毛细血管（新生血管形成），经CD31和α-平滑肌肌动蛋白（SMA）免疫标记证实。功能恢复良好，肢体活动度保留且步态正常。这些发现提供了局部生物相容性的初步证据，并支持将BGAuSP-Alg-MCC复合材料纳入犬类患者广泛性体积性肌肉丢失的多模式手术管理方案的可行性。
图形摘要
[图片内容请查看原文]
补充信息
在线版本包含补充材料，可在 https://doi.org/10.1007/s11259-026-11557-x 获取。
关键词：生物活性玻璃，海藻酸盐，纤维素，体积性肌肉丢失
背景
骨骼肌损伤后的再生是一个复杂的动态过程，主要由卫星细胞驱动（Järvinen et al. 2007; Collins et al. 2007; Lin et al. 2007）。在严重创伤中，内在愈合能力可能被压倒，导致体积性肌肉丢失（VML）、纤维化和功能损害。创伤性肢体损伤和保肢手术可导致VML，尽管肌肉瓣或组织转位等手术技术常被采用，但很少有临床研究专门探讨治疗结局。再生医学为增强修复和恢复这些棘手病例的肌肉结构与功能提供了有前景的策略（Perez-Terzic and Childers 2014）。
已有多种治疗策略在体内研究中用于VML的治疗，包括自体组织移植、手术再植入、结合细胞或生长因子的生物材料方法以及干细胞移植（Shi et al. 2009; Corona et al. 2013, 2017; Oshima et al. 2014; Goldman et al. 2018; Qiu et al. 2018）。其中许多方法依赖于使用细胞或分子因子来增强组织再生。相比之下，不添加额外细胞或生长因子的生物材料的再生潜力似乎较为有限。例如，去活化的骨骼肌细胞外基质（ECM）支架单独使用时对大型肌肉缺损的恢复不充分，部分原因是它们不能充分促进内源性卫星细胞的募集和活化（Garg et al. 2014; Aurora et al. 2015）。
具有定制组成、结构和力学性质的生物材料被广泛研究用于软组织再生，因为它们可以影响细胞黏附、分化和组织重塑。理想的软组织支架应具有生物相容性、支持细胞黏附、引起最小的炎症和毒性、经历适当的生物降解并提供合适的孔隙率（Sindhi et al. 2025）。
天然碳水化合物基聚合物，包括纤维素、壳聚糖、海藻酸盐和透明质酸，因其生物相容性和生物可降解性而被广泛用于生物医学应用（Serafin et al. 2023; Sindhi et al. 2025）。纤维素是一种存在于植物和多种其他生物体中的线性多糖，因其力学性质、生物相容性、低毒性和相对低成本而被用于多功能支架（Seddiqi et al. 2021）。海藻酸盐来源于褐藻，形成离子交联水凝胶，可维持湿润环境，用于细胞封装、组织修复和药物递送（Ahmad Raus et al. 2021）。生物活性玻璃最初为骨再生而开发，目前也因其支持血管生成和再生反应的潜力而被研究用于软组织修复（Zhu et al. 2025; Zăvoi et al. 2025）。然而，支架介导的重塑可能因解剖部位而异，特别是在涉及骨、肌肉和肌腱组合的损伤中。伴侣动物广泛的创伤后皮肤缺损仍然具有挑战性，可能需要系列清创、感染控制、深层结构保护以及移植或皮瓣等重建技术（Williams 2009）。
在广泛性创伤中，供区可用性可能有限（Beier et al. 2006; Mase et al. 2010），且一些保肢失败最终需要截肢和假肢康复（Turner and Badylak 2012）。这些局限性支持继续研究旨在辅助大型缺损骨骼肌重建的即用型仿生材料（Mase et al. 2010）。
基于既往实验表征及已报道的细胞浸润、血管生成和肌管形成的研究结果，选择了金纳米颗粒–生物活性玻璃–海藻酸盐–纤维素（BGAuSP-Alg-MCC）复合材料，且无细胞毒性、生物材料诱导的组织坏死或中性粒细胞性炎症的证据，支持该材料在肌肉组织中的生物相容性（Feraru et al. 2025）。在本临床病例中，该复合材料被用作重建手术的辅助材料，用于涉及三角肌、肱三头肌外侧头和冈下肌的复杂缺损。据我们所知，这是首例报道的犬病例，将此种复合材料与转位皮瓣联合应用于不适合一期闭合的广泛多肌肉缺损。目的是描述手术方法及短期临床、计算机断层扫描、组织学和免疫组织化学发现；单病例设计并非旨在确立生物材料特异性疗效。
病例介绍
病史与临床发现
一只3岁、既往健康的已绝育混种犬（30 kg）在被割草机严重致伤约1小时后，被送至克卢日-纳波卡农业科学与兽医大学急诊服务处。该动物由一家非政府组织（NGO）运送至医院，因为主人无力承担所需的急诊治疗费用。
到达急诊服务处时，患犬呈侧卧位，反应极弱。黏膜苍白，毛细血管再充盈时间无法测出。心率为200次/分，节律规整，呼吸频率为80次/分，体温为36.6 °C。发现右前肢上段严重创伤，特征为多处穿刺伤、深部撕裂伤和活动性出血。
初始复苏包括使用等渗晶体液进行液体治疗（30 mL/kg IV；0.9% NaCl），并使用强制空气加温装置主动保温直至体温达到37.5 °C。在培养结果可用之前，开始经验性抗菌治疗，使用阿米卡星（15 mg/kg IV q24h）。阿米卡星单药治疗不能提供全面的链球菌或厌氧菌覆盖；因此，本病例中的初始抗生素治疗不应被解释为广谱覆盖。镇痛采用芬太尼单次推注（3 µg/kg IV；Fentadon 50 µg/mL，Dechra，荷兰），随后以恒定速率输注（5 µg/kg/h IV）。初始血液学和生化检查包括测定PCV、总血浆蛋白和活化凝血时间，以及静脉生化谱，涵盖血气值、主要电解质（钠、钾、氯、离子钙、离子镁）、葡萄糖、乳酸、BUN和肌酐浓度。血液学分析显示轻度白细胞增多伴中性粒细胞增多，血浆乳酸浓度升高至3 mmol/L（27 mg/dL；参考区间< 2.5 mmol/L [< 22.5 mg/dL]），而所有其他血液学参数报告在正常范围内。
手术过程
本研究经罗马尼亚克卢日-纳波卡农业科学与兽医大学伦理委员会批准（批准号423/22.01.2024），并按照当地法规进行。已获得主人和NGO的书面知情同意。由于患犬居住地偏远且NGO缺乏合适的临时住所，该犬在整个恢复期间一直住院。
经过24小时的稳定和密集监测后，患犬在全身麻醉下接受了手术干预。头孢噻肟（25 mg/kg IV q12h；Cefotax 1 g，The Egyptian International Pharmaceutical Industries Company，埃及）在手术前30分钟给药作为围手术期抗菌覆盖，随后继续与正在进行的阿米卡星治疗（15 mg/kg IV q24h）联合使用。美洛昔康（0.2 mg/kg IV；Metacam，Boehringer Ingelheim）用于围手术期镇痛。美沙酮（0.2 mg/kg IM；Insistor 10 mg/mL，Richter Pharma，Wels，奥地利）作为术前用药，20分钟后给予右美托咪定（0.02 mg/kg IV；Dexdomitor 0.5 mg/mL，Orion Pharma）和氯胺酮（2 mg/kg IV；Ketomidor 10%，Richter Pharma，奥地利）。麻醉诱导以丙泊酚（Propofol Lipuro 10 mg/mL；B. Braun，德国）静脉给药至效应完成，麻醉以异氟烷（Isoflutek 1,000 mg/g；Laboratorios Karizoo S.A.，巴塞罗那，西班牙）在70%氧气中输送维持。术中持续监测包括脉搏血氧饱和度、呼气末CO2、直肠温度、心电图和多普勒收缩压。
对缺血肌肉和筋膜进行了手术清创（图1A、B）。尽管伤口不足6小时且归类为污染伤口，但冲洗和机械清除未能去除所有碎屑，因此不适合一期闭合。局部应用了少量糖，因为糖有助于减少污染性渗出伤口的细菌负荷（Mphande等，2007）。其强渗透效应可抑制大肠杆菌、铜绿假单胞菌和链球菌等微生物的生长。此外，糖可促进轻度浅表清创、减轻恶臭，并支持肉芽组织早期形成（Chirife等，1983）。这些特性使其成为一种简单、廉价且易得的临时改善伤口状况的选择。由于前肢近端存在大面积缺损，该区域常规绷带容易滑脱，因此采用了褥式包扎（tie-over bandage）以实现更稳定的覆盖（图1C）。该技术确保了伤口的可靠保护，并维持了有利于持续护理的最佳环境。犬从麻醉中顺利恢复，转入ICU进行密集监测和术后管理。住院期间持续进行抗菌和镇痛治疗。在最初四天内，犬每天再次麻醉以进行伤口评估和褥式包扎更换，随后一周内每三天进行一次。
[图：A 就诊时的创伤性伤口，显示广泛的组织失活和污染。B 伤口外观]
一旦伤口污染程度明显降低且局部组织状况被认为令人满意，即实现了确定性伤口闭合（图2）。随后微生物学样本检测为铜绿假单胞菌阳性，该菌对阿米卡星敏感但对头孢噻肟耐药。根据抗菌药物敏感性结果，停用头孢噻肟，维持阿米卡星作为唯一抗菌治疗。在全身麻醉下，采用前述相同方案，用BGAuSP-Alg-MCC复合材料填充死腔，随后应用Hydrosorb® Gel以维持伤口湿润并支持早期肉芽形成。然后用移位皮瓣覆盖缺损（图2C、D）。放置Penrose引流管24小时以防止液体积聚，未观察到明显引流。麻醉和恢复顺利，犬在次日临床状况保持稳定。
[图：A、B 移位皮瓣手术。C 在缺损内放置BGAuSP-Alg-MCC复合材料。D 缺损闭合]
复合材料制备
首先合成球形金纳米颗粒（AuSP），然后分别采用Turkevich–Frens法（Frens，1973）和溶胶–凝胶法合成BGAuSP（60SiO2·31.85CaO·8P2O5·0.15Au2O mol%），如先前研究所述（Magyari等，2017；Tóth等，2022）。引入玻璃样品中的金含量对应于BGAuSP中的0.09 at%。采用先前研究所述的合成方法将BGAuSP引入Alg-MCC生物聚合物复合材料中（Feraru等，2025）。藻酸盐-纤维素的重量比为1：0.15，BGAuSP在Alg-MCC复合材料中的重量比为12 wt%。有关合成细节、结构和形态学特性，可参阅补充材料。
计算机断层扫描方案与评估
在确定性重建后立即以及术后1个月和3个月，使用Siemens Somatom Scope系统进行计算机断层扫描（CT）检查。视野包括右前肢和肩部区域。采集的图像以医学数字成像和通信（DICOM）格式存储在Siemens工作站上，并归档于PACS服务器。对于本病例报告，CT评估为定性评估，考虑了重建/植入区域的位置和清晰度、相对于邻近肌肉的衰减、周围软组织变化以及并发症证据。标准化感兴趣区Hounsfield单位（HU）测量和体积分割无法进行分析；因此，未报告平均HU值和定量植入物体积变化。
组织学和免疫组织化学
植入后3个月，采用打孔活检技术获取组织样本。标本在10%中性缓冲福尔马林中固定24小时，并使用Thermo Scientific Citadel 2000组织处理仪进行常规处理。石蜡包埋样品以3 μm厚度切片，并进行苏木精和伊红（H&E）及Masson三色染色。对于免疫组织化学评估，使用Novocastra™抗原修复液（1：10稀释）在pH 9条件下进行热诱导抗原修复30分钟。按照制造商说明，使用Dako EnVision Flex + Kit（小鼠，高pH）进行免疫标记。使用的一抗为α-平滑肌肌动蛋白（α-SMA；1：100稀释，多克隆，ab5694，Abcam）和CD31（即用型，克隆号：JC70A，Leica Biosystems）。使用Olympus SP-350相机和Stream Basic成像软件1.5.1版（Olympus Corporation，东京，日本）采集图像。
术后病程与随访
在放置移位皮瓣和复合材料七天后，观察到皮瓣上部出现一小块浅表坏死区域（图3A）。随后的微生物学样本培养为阴性，使得活动性细菌污染的可能性较小。临床上，该病变被认为更符合因血管化不足导致的局部缺血。对受损区域进行了手术清创，并让其通过二期愈合（图3B-D）。未观察到进一步感染、坏死或伤口裂开。骨科评估包括视觉步态观察和手法关节活动度评估，未检测到明显异常。
[图：放置移位皮瓣和BGAuSP-Alg-MCC复合材料后7天伤口的术后外观，s]
在确定性重建后立即进行的CT显示，BGAuSP-Alg-MCC复合材料位于右肩肌肉缺损内，表现为局灶性高衰减区域（图4A）。在轴位和矢状位重建图像上，该材料在残余肌肉边缘之间清晰可辨。未发现早期液体积聚、异常气体模式或植入物移位。在1个月时，重建/植入区域相对于邻近肌肉仍呈轻度高衰减（图4B；绿色箭头），但边界不如术后即刻清晰。这种定性变化符合渐进性整合或重塑，但不足以量化材料的存留或吸收。覆盖其上的皮下组织中可见一条薄而边界清晰的高衰减带（图4B；红色箭头），被解释为轻度局限性术后脂膜炎，未向深部延伸。
[图：A 确定性重建后立即获得的轴位和矢状位CT图像，显示局灶性高衰减]
在3个月时，修复的肌肉区域（图4C；绿色箭头）外观更加均匀，其衰减值接近邻近肌肉。CT的定性表现符合持续的组织重塑。然而，在缺乏标准化HU测量和体积分割的情况下，CT无法确定组织整合、生物材料存留和生物材料吸收的相对贡献。在3个月的CT检查中，未发现脓肿、血清肿、广泛的周围软组织炎症或持续感染的影像学证据。
植入后3个月，组织学检查显示伤口相关肉芽组织伴有中度炎症细胞浸润。肉芽组织含有丰满的间充质细胞、中度胶原沉积和新形成的小血管通道（图5A）。炎症细胞由中等数量的巨噬细胞和中性粒细胞组成，并散在少量小淋巴细胞和浆细胞。免疫组化分析证实了血管生成，表现为内皮细胞中强烈的细胞质CD31表达（图5B），其周围环绕着α-SMA阳性细胞，被解释为周细胞（图5C）。对CD31和α-SMA染色组织的分析显示存在成熟、形态良好的血管。
[图：BGAuSP-Alg-MCC处理伤口的显微特征。A 由胶原纤维组成的肉芽组织，丰满的m]
出院时，开具口服阿莫西林-克拉维酸钾（14 mg/kg PO q12h；Clavaseptin，Biotur）用于抗菌治疗，同时给予曲马多（4 mg/kg PO q8h；Tramvetol，Altius）和美洛昔康（0.1 mg/kg PO q24h；Metacam，Boehringer Ingelheim）为期5天用于镇痛。残余的浅表伤口在家中用无菌生理盐水每日清洗进行管理，并开具伊丽莎白项圈直至完全愈合。由于主人无法返回诊所进行每周随访，患者的临床进展通过每日电话联系进行监测，直至伤口完全闭合。向主人询问了食欲、发声、活动能力、触诊反应、精神状态、一般活动和伤口状况。未报告进一步的伤口裂开。
讨论
我们先前的研究（Feraru et al. 2025）证明了BGAuSP-Alg-MCC促进骨骼肌再生的潜力，在体外和体内均观察到再生效应。基于这些发现，选择该复合材料作为VML治疗的候选生物材料进行进一步研究。CD31阳性内皮细胞和α-SMA阳性血管周细胞的存在支持形成具有壁细胞包被的血管化组织，这与血管发育和持续的组织重塑相一致。充分的血管化是骨骼肌修复的重要组成部分，因为血管的存在可确保足够的血管支持，并有助于在肌肉组织生长和结构组织化过程中维持细胞活力（Levenberg et al. 2005; Forbes and Rosenthal 2014）。因此，这些发现为重建区域内血管化修复反应提供了令人鼓舞的证据。然而，CD31和α-SMA并非肌生成特异性标志物，不能区分新再生的肌肉与植入材料周围的组织整合、肉芽形成或重塑。由于未进行肌纤维特异性免疫组化标志物检测和定量肌纤维分析，目前的研究结果应被解释为血管化组织修复的证据，而非功能性新肌纤维形成的确定性证据。虽然先前的研究表明，当支架缺乏卫星细胞或生长因子时，VML修复可能受限，但本研究发现提示BGAuSP-Alg-MCC复合材料可能支持骨骼肌中的血管化修复反应和组织重塑，尽管其促进从头肌肉再生的能力仍需进一步研究（Garg et al. 2014; Aurora et al. 2015）。
金纳米颗粒因其理化性质和生物学特性以及作为治疗药物递送系统的潜力，在组织修复中受到越来越多的关注（Nukaly and Ansari 2023）。动物模型实验研究报告称，与对照组相比，基于金纳米颗粒-生物活性玻璃的干预可改善伤口愈合（Mârza et al. 2019, 2021）。推测的机制包括调节转化生长因子、炎性细胞因子和血管生成相关通路，以及抗菌和抗氧化作用（Chen et al. 2023; Zhang et al. 2024）。此外，金纳米颗粒与增强的胶原形成和组织化相关，这可能有助于改善伤口闭合和减少瘢痕形成（Liu et al. 2012）。
分期临床策略结合了序列清创、糖敷料、Hydrosorb®凝胶、转位皮瓣和BGAuSP-Alg-MCC复合材料。因此，良好的临床病程应被解释为这种联合重建方法的结果，而非单独生物材料的效果。尽管如此，BGAuSP-Alg-MCC的加入与重建区域内渐进性伤口愈合和组织重塑相关。3个月时观察到的中度炎性浸润也需要谨慎解读。在缺乏对照组和定量炎症评分的情况下，它可能反映持续的伤口重塑、宿主对植入材料的反应，或两者兼有。
局限性包括单病例设计、缺乏对照组、12周后长期结局未知，以及在没有先进步态分析的情况下可能未检测到的细微功能缺陷。支架降解、机械稳定性和离子释放动力学的变异性也可能影响再生反应。
未来的研究应侧重于更大规模、对照队列以验证这些发现，并更好地界定BGAuSP-Alg-MCC复合材料在VML修复中的作用。建议进行长期随访和客观功能评估，以进一步表征再生结局和持久性。此外，优化支架组成并与支持性生物疗法整合可能增强广泛缺损中的肌肉再生。
结论
总之，本单一病例证明了使用BGAuSP-Alg-MCC复合材料作为转位皮瓣重建辅助手段，在患有复杂体积性肌肉缺失的犬类患者中具有技术可行性（见补充视频）。在12周内，联合治疗与渐进性伤口愈合、符合重建区域重塑的定性CT表现以及组织学上的血管化肉芽组织相关。3个月时仍存在中度炎症。由于患者接受了多种同时进行的干预措施，且没有对照治疗，观察到的结果反映了联合重建策略，使得难以确定BGAuSP-Alg-MCC复合材料的独立贡献。尽管如此，联合方法与渐进性伤口愈合、良好的临床恢复、符合组织重塑的定性CT变化以及3个月时的血管化肉芽组织相关。这些发现支持将BGAuSP-Alg-MCC纳入基于皮瓣的严重肌肉缺损重建的临床可行性，并为其在组织修复中潜在作用的进一步研究提供了基础。尽管该复合材料此前已在对照大鼠VML模型中进行过研究，但仍需要在临床相关的大动物模型中进行更大队列和更长随访的进一步研究，以评估材料的持久性和降解、宿主反应、功能恢复以及该复合材料对组织再生的具体贡献。
补充信息
以下是电子补充材料的链接。
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致谢
我们要感谢兽医学院外科与重症监护室以及诊断影像团队提供的重要支持和基础设施。我们还感谢Andrei Mariuțeac在示意图表示方面的协助。
作者贡献
Alina Zăvoi - 调查、方法学、写作——原始草稿、写作——审查与编辑。Tom Ablassmaier - 调查、方法学。Bianca Pop - 调查、方法学。Ștefana Mureșan - 调查、方法学。Andrada Negoescu - 调查、方法学。Felix Lucaci - 调查、方法学。Robert Purdoiu - 概念化、调查、方法学、资源、写作——原始草稿、写作——审查与编辑。Alexandra Feraru - 调查、方法学。Klara Magyari - 概念化、调查、方法学、资源、写作——原始草稿、写作——审查与编辑。Lucia Bel - 概念化、监督、写作——审查与编辑。Mădălina Dragomir - 调查、方法学。Liviu Oana - 调查、概念化、监督、写作——审查与编辑。Marian Taulescu - 概念化、监督、写作——审查与编辑。Alexandra Dreancă - 概念化、数据管理、调查、方法学、写作——原始草稿、写作——审查与编辑。
资助
本研究由欧洲投资与项目部（MIPE）作为2021–2027智能增长、数字化和金融工具计划的管理机构以及研究、创新和数字化部（MCID）作为中介研究机构资助，项目代码SMIS 2021 + 324,771，合同MIPE编号G-2024–71962/23.10.2024和合同MCID编号390,005/23.10.2024，项目缩写INSPIRE-II。
数据可用性
研究中呈现的原始贡献包含在文章中；进一步咨询可联系通讯作者。
声明
伦理批准
已获得动物主人的书面知情同意，同意其动物参与本研究。
利益冲突
Klara Magyari、Alexandra Dreancă和Marian Tulescu拥有待批专利#CBI 2024 A 00476，归属于OSIM。其他作者声明他们没有已知的竞争性财务利益或个人关系可能影响本文报告的工作。
脚注
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参考文献
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关联数据
补充材料
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数据可用性声明
研究中呈现的原始贡献包含在文章中；进一步咨询可联系通讯作者。
查看英文原文▾
Abstract
This case report describes the management of a complex volumetric muscle loss (VML) injury in a 3-year-old mixed-breed dog following severe trauma to the right forelimb, which resulted in extensive multi-muscle defects that were unsuitable for primary closure. After initial stabilization and surgical debridement, reconstruction was achieved using a transpositional flap, while a gold nanoparticles–bioactive glass–alginate–cellulose (BGAuSP-Alg-MCC) composite was implanted to fill the dead space and support tissue regeneration. Postoperative healing was evaluated through clinical follow-up, computed tomography performed immediately and at 1 and 3 months postoperatively, as well as histological and immunohistochemical analyses. Imaging demonstrated progressive integration of the biomaterial, restoration of tissue density comparable to adjacent muscle, and absence of major complications. Histological evaluation demonstrated moderate inflammation and granulation tissue formation, containing numerous blood capillaries (neoangiogenesis), confirmed by CD31 and α-Smooth muscle actin (SMA) immunolabelling. Functional recovery was favorable, with preserved limb mobility and normal gait. These findings provide preliminary evidence of local biocompatibility and support the feasibility of incorporating the BGAuSP-Alg-MCC composite into a multimodal surgical management approach for extensive volumetric muscle loss in canine patients.
Graphical Abstract
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Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1007/s11259-026-11557-x.
Keywords: Bioactive glass, Alginate, Cellulose, Volumetric muscle loss
Background
Skeletal muscle regeneration after injury is a complex and dynamic process driven primarily by satellite cells (Järvinen et al. 2007; Collins et al. 2007; Lin et al. 2007). In severe trauma, the intrinsic healing capacity can be overwhelmed, resulting in volumetric muscle loss (VML), fibrosis, and functional impairment. Traumatic extremity injuries and limb-salvage procedures can cause VML, and although surgical techniques such as muscle flaps or tissue transposition are commonly employed, few clinical studies specifically address treatment outcomes. Regenerative medicine offers promising strategies to enhance repair and restore muscle structure and function in these challenging cases (Perez-Terzic and Childers 2014).
Several therapeutic strategies have been investigated for the treatment of VML in vivo, including autologous tissue grafting, surgical reimplantation, biomaterial-based approaches incorporating cells or growth factors, and stem cell transplantation (Shi et al. 2009; Corona et al. 2013, 2017; Oshima et al. 2014; Goldman et al. 2018; Qiu et al. 2018). Many of these approaches rely on the use of cellular or molecular factors to enhance tissue regeneration. In contrast, the regenerative potential of biomaterials used without additional cells or growth factors appears to be more limited. For example, devitalized skeletal muscle extracellular matrix (ECM) scaffolds have shown insufficient restoration of large muscle defects when used alone, partly because they do not adequately promote the recruitment and activation of endogenous satellite cells (Garg et al. 2014; Aurora et al. 2015).
Biomaterials with tailored composition, structure, and mechanical properties are widely investigated for soft-tissue regeneration because they can influence cell adhesion, differentiation, and tissue remodeling. An ideal soft-tissue scaffold should be biocompatible, support cell adhesion, elicit minimal inflammation and toxicity, undergo appropriate biodegradation, and provide suitable porosity (Sindhi et al. 2025).
Natural carbohydrate-based polymers, including cellulose, chitosan, alginate, and hyaluronic acid, are widely used in biomedical applications because of their biocompatibility and biodegradability (Serafin et al. 2023; Sindhi et al. 2025). Cellulose is a linear polysaccharide present in plants and several other organisms and is used in multifunctional scaffolds because of its mechanical properties, biocompatibility, low toxicity, and relatively low cost (Seddiqi et al. 2021). Alginate, derived from brown seaweed, forms ionically cross-linked hydrogels that maintain a moist environment and are used for cell encapsulation, tissue repair, and drug delivery (Ahmad Raus et al. 2021). Bioactive glasses, initially developed for bone regeneration, are also being investigated for soft-tissue repair because of their potential to support angiogenic and regenerative responses (Zhu et al. 2025; Zăvoi et al. 2025). Nevertheless, scaffold-mediated remodeling may differ across anatomical sites, particularly in injuries involving combinations of bone, muscle, and tendon. Extensive post-traumatic skin defects in companion animals remain challenging and may require serial debridement, infection control, protection of deeper structures, and reconstructive techniques such as grafts or flaps (Williams 2009).
In extensive traumatic injuries, donor-site availability may be limited (Beier et al. 2006; Mase et al. 2010), and some limb-salvage failures ultimately require amputation and prosthetic rehabilitation (Turner and Badylak 2012). These limitations support continued investigation of off-the-shelf biomimetic materials intended to assist skeletal-muscle reconstruction in large defects (Mase et al. 2010).
The gold nanoparticles–bioactive glass–alginate–cellulose (BGAuSP-Alg-MCC) composite was selected on the basis of previous experimental characterization and reported findings of cellular infiltration, angiogenesis, and myotube formation, with no evidence of cytotoxicity, biomaterial-induced tissue necrosis, or neutrophilic inflammation, supporting the biocompatibility of the material in muscle tissue (Feraru et al. 2025). In the present clinical case, the composite was used as an adjunct to reconstructive surgery in a complex defect involving the deltoid, lateral triceps brachii, and infraspinatus muscles. To our knowledge, this is the first reported canine case combining this composite with a transpositional flap for an extensive multi-muscle defect unsuitable for primary closure. The aim was to describe the surgical approach and short-term clinical, computed tomography, histological, and immunohistochemical findings; the single-case design was not intended to establish biomaterial-specific efficacy.
Case presentation
History and clinical findings
A 3-year-old, previously healthy, spayed mixed-breed dog (30 kg) was presented to the emergency service at the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca approximately 1 h after being severely injured by a weed trimmer. The animal was transported to the hospital by a Non-Governmental Organization (NGO), as the owner was unable to afford the required emergency care.
Upon presentation to the emergency service, the dog was in lateral recumbency and minimally responsive. The mucous membranes were pale, and the capillary refill time was undetectable. The heart rate was 200 beats/min with a regular rhythm, the respiratory rate was 80 breaths/min, and the body temperature was 36.6 °C. Severe trauma of the right upper forelimb was identified, characterized by multiple puncture wounds, deep lacerations, and active hemorrhage.
Initial resuscitation included fluid therapy with isotonic crystalloids (30 mL/kg IV; 0.9% NaCl) and active warming with a forced-air device until body temperature reached 37.5 °C. Empirical antimicrobial therapy with amikacin (15 mg/kg IV q24h) was initiated before culture results were available. Amikacin monotherapy does not provide comprehensive streptococcal or anaerobic coverage; therefore, the initial antibiotic treatment in this case should not be interpreted as broad-spectrum coverage. Analgesia was provided with a fentanyl bolus (3 µg/kg IV; Fentadon 50 µg/mL, Dechra, Netherlands) followed by a constant-rate infusion (5 µg/kg/h IV). The initial hematologic and biochemical workup included determination of PCV, total plasma protein, and activated clotting time, along with a venous biochemistry profile encompassing blood gas values, major electrolytes (sodium, potassium, chloride, ionized calcium, ionized magnesium), glucose, lactate, BUN, and creatinine concentrations. Hematological analysis revealed a mild leukocytosis with neutrophilia and an elevated plasma lactate concentration of 3 mmol/L (27 mg/dL; reference interval < 2.5 mmol/L [< 22.5 mg/dL]), while all other hematological parameters were reported within normal limits.
Surgical procedure
The study was approved by the Ethics Committee of the University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania (approval no. 423/22.01.2024) and conducted in accordance with local regulations. Written informed consent was obtained from the owner and the NGO. The dog remained hospitalized throughout recovery due to its remote home location and the NGO’s lack of suitable temporary housing.
After 24 h of stabilization and intensive monitoring, the dog underwent surgical intervention under general anesthesia. Cefotaxime (25 mg/kg IV q12h; Cefotax 1 g, The Egyptian International Pharmaceutical Industries Company, Egypt) was administered 30 min before surgery as perioperative antimicrobial coverage and was subsequently continued in combination with the ongoing amikacin therapy (15 mg/kg IV q24h). Meloxicam (0.2 mg/kg IV; Metacam, Boehringer Ingelheim) was administered for perioperative analgesia. Methadone (0.2 mg/kg IM; Insistor 10 mg/mL, Richter Pharma, Wels, Austria) was administered as premedication, followed 20 min later by dexmedetomidine (0.02 mg/kg IV; Dexdomitor 0.5 mg/mL, Orion Pharma) and ketamine (2 mg/kg IV; Ketomidor 10%, Richter Pharma, Austria). Anesthetic induction was completed with propofol (Propofol Lipuro 10 mg/mL; B. Braun, Germany) administered intravenously to effect, and anesthesia was maintained with isoflurane (Isoflutek 1,000 mg/g; Laboratorios Karizoo S.A., Barcelona, Spain) delivered in 70% oxygen. Continuous intraoperative monitoring included pulse oximetry, end-tidal CO2, rectal temperature, electrocardiography, and Doppler systolic arterial pressure.
Ischemic muscle and fascia were surgically debrided (Fig. 1A, B). Although the wound was less than 6 h old and classified as contaminated, lavage and mechanical removal did not eliminate all debris, making primary closure inappropriate. A small amount of sugar was applied, as it can help reduce bacterial load in contaminated, exudative wounds (Mphande et al. 2007). Its strong osmotic effect inhibits the growth of organisms such as Escherichia coli, Pseudomonas aeruginosa, and Streptococcus. In addition, sugar promotes mild superficial debridement, decreases malodor, and supports the early formation of granulation tissue (Chirife et al. 1983). These properties make it a simple, inexpensive, and accessible option for temporarily improving wound conditions. Due to the extensive defect on the proximal forelimb, an area where conventional bandages tend to slip, a tie-over bandage was applied for more stable coverage (Fig. 1C). This technique ensured secure wound protection and maintained an optimal environment for ongoing care. The dog recovered uneventfully from anesthesia and was transferred to the ICU for intensive monitoring and postoperative management. Antimicrobial and analgesic therapy continued throughout hospitalization. The dog was re-anesthetized for daily wound evaluation and tie-over bandage replacement during the first four days, followed by every three days for an additional week.
[图：A Traumatic wound at presentation, showing extensive tissue devitalization and contamination. B Appearance of the wound ]
Once the degree of wound contamination had markedly decreased and the local tissue conditions were considered satisfactory, definitive wound closure was achieved (Fig. 2). A microbiological sample subsequently tested positive for Pseudomonas aeruginosa, which was susceptible to amikacin but resistant to cefotaxime. Based on the antimicrobial susceptibility results, cefotaxime was discontinued, and amikacin was maintained as the sole antimicrobial therapy. Under general anesthesia, using the same protocol described previously, the dead space was filled with BGAuSP-Alg-MCC composite, followed by application of Hydrosorb® Gel to maintain wound hydration and support early granulation. The defect was then covered with a transpositional skin flap (Fig. 2C, D). A Penrose drain was placed for 24 h to prevent fluid accumulation, with no significant drainage observed. Anesthesia and recovery were uneventful, and the dog remained clinically stable during the following day.
[图：A , B Transpositional flap procedure. C Placement of BGAuSP-Alg-MCC composite within the defect. D Closure of the defect]
Composite preparation
Firstly, the spherical gold nanoparticles (AuSP) was synthesized, followed by BGAuSP (60SiO2·31.85CaO·8P2O5·0.15Au2O mol%), using the Turkevich–Frens method (Frens 1973) and the sol–gel method, respectively, as described in previous studies (Magyari et al. 2017; Tóth et al. 2022). The gold amount introduced into the glass sample corresponded to 0.09 at% in the BGAuSP. The BGAuSP was introduced into Alg-MCC biopolymer composites using a synthesis described in a previous study (Feraru et al. 2025). The weight ratio of alginate-cellulose is 1:0.15, and the weight ratio of BGAuSP in the Alg-MCC composite is 12 wt%. For the synthesis details, structural and morphological properties, the Supplementary Materials can be consulted.
Computed tomography protocol and assessment
Computed tomography (CT) examinations were performed immediately after definitive reconstruction and at 1 and 3 months postoperatively using a Siemens Somatom Scope system. The right forelimb and shoulder region were included in the field of view. The acquired images were stored in Digital Imaging and Communications in Medicine (DICOM) format on the Siemens workstation and archived on the PACS server. For this case report, CT assessment was qualitative and considered the position and conspicuity of the reconstructed/implant region, attenuation relative to adjacent muscle, surrounding soft-tissue changes, and evidence of complications. Standardized region-of-interest Hounsfield unit (HU) measurements and volumetric segmentation were not available for analysis; therefore, mean HU values and quantitative implant-volume changes are not reported.
Histology and immunohistochemistry
At 3 months post-implantation, tissue samples were obtained using a punch biopsy technique. The specimens were fixed in 10% neutral-buffered formalin for 24 h and routinely processed using a Thermo Scientific Citadel 2000 tissue processor. Paraffin-embedded samples were sectioned at a thickness of 3 μm and stained with hematoxylin and eosin (H&E) and Masson’s Trichrome. For immunohistochemical evaluation, heat-induced epitope retrieval was performed at pH 9 using Novocastra™ Epitope Retrieval Solution (1:10 dilution) for 30 min. Immunolabeling was performed using the Dako EnVision Flex + Kit (Mouse, high pH) according to the manufacturer’s instructions. The primary antibodies used were α-smooth muscle actin (α-SMA; 1:100 dilution, polyclonal, ab5694, Abcam) and CD31 (ready-to-use, clone: JC70A, Leica Biosystems). Images were acquired using an Olympus SP-350 camera and Stream Basic imaging software, version 1.5.1 (Olympus Corporation, Tokyo, Japan).
Postoperative course and follow-up
Seven days after placement of the transposition flap and composite, a small area of superficial necrosis was observed at the upper portion of the flap (Fig. 3A). A subsequent microbiological sample was culture-negative, making active bacterial contamination less likely. Clinically, the lesion was considered more consistent with local ischemia due to insufficient vascularization. The compromised region was surgically debrided and allowed to heal by secondary intention (Fig. 3B-D). No further infection, necrosis, or wound dehiscence was observed. Orthopedic assessment included visual gait observation and manual range-of-motion evaluation, with no obvious abnormalities detected.
[图：A Postoperative appearance of the wound 7 days after placement of the transposition flap and BGAuSP-Alg-MCC composite, s]
Immediately after definitive reconstruction, CT demonstrated the BGAuSP-Alg-MCC composite within the right shoulder muscular defect as a focal hyperattenuating region (Fig. 4A). On axial and sagittal reconstructions, the material was clearly delineated between the remaining muscle margins. No early fluid collection, abnormal gas pattern, or implant displacement was identified. At 1 month, the reconstructed/implant region remained mildly hyperattenuating relative to adjacent muscle (Fig. 4B; green arrows) but was less sharply delineated than immediately after surgery. This qualitative change was compatible with progressive incorporation or remodeling but was not sufficient to quantify material persistence or resorption. A thin, well-defined hyperattenuating band in the overlying subcutaneous tissue (Fig. 4B; red arrows) was interpreted as mild localized postoperative panniculitis without deeper extension.
[图：A Axial and sagittal CT images obtained immediately after definitive reconstruction, showing the focal hyperattenuating ]
At 3 months, the repaired muscle region (Fig. 4C; green arrow) appeared more homogeneous, and its attenuation approached that of adjacent muscle. The qualitative CT appearance was compatible with ongoing tissue remodeling. However, in the absence of standardized HU measurements and volumetric segmentation, CT could not determine the relative contributions of tissue integration, biomaterial persistence, and biomaterial resorption. No abscess, seroma, extensive surrounding soft-tissue inflammation, or imaging evidence of ongoing infection was identified at the 3-month CT examination.
At 3 months post-implantation, histological examination revealed wound-associated granulation tissue with a moderate inflammatory infiltrate. The granulation tissue contained plump mesenchymal cells, moderate collagen deposition, and small, newly formed blood channels (Fig. 5A). The inflammatory cells consisted of moderate numbers of macrophages and neutrophils, along with scattered small lymphocytes and plasma cells. Immunohistochemical analysis confirmed angiogenesis, demonstrated by strong cytoplasmic CD31 expression in endothelial cells (Fig. 5B), which were surrounded by α-SMA-positive cells interpreted as pericytes (Fig. 5C). Analysis of CD31 and α-SMA-stained tissues revealed the presence of mature, well-formed blood vessels.
[图：Microscopic features of the wound treated with BGAuSP-Alg-MCC. A Granulation tissue composed of collagen fibers, plump m]
At discharge, oral amoxicillin-clavulanate (14 mg/kg PO q12h; Clavaseptin, Biotur) was prescribed for antimicrobial therapy, together with tramadol (4 mg/kg PO q8h; Tramvetol, Altius) and meloxicam (0.1 mg/kg PO q24h; Metacam, Boehringer Ingelheim) for 5 days for analgesia. The remaining superficial wound was managed at home with daily cleansing using sterile saline, and an Elizabethan collar was prescribed until complete healing. As the owner was unable to return to the clinic for weekly follow-up visits, the patient’s clinical progress was monitored through daily telephone contact until complete wound closure. The owner was asked about appetite, vocalization, mobility, response to palpation, demeanor, general activity, and wound status. No further wound dehiscence was reported.
Discussion
Our previous study (Feraru et al. 2025) demonstrated the potential of BGAuSP-Alg-MCC to promote skeletal muscle regeneration, with regenerative effects observed both in vitro and in vivo. Based on these findings, this composite was selected for further investigation as a candidate biomaterial for VML treatment. The presence of CD31-positive endothelial cells and α-SMA-positive perivascular cells supports the formation of vascularized tissue with mural-cell investment, consistent with vascular development and ongoing tissue remodeling. Adequate vascularization is an important component of skeletal-muscle repair, as the presence of blood vessels ensures sufficient vascular support and helps maintain cell viability during muscle tissue growth and structural organization (Levenberg et al. 2005; Forbes and Rosenthal 2014). These findings therefore provide encouraging evidence of a vascularized reparative response within the reconstructed region. However, CD31 and α-SMA are not myogenesis-specific markers and do not distinguish newly regenerated muscle from tissue integration, granulation, or remodeling around the implanted material. Because myofiber-specific immunohistochemical markers and quantitative myofiber analysis were not performed, the present findings should be interpreted as evidence of vascularized tissue repair rather than definitive proof of the formation of functional new muscle fibers. While previous studies suggest that VML repair may be limited when scaffolds lack satellite cells or growth factors, the present findings suggest that the BGAuSP-Alg-MCC composite may support a vascularized reparative response and tissue remodeling in skeletal muscle, although its capacity to promote de novo muscle regeneration requires further investigation (Garg et al. 2014; Aurora et al. 2015).
Gold nanoparticles have attracted increasing interest in tissue repair because of their physicochemical and biological properties and their potential to act as delivery systems for therapeutic agents (Nukaly and Ansari 2023). Experimental studies in animal models have reported improved wound healing following gold nanoparticles-bioactive glass-based interventions compared with controls (Mârza et al. 2019, 2021). Proposed mechanisms include modulation of transforming growth factors, inflammatory cytokines, and angiogenesis-related pathways, together with antibacterial and antioxidant effects (Chen et al. 2023; Zhang et al. 2024). In addition, gold nanoparticles have been associated with enhanced collagen formation and organization, which may contribute to improved wound closure and reduced scar formation (Liu et al. 2012).
The staged clinical strategy combined serial debridement, sugar dressing, Hydrosorb® gel, a transposition skin flap, and the BGAuSP-Alg-MCC composite. The favorable clinical course should therefore be interpreted as the result of this combined reconstructive approach, rather than as an effect of the biomaterial alone. Nevertheless, the incorporation of BGAuSP-Alg-MCC was associated with progressive wound healing and tissue remodeling within the reconstructed region. The moderate inflammatory infiltrate observed at 3 months also requires cautious interpretation. In the absence of a control group and quantitative inflammatory scoring, it may reflect ongoing wound remodeling, a host response to the implanted material, or a combination of both.
Limitations include the single-case design, absence of a control group, unknown long-term outcomes beyond 12 weeks, and possible subtle functional deficits undetected without advanced gait analysis. Variability in scaffold degradation, mechanical stability, and ion-release kinetics may also influence regenerative responses.
Future investigations should focus on larger, controlled cohorts to validate these findings and to better define the role of the BGAuSP-Alg-MCC composite in VML repair. Long-term follow-up and objective functional assessments are recommended to further characterize regenerative outcomes and durability. Additionally, optimization of scaffold composition and integration with supportive biological therapies may enhance muscle regeneration in extensive defects.
Conclusion
In conclusion, this single case demonstrates the technical feasibility of using BGAuSP-Alg-MCC composite as an adjunct to transpositional flap reconstruction in a canine patient with complex volumetric muscle loss (see Supplementary Video). Over 12 weeks, the combined treatment was associated with progressive wound healing, a qualitative CT appearance compatible with remodeling of the reconstructed region, and vascularized granulation tissue at histology. Moderate inflammation remained present at 3 months. Because the patient received multiple concurrent interventions and no control treatment was available, the observed outcome reflects the combined reconstructive strategy, making the independent contribution of the BGAuSP-Alg-MCC composite difficult to determine. Nevertheless, the combined approach was associated with progressive wound healing, favorable clinical recovery, qualitative CT changes compatible with tissue remodeling, and vascularized granulation tissue at 3 months. These findings support the clinical feasibility of incorporating BGAuSP-Alg-MCC into flap-based reconstruction of severe muscular defects and provide a basis for further investigation of its potential role in tissue repair. Although the composite has been previously investigated in a controlled rat VML model, further studies in clinically relevant large-animal models with larger cohorts and longer follow-up are warranted to evaluate material persistence and degradation, host response, functional recovery, and the specific contribution of the composite to tissue regeneration.
Supplementary Information
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Acknowledgements
We would like to thank the Surgery and Intensive Care Unit of the Faculty of Veterinary Medicine, as well as the Diagnostic Imaging team, for providing essential support and infrastructure. We also thank Andrei Mariuțeac for his assistance with the schematic representations.
Author contributions
Alina Zăvoi - investigation, methodology, writing – original draft, writing – review and editing. Tom Ablassmaier - investigation, methodology. Bianca Pop - investigation, methodology. Ștefana Mureșan - investigation, methodology. Andrada Negoescu - investigation, methodology. Felix Lucaci - investigation, methodology. Robert Purdoiu - conceptualization, investigation, methodology, resources, writing – original draft, writing – review and editing. Alexandra Feraru - investigation, methodology. Klara Magyari - conceptualization, investigation, methodology, resources, writing – original draft, writing – review and editing. Lucia Bel - conceptualization, supervision, writing – review and editing. Mădălina Dragomir - investigation, methodology. Liviu Oana - investigation, conceptualization, supervision, writing – review and editing. Marian Taulescu - conceptualization, supervision, writing – review and editing. Alexandra Dreancă - conceptualization, data curation, investigation, methodology, writing – original draft, writing – review and editing.
Funding
This study was funded by the Ministry of European Investment and Projects (MIPE) as Managing Authority for the Smart Growth, Digitalization and Financial Instruments Programme 2021–2027 and the Ministry of Research, Innovation and Digitalization (MCID) as Intermediary Research Body, project code SMIS 2021 + 324,771 contract MIPE no. G-2024–71962/23.10.2024 and contract MCID no.390,005/23.10.2024, project acronym INSPIRE-II.
Data availability
The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author/s.
Declarations
Ethical approval
Written informed consent was obtained from the owner for the participation of their animal in this study.
Competing interests
Klara Magyari, Alexandra Dreancă, and Marian Tulescu have a patent #CBI 2024 A 00476 pending to OSIM. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Footnotes
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References
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Associated Data
Supplementary Materials
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Data Availability Statement
The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author/s.