兽研目的
肩袖修复术后再撕裂率高，尤其是在中大型撕裂和肌腱质量较差的患者中，传统缝线锚钉固定往往无法在愈合过程中提供足够的机械支撑。本研究旨在系统评价猪源脱细胞真皮基质（pADM）补片在大动物模型中用于肩袖增强修复的体内安全性、降解特性及修复疗效。
方法
在24只成年比格犬中建立双侧冈下肌腱止点损伤模型。试验组动物接受pADM补片增强联合缝线锚钉固定，对照组仅接受常规锚钉修复。于术后2、4、6和13周进行包括血液学分析、大体观察、CT/MRI影像、组织病理学（HE和Masson染色）及生物力学测试在内的综合评估。
结果
植入pADM补片未引起异常炎症或免疫反应，表明其具有良好的生物相容性。影像学和组织学分析显示，试验组肌腱结构恢复更优，腱-骨整合增强。补片发生渐进性降解并实现有效的宿主组织整合。生物力学测试显示，试验组在所有时间点的最大失败载荷均显著更高，表明修复肌腱具有更优的机械完整性（P < 0.05）。
结论
pADM补片具有良好的生物安全性和生物相容性，可促进肌腱结构恢复和腱-骨愈合，并增强生物力学强度。其降解动力学与宿主组织重塑过程相匹配，至13周时完全吸收。该脱细胞真皮基质补片是一种安全有效的肩袖修复增强材料，具有良好的临床转化潜力。
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引言
肩袖损伤是肩部最常见的肌肉骨骼疾病之一，是疼痛、功能受限和生活质量下降的主要原因（Oh et al., 2018）。其发病机制涉及多种因素，包括急性创伤、慢性肌腱退变、重复性机械过载和年龄相关组织退化（Oh et al., 2018; Derwin et al., 2010）。尽管关节镜锚钉修复已成为标准治疗方法，但对于中重度撕裂、组织质量差或退变性损伤，单纯锚钉修复往往无法提供足够的机械稳定性，并导致腱-骨愈合缓慢，术后再撕裂率高达20–90%（Derwin et al., 2010; Rhee and Oh, 2017; Cooper and Mirzayan, 2016）。因此，在提高修复结构机械稳定性的同时促进生物学有效的肌腱再生，已成为肩袖手术中的关键挑战（Jiang et al., 2024）。
为解决上述局限性，补片增强策略越来越多地被引入肩袖修复。理想的增强材料除提供机械增强和降低局部应力集中外，还应通过促进宿主细胞浸润、胶原沉积、血管生成和细胞外基质（ECM）重塑来主动调节局部愈合微环境。已有多种生物材料被探索用于肩袖增强，包括自体移植物、合成支架和ECM源性生物补片（Derwin et al., 2010; Jiang et al., 2024）。研究表明，自体移植物（如肱二头肌长头腱）具有良好的生物相容性和组织相容性，但受限于供区并发症和可获得性（Rhee and Oh, 2017）。同种异体或异种补片（如猪小肠黏膜下层，SIS）具有一定的生物活性，但部分研究报道其可能引起强烈炎症反应甚至导致修复失败（Walton et al., 2007）。因此，兼具机械支撑和生物调控功能的生物补片已成为肌腱组织工程和再生修复领域的主要研究焦点。
在这些生物材料中，脱细胞真皮基质（ADM）因其独特的结构和生物学特性，已成为软组织增强中特别有前景的候选材料。作为一种脱细胞ECM源性支架，ADM保留了天然三维胶原架构、生物活性黏附基序和基质相关信号分子，同时基本消除了免疫原性细胞成分（Chen et al., 2022）。这些特性使ADM能够支持细胞迁移、血管长入、胶原重塑和建设性组织再生（Chen et al., 2022; Bush and Gertzman, 2016; Mohamma et al., 2023）。特别是猪源ADM（pADM）来源丰富、机械性能良好且生物降解可控，使其适用于肩袖增强。
尽管具有这些有前景的特征，关于异种ADM在肩袖修复中的报道仍然稀少，且大多数现有研究——甚至包括在大动物模型中的研究——在很大程度上局限于短期组织学观察或孤立的生物力学测量，未能在较长时间内系统阐明体内降解动力学、组织整合模式或机械增强机制（Cooper and Mirzayan, 2016; Adams et al., 2006）。此外，目前文献中占主导地位的自体或异体真皮基质的使用受到供体可用性、潜在继发并发症和批次间变异性等因素的限制。这些空白凸显了在临床相关大动物模型中对异种pADM进行全面、长期评估的必要性。因此，本研究利用比格犬冈下肌腱止点损伤建立了标准化的肩袖修复模型。采用自身对照设计，将CT/MRI成像、定量组织病理学、体内降解分析和生物力学测试整合于单一实验框架内，我们在多个时间点（第2、4、6和13周）进行了多维度、多时间点的综合评估，以系统阐明pADM补片的安全性、降解动力学、组织整合模式和机械增强机制。
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材料与方法
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实验材料
生物型pADM补片使用（BP-01，50 mm × 50 mm，由博碧（重庆）生物科技有限公司提供）。该补片通过一系列脱细胞、纯化和灭菌程序制造，旨在保留天然细胞外基质结构的同时最大限度降低免疫原性。pADM补片在体内植入前进行了系统的体外表征，包括脱细胞效率（残留DNA和总蛋白含量）、力学性能、细胞相容性和体外降解动力学。缝合锚钉用作对照修复材料。
24只成年比格犬（普通级，雌雄比=1:1，体重范围13–15 kg）由湖南恒信旺生物科技有限公司提供。所有动物实验方案均经机构动物护理与使用委员会（IACUC）批准。本研究在设计、实施和报告动物实验方面严格遵循ARRIVE 2.0指南（补充表S1）。动物护理和实验程序符合实验动物护理的相关指南，方案经机构动物护理与使用委员会批准（EP-I-D-250205-00-01）。
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实验设计与分组
采用自身对照设计。经过7天检疫期后，动物被随机分组。每只犬的左肩关节分配至试验组（pADM补片联合缝合锚钉修复），右肩关节作为对照组（仅缝合锚钉修复）。根据观察时间点，动物分为2W、4W、6W和13W组（每组n = 6）。在所有时间点，检查均在丙泊酚麻醉下进行以确保动物福利。
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动物模型制备与手术操作
犬术前禁食8 h。采用静脉注射丙泊酚（10 mg/mL，1 mL/kg）诱导麻醉，异氟烷（2.0%–2.5%，氧气为载体）维持麻醉，持续监测呼吸和心率。进行标准皮肤准备、消毒和铺巾。沿肩胛冈下缘做切口；依次切开皮肤和皮下组织，钝性分离肌肉层以暴露冈下肌腱在肱骨大结节上的止点。
将冈下肌腱在其止点处切断，切除约5 mm × 5 mm的肌腱段以模拟临床中型撕裂（图1）。对足印区软组织进行清创，使用丝锥制备骨隧道，依次植入1枚内排锚钉（3.5 × 16 mm）和2枚外排锚钉（3.4 × 14 mm）。在试验组中，生物补片用生理盐水复水，修剪至最终尺寸约18 mm × 10 mm（保留其原始厚度），置于腱-骨界面，采用缝线桥技术固定于大结节外侧面。在对照组中，采用相同类型的锚钉和缝线桥技术进行肌腱缝合和锚钉固定，但不植入补片。所有手术操作均由同一名经验丰富的外科医生完成。切口分层闭合。术后动物置于恢复室，苏醒后返回饲养笼。术后给予美洛昔康（第1天0.2 mL/kg，之后每天0.1 mL/kg，皮下注射）镇痛5天，头孢噻呋钠（2.2 mg/kg，肌内注射）预防感染5天。在指定时间点（术后2、4、6和13周），动物经肌内注射Zoletil 2.5 mg/kg麻醉，随后静脉注射丙泊酚3 mg/kg。确认角膜反射和踏板回缩反射消失后，在深麻醉状态下通过放血实施安乐死。简言之，在股三角处做10 cm横向切口，完全切断股动静脉。用湿纱布持续擦拭切口处的血液和血凝块，同时用流水保持股动脉开口通畅。动物在3~5 min内因急性失血性休克死亡，并通过听诊无心跳及瞳孔散大对光无反应进一步确认。
[图：比格犬肩袖损伤补片植入手术过程，(a) 切口与组织暴露，(b)]
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观察与评价指标
术后每日进行一般健康状况评估，包括精神状态、采食情况、活动水平、体重、伤口愈合及行为表现。观察期间记录任何异常发现或术后并发症，包括感染、血肿、跛行或其他局部和全身不良反应。
术前及术后2、4、6和13周采集静脉血，进行全血细胞计数、血液生化和凝血功能检测。为进一步评估对植入材料的全身炎症和免疫反应，检测血清免疫球蛋白G（IgG）、免疫球蛋白M（IgM）和超敏C反应蛋白（CRP）水平。
术前、术后即刻及各时间点进行CT扫描，评估锚钉位置、骨隧道状态、骨吸收及软组织肿胀情况。术后即刻及各时间点进行MRI检查，观察肌腱连续性、信号强度、软组织修复及关节腔状态。
术前及各时间点在MRI图像上测量三个感兴趣区（ROIs）的肌腱厚度，以评估形态学恢复情况。三个ROIs定义如下：撕裂部位——在撕裂缘垂直于肌腱纤维测量，位于内排锚钉内侧10 mm处；固定部位——在补片-肌腱界面处测量，对应内排锚钉中轴；固定下游部位——在锚钉边缘外侧15 mm的正常肌腱区域测量，用于与修复部位比较。
所有测量均在标准化平面上获取，避免肌腱折叠或拉伸，由同一名经过培训的工程师完成，每个部位重复测量三次，取平均值进行分析。安乐死后进行大体解剖，检查主要器官（心、肝、脾、肺、肾）的形态、大小、颜色和质地，并检查手术区域、补片状况及腱-骨界面。
手术区域组织经固定、脱钙、脱水、包埋后制备病理切片。进行苏木精-伊红染色和Masson染色，以评估炎症和组织反应，采用既往建立的六项评分系统（0~3分）（Wang and He, 2022）对肌腱修复质量进行半定量评估，并对补片体内降解进行分级（De Jong et al., 2005）。
将完整的肌腱-肱骨标本置于万能试验机（型号E43.104；MTS工业系统（中国）有限公司）上，配备0–5 KN载荷传感器进行拉伸测试。每个标本的两端沿肌腱加载方向牢固固定于夹具中，确保补片-肌腱修复界面精确居中于测试夹具内，无偏心加载或压缩损伤。所有时间点和组别的所有标本均以30 mm/min的恒定加载速率进行测试（标距：15 mm）。最大失效载荷（N）确定为载荷-位移曲线的峰值点：上升阶段代表肌腱拉伸和弹性变形，峰值代表修复标本承受的最大载荷，随后的急剧下降表明修复界面失效。所有样本制备和测试程序均由同一操作者执行以确保一致性。每个时间点和组别测试三个平行标本，取平均值用于统计分析。记录肌腱-骨交界处的最大失效载荷以评估修复部位的机械强度。
盲法实施如下：影像学和生物力学评估采用单盲法，组织病理学评估采用双盲法（切片重新编码，全部读片后揭盲）。
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统计分析
数据采用SPSS统计软件分析。计量数据以均数±标准差（x̄ ± s）表示。组间比较采用配对Student's t检验或Wilcoxon符号秩检验。不同时间点间比较采用单因素方差分析（ANOVA）。P值<0.05认为具有统计学意义。基于第2周肌腱厚度数据（数据变异性最大，代表最差情况），使用G*Power 3.1（α = 0.05）进行事后功效分析。获得的功效值为：断裂部位0.987、固定部位0.931、下游部位>0.999，证实样本量（n = 6）提供了足够的统计功效。
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结果
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一般状况与安全性
所有犬均平稳度过术后恢复期，恢复顺利。观察期间未检测到精神状态、饮食、呼吸、排便或自主活动异常，未发生不良事件（如死亡、癫痫发作、感染、血肿、脂肪液化或跛行）。手术切口均实现一期愈合，无红肿、渗出或溃疡。各时间点体重均呈正常增长趋势，无异常减轻或波动。
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血液学与免疫学结果
术后各时间点全血细胞计数、血液生化和凝血参数均维持在正常参考范围内（补充表S2、S3）。尽管部分指标显示统计学显著变化，但无生理学意义，不提示器官损伤、造血异常或凝血功能障碍。
关于免疫炎症标志物（图2），CRP在术后出现一过性升高，于术后第3天（23.546 ± 17.367 μg/mL）和第2周达到峰值，随后迅速下降，至第4周恢复至接近基线水平（5.550 ± 2.358 μg/mL），反映创伤后典型的急性炎症反应。相比之下，IgG和IgM水平在任何时间点均未见异常波动，提示体液免疫功能未受显著影响，材料未引起异常免疫反应。
[图：术后3天、2、4、6和13周测定的血清CRP、IgG和IgM水平。]
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影像学评估
CT检查显示肱骨近端骨隧道形态规则，锚钉位置稳定，无移位、松动或穿破，骨隧道周围无骨吸收或破坏（图3）。术后即刻手术区域可见少量气体和软组织肿胀；2周时肿胀减轻，4、6和13周时软组织形态基本恢复正常。
[图：试验组和对照组术前、术中及术后2、4、6和13周的Micro-CT分析结果。红框i]
MRI检查显示术后即刻手术区域冈下肌腱连续性中断、结构紊乱（图4；补充图S1）。2周时肌腱连续性恢复，呈中等信号强度。4和6周时肌腱信号逐渐降低，结构规则性改善。13周时肌腱信号低，形态和信号接近正常组织；腱-骨愈合满意，未见明显异常关节积液。
[图：T1加权磁共振成像图像。红色箭头：断裂部位（内侧锚钉内侧10 mm）；黄色箭头]
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肌腱厚度变化
术后早期（2和4周），所有测量部位的肌腱厚度较术前值减小，随后逐渐恢复（图5）。
[图：术后13周内（a）断裂部位、（b）固定部位和（c）固定部位下游的肌腱厚度。]
在断裂部位：对照组术前厚度为2.2 ± 0.1 mm，试验组为2.1 ± 0.1 mm。第4周时，两组均达到最低值（对照组：1.6 ± 0.1 mm；试验组：1.8 ± 0.1 mm）。此后厚度逐渐增加，至第13周时两组恢复至相近水平（对照组：2.1 ± 0.6 mm；试验组：2.1 ± 0.1 mm）。
在固定部位：对照组从2.1 ± 0.1 mm逐渐降至第4周的1.7 ± 0.1 mm；试验组从2.2 ± 0.1 mm缓慢降至第4周的1.9 ± 0.1 mm。该部位的下降幅度小于残端部位，且试验组厚度略高于对照组。
固定部位下游：对照组从2.2 ± 0.2 mm降至第4周的1.8 ± 0.1 mm；试验组从2.2 ± 0.2 mm降至第4周的1.9 ± 0.2 mm。第6周时，试验组已恢复至术前水平（与术前值无显著差异），而对照组仍低于术前水平。第13周时，两组均恢复至正常水平，组间无统计学差异。
总体而言，与术前值相比，两组术后第2周和第4周的肌腱厚度均显著减小。在各对应时间点，试验组冈下肌腱厚度始终大于对照组。术后第6周时，试验组肌腱结构恢复优于对照组，可见近乎完全的组织重建。至术后第13周时，两组均显示冈下肌腱结构完整性恢复。
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大体解剖观察
各时间点大体解剖显示，主要器官（心脏、肝脏、脾脏、肺脏、肾脏）的位置、形态、颜色和质地均正常，未见坏死、结节、积液或异常粘连。手术区域未见明显积液、化脓或组织坏死。补片与周围组织黏附良好，界面清晰，未见异物性肉芽肿或异常组织增生。
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组织病理学评价
试验组与对照组在各时间点的炎症及组织反应评分相似，无统计学显著差异（表1；补充表S4）。两组术后炎症均升高，于第4周达峰值（加权总评分：13），随后逐渐下降，符合伤口愈合的生理性炎症过程。
TABLE 1. Histopathological inflammatory reaction scores.
| Group | Statistical analysis of inflammation and tissue response | |||
|---|---|---|---|---|
| 2W M(P 25 ,P 75 ) | 4W M(P 25 ,P 75 ) | 6W M(P 25 ,P 75 ) | 13W M(P 25 ,P 75 ) | |
| Test group | 11.00 (11.00,11.50) | 13.00 (12.50,13.50) | 9.00 (9.00,9.50) | 7.00 (6.00,7.50) |
| Control group | 11.00 (11.00,12.00) | 13.00 (13.00,13.50) | 10.00 (9.50,10.50) | 7.00 (7.00,7.50) |
| Mean of test group | 11.33 | 13.00 | 9.33 | 6.67 |
| Mean of control group | 11.67 | 13.33 | 10.00 | 7.33 |
| Z | 0.258 | 0.471 | 0.943 | 0.471 |
| P | 0.796 | 0.637 | 0.346 | 0.637 |
如表2和补充表S5所示，肌腱修复的半定量评分表明，试验组在各时间点的修复质量均显著优于对照组，表现为纤维结构更连续、纤维排列更规则、细胞核形态接近正常肌腱、炎症细胞浸润更轻微以及新生血管化更适度。
TABLE 2. Semi-quantitative scores for tendon repair.
| Group | Statistical analysis of semi-quantitative tendon repair scores | |||
|---|---|---|---|---|
| 2W M(P 25 ,P 75 ) | 4W M(P 25 ,P 75 ) | 6W M(P 25 ,P 75 ) | 13W M(P 25 ,P 75 ) | |
| Test group | 12.00 (11.50,12.00) | 8.00 (8.00,8.00) | 4.00 (4.00,4.00) | 2.00 (1.50,2.00) |
| Control group | 16.00 (15.50,16.50) | 10.00 (9.50,10.00) | 6.00 (5.50,6.50) | 4.00 (3.50,4.50) |
| Mean of test group | 11.67 | 8.00 | 4.00 | 1.67 |
| Mean of control group | 16.00 | 9.67 | 6.00 | 4.00 |
| Z | 1.993 | 2.121 | 2.087 | 1.993 |
| P | 0.046 | 0.034 | 0.037 | 0.046 |
连续时间点对腱-骨界面的组织学评价显示，试验组与对照组之间的修复模式存在明显差异（图6、7）。第2周时，对照组表现为急性炎症，多形核白细胞丰富（>30%），新形成的胶原纤维广泛不连续。试验组显示较轻的淋巴细胞浸润（炎症范围和新生血管化限于20–30%），补片结构完整，仅边缘轻度溶解；胶原纤维总体保持连续性，伴局灶性中断，新生纤维已开始与残留的腱-骨界面纤维融合。
[图：对照组和试验组第2、4、6和13周的HE染色。红色箭头：植入锚钉区域；黑色箭头：补片；绿色]
[图：对照组和试验组第2、4、6和13周的Masson染色。绿色箭头：新形成纤维与……之间的界面]
第4周时，试验组更早过渡至增殖/重塑期，多形核白细胞和淋巴细胞浸润降至最低水平，并可见巨噬细胞。胶原连续性明显改善，仅遗留少量不连续区域，补片保持完整。对照组仍保留部分多形核白细胞，纤维排列呈中度疏松。
在6周时，试验组表现出同步的降解-再生过程，炎症浸润和新生血管化降至10–19%，细胞密度首先恢复正常。补片显示中度降解（裂纹/碎片），并伴有宿主胶原长入。在对照组中，尽管纤维变得连续且轻度疏松，但巨噬细胞浸润和新生血管化仍处于较高水平（20–30%），表明处于更活跃的重塑状态。
在13周时，试验组的补片已完全降解，被连续的自体组织替代，细胞核呈成熟的细长梭形；炎症浸润<10%，未观察到锚定部位炎症。对照组显示连续的纤维，但细胞密度轻度升高且细胞核更圆，提示成熟延迟。两组均可见广泛的纤维-界面整合。
总之，该生物补片表现出三个核心优势：减轻早期炎症、可控降解并有序再生，以及促进晚期生理性成熟。组织学证实其被自体组织完全、同步替代且无残留，支持其作为腱-骨愈合功能性支架的可行性和优越性。
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生物力学测试
在术后2、4、6和13周，试验组腱-骨交界处的最大失效载荷显著高于对照组（图8）。2、4、6和13周的P值分别为0.013、0.049、0.014和0.036。
[图：2、4、6和13周后最大失效力的力学测试数据。星号表示显著差异]
随着术后时间延长，两组的机械强度逐渐改善，试验组增强更为明显，表明该生物补片有效增强了修复部位的机械稳定性并支持早期康复。
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讨论
巨大肩袖撕裂修复后的高再撕裂率归因于多种因素，包括肌腱退变、血供不足、腱-骨界面生物学愈合能力有限以及持续的复杂机械负荷（Mcelvany等，2015）。传统单纯缝合修复在一定程度上恢复了解剖连续性，但无法维持修复界面的长期机械稳定性，也无法有效促进大型或严重退变性肩袖撕裂的腱-骨交界处组织学愈合（Mandaleson，2021；Longo等，2021）。因此，基于生物材料的增强修复已成为肩袖修复的主要研究方向。在本研究中，我们采用比格犬肩袖损伤模型和13周多维度观察方案，系统评估了pADM补片用于肩袖增强修复的安全性、降解行为及促愈合效果。
安全性是生物补片临床转化的首要前提。在本研究中，ADM补片植入未引发异常炎症或免疫反应；血液学、生化学和免疫学指标均保持在正常范围内，主要器官大体完整，组织相容性良好。两组的局部炎症反应均表现为早期升高、后期降低，表明ADM补片未加重局部炎症负担。这一安全性特征具有重要临床意义，因为一些常用的生物材料，例如SIS补片，尽管因其易获得而被广泛研究，但已被证明在早期移植后会引起排斥反应和明显炎症（Cooper和Mirzayan，2016；Walton等，2007；Toth等，2025）。相比之下，本研究使用的ADM补片在炎症控制方面表现出明显优势。
ADM的核心优势在于其保留了三维ECM网络，为成纤维细胞和腱细胞的黏附、迁移和增殖提供了支架（Brown和Badylak，2014；Gadre等，2024）。与合成材料相比，ADM在脱细胞过程中保留了关键的ECM成分，包括I型和III型胶原、糖胺聚糖（GAGs）、弹性蛋白以及多种基质结合蛋白（Brown和Badylak，2014）。这些成分不仅维持微结构稳定性，还通过释放生物活性信号调节细胞行为和局部再生，从而促进有序的胶原沉积和组织再生（Rijal，2017；Liu等，2025）。天然ECM材料还具有调节局部免疫微环境的能力。一些研究表明，此类材料可促进巨噬细胞从M1向M2表型极化，从而降低慢性炎症和纤维化风险并增强组织再生能力（Cornelison等，2018；Zhang等，2022）。在本研究中，试验组未出现持续性炎症且组织愈合更优，间接支持了ADM补片可能通过此类免疫调节机制改善修复环境的假说。
在肌腱-骨愈合过程中，胶原纤维的排列和密度是组织成熟度的重要形态学指标（Thomopoulos等，2015）。正常肌腱组织表现为高度有序、致密且沿应力方向排列的胶原纤维结构，而早期修复组织通常呈现为排列紊乱、疏松的胶原网络（Darrieutort-Laffite等，2024）。随着组织重塑的进展，胶原纤维逐渐重新排列，形成更连续、致密且定向的成熟结构（Kanazawa等，2016；Sohn和Cho，2025）。本研究的Masson染色显示，与对照组相比，试验组胶原纤维排列更连续、致密且定向，提示ADM补片有效促进了ECM重塑和组织成熟，使修复组织更接近正常肌腱结构。Hurley等对五项随机对照试验（307例患者）的荟萃分析显示，脱细胞胶原基质补片增强组的再撕裂率为11%，而对照组为34%（Hurley等，2024）。该临床证据从定量角度支持了ADM补片促进胶原重塑的组织学基础。这些发现与既往研究一致，表明ADM不仅提供被动机械支撑，还通过调控ECM重建和局部微环境主动改善肌腱-骨愈合质量（Flury等，2018；Gillespie等，2016）。
血管生成是影响肌腱-骨愈合的另一重要因素（Brown和Badylak，2014；Liu等，2025）。适度的新生血管化为修复区域提供氧气、营养物质和细胞来源，促进炎症消退和组织再生（Zhang等，2022；Neishabouri等，2022）。ADM保留的天然ECM结构可作为内皮细胞迁移和微血管长入的支架，部分基质降解产物也可能发挥促血管生成作用（Darrieutort-Laffite等，2024）。Xu等（Xu等，2012）在灵长类肩袖修复模型中植入猪源α-gal清除ADM补片，观察到3个月时血管结构丰富，至6个月时血管密度降至与正常肌腱相似的水平。该研究还通过免疫化学和血清分析证实无显著炎症，组织学上重塑为平行的胶原纤维排列。这些数据与本试验组“早期适度新生血管化，随后逐渐减少并成熟”的观察结果高度一致，表明ADM有助于建立稳定有序的修复微环境。
材料降解行为与组织愈合过程的匹配性是评价生物补片性能的核心参数（Liu等，2025）。理想的补片应随着新组织的成熟而逐渐降解，同时提供足够的机械支撑，实现动态替代。本研究通过多时间点组织学观察，揭示了ADM补片在比格犬肩袖修复模型中的渐进性降解模式：“早期结构完整—中期降解伴纤维组织长入—晚期完全吸收”，完全降解约发生在13周（Xu等，2012）。该时间进程与肌腱-骨愈合的关键重塑期基本吻合，表明补片的降解动力学与组织修复需求匹配良好。Ide等（Ide等，2009）在大鼠ADM重建肩袖缺损模型中，于术后2、6和12周进行组织学和生物力学评价，发现12周内ADM补片已在组织学上完全整合为类似正常对照的结构，尽管胶原排列仍有些不规则。Nicholson等（Nicholson等，2007）在绵羊肩袖修复模型中比较了交联pADM补片（Zimmer Collagen Repair Patch）和猪SIS补片的整合行为。9周时，大部分SIS材料已被吸收，而ADM补片表面仍有巨噬细胞附着；至24周时，ADM组巨噬细胞消失，补片完全整合入周围组织，伴有血管和成纤维细胞侵入（Nicholson等，2007）。本研究13周时补片完全吸收可能反映了物种差异或补片交联程度对降解速率的影响。
生物力学测试进一步证实，试验组在各时间点的肌腱-骨交界处强度均显著更高，术后早期优势尤为突出。这直接降低了早期再撕裂的风险，为更积极的早期康复提供了力学基础，具有明确的临床意义。结合组织学观察可以得出结论：ADM补片通过增强修复结构的机械完整性、促进胶原成熟以及促进肌腱-骨界面整合，实现了修复性能的持续改善。
在临床实践中，对于大型、退变性或软组织质量较差的肩袖撕裂，单纯缝线修复往往难以获得满意结果。ADM补片可作为覆盖修复界面的增强层，以降低张力、改善愈合环境并提高修复成功率。本研究提供了来自大动物模型的系统性临床前证据，全面验证了ADM补片用于肩袖增强的安全性和有效性，并支持其进一步的临床转化。
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结论
本研究在比格犬肩袖损伤模型中系统评价了pADM补片用于肩袖增强的安全性和有效性。结果表明，该补片具有良好的生物相容性和体内安全性，未诱导异常炎症反应、免疫排斥或主要器官毒性。在组织愈合方面，ADM补片显著促进肌腱结构恢复，加速腱-骨愈合，并提高修复部位的生物力学强度。此外，该补片表现出逐步降解特性，其降解动力学与宿主组织重塑过程相匹配，至13周时完全吸收。总之，pADM补片在该急性犬肩袖损伤模型中表现出良好的安全性和有效性，但仍需进一步研究以验证其在人类慢性退变性肩袖病变中的适用性。
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局限性与未来方向
本研究为大动物自身对照实验，样本量有限。观察期设定为13周，虽覆盖主要愈合阶段，但仍需进一步研究更长期的体内反应和长期安全性。实验基于比格犬冈下肌损伤模型，该模型在病理特征、力学环境和愈合能力方面与人类肩袖损伤存在固有差异；因此，将结果外推至临床实践需谨慎，且必须辅以人体研究。
此外，本研究尚存在若干局限性应予说明。本研究采用健康、急性肩袖损伤模型，与人类慢性退变性肩袖撕裂存在根本差异，后者以肌腱退变、脂肪浸润、肌肉萎缩及内在愈合能力受损为特征，因此本研究结果可能无法直接转化至这一更具临床代表性的情境。未进行功能结局评估（如关节活动度、步态分析），限制了我们全面评价修复后功能恢复的能力。缺乏腱-骨界面的分子水平评估（如生长因子表达、基因调控网络），限制了我们对pADM促进腱-骨愈合潜在机制的理解。此外，本研究仅在单一动物物种中进行，未与其他市售生物补片进行头对头比较，难以全面评估pADM相对于现有产品的优越性。
未来研究可包括多中心、前瞻性临床试验，以进一步评价该补片在人体中的安全性和有效性。同时，应结合影像学定量评价、分子生物学检测等手段，深入阐明脱细胞基质调控腱-骨愈合的分子机制，为材料优化和临床方案改进提供更精确的理论依据。
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Objective
Rotator cuff repair remains challenged by a high postoperative retear rate, particularly in moderate-to-large tears and in patients with poor tendon quality, where conventional suture-anchor fixation often fails to provide sufficient mechanical support during the healing process. This study aimed to systematically evaluate the in vivo safety, degradation characteristics, and reparative efficacy of a porcine-derived acellular dermal matrix (pADM) patch for rotator cuff augmentation in a large animal model.
Methods
Bilateral infraspinatus tendon insertion injury models were established in 24 adult beagle dogs. Animals in the test group received pADM patch augmentation in combination with suture-anchor fixation, whereas those in the control group underwent conventional anchor repair alone. Comprehensive assessments including hematological analysis, gross observation, CT/MRI images, histopathology (HE and Masson staining), and biomechanical testing were performed at 2, 4, 6, and 13 weeks postoperatively.
Results
Implantation of the pADM patch elicited no abnormal inflammatory or immune responses, indicating favorable biocompatibility. Imaging and histology analyses demonstrated superior tendon structural restoration and enhanced tendon-bone integration in the test group. The patch underwent progressive degradation and achieved effective host-tissue integration. Biomechanical testing revealed significantly higher maximum failure loads in the test group at all time points, indicating superior mechanical integrity of the repaired tendon (P < 0.05).
Conclusion
The pADM patch exhibits favorable biosafety and biocompatibility, promotes tendon structure recovery and tendon-bone healing, and enhances biomechanical strength. Its degradation kinetics match the host tissue remodeling process, with complete resorption by 13 weeks. This acellular dermal matrix patch represents a safe and effective augmentation material for rotator cuff repair, with promising potential for clinical translation.
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Introduction
Rotator cuff injury is one of the most prevalent musculoskeletal disorders of the shoulder and represents a major cause of pain, functional limitation, and reduced quality of life (Oh et al., 2018). Its pathogenesis is multifactorial and involves acute trauma, chronic tendon degeneration, repetitive mechanical overload, and age-related tissue deterioration (Oh et al., 2018; Derwin et al., 2010). Although arthroscopic anchor repair has become the standard treatment, for moderate-to-severe tears, poor tissue quality, or degenerative injuries, simple anchor repair often fails to provide adequate mechanical stability and results in slow tendon-bone healing, with postoperative retear rates as high as 20–90% (Derwin et al., 2010; Rhee and Oh, 2017; Cooper and Mirzayan, 2016). Therefore, improving the mechanical stability of the repair construct while simultaneously promoting biologically effective tendon regeneration has become a critical challenge in rotator cuff surgery (Jiang et al., 2024).
To address these limitations, patch augmentation strategies have been increasingly introduced into rotator cuff repair. In addition to providing mechanical reinforcement and reducing local stress concentration, an ideal augmentation material should also actively regulate the local healing microenvironment by promoting host cell infiltration, collagen deposition, angiogenesis, and extracellular matrix (ECM) remodeling. Various biomaterials have been explored for rotator cuff augmentation, including autologous grafts, synthetic scaffolds, and ECM-derived biological patches (Derwin et al., 2010; Jiang et al., 2024). Studies have shown that autografts (e.g., long head of biceps tendon) exhibit good biocompatibility and histocompatibility but are limited by donor-site morbidity and availability (Rhee and Oh, 2017). Allogeneic or xenogeneic patches (e.g., porcine small intestinal submucosa, SIS) possess some biological activity, but some studies report that they may elicit strong inflammatory reactions and even lead to repair failure (Walton et al., 2007). Consequently, biological patches capable of mechanical support and biological regulation have emerged as a major focus in tendon tissue engineering and regenerative repair.
Among these biomaterials, acellular dermal matrix (ADM) has emerged as a particularly promising candidate for soft-tissue augmentation due to its unique structural and biological characteristics. As a decellularized ECM-derived scaffold, ADM preserves the native three-dimensional collagen architecture, bioactive adhesion motifs, and matrix-associated signaling molecules while largely eliminating immunogenic cellular constituents (Chen et al., 2022). These properties enable ADM to support cell migration, vascular ingrowth, collagen remodeling, and constructive tissue regeneration (Chen et al., 2022; Bush and Gertzman, 2016; Mohamma et al., 2023). In particular, porcine-derived ADM (pADM) provides abundant availability, favorable mechanical properties, and controllable biodegradation, making it suitable for rotator cuff augmentation.
Despite these promising features, reports on xenogeneic ADM in rotator cuff repair remain scarce, and most existing studies-even those in large animal models-have been largely confined to short-term histological observations or isolated biomechanical measurements, without systematic elucidation of in vivo degradation kinetics, tissue integration pattern, or mechanical augmentation mechanism over an extended period (Cooper and Mirzayan, 2016; Adams et al., 2006). In addition, the use of autologous or allogeneic dermal matrices, which dominate the current literature, is constrained by donor availability, potential secondary morbidity, and batch-to-batch variability. These gaps highlight the need for a comprehensive, long-term evaluation of xenogeneic pADM in a clinically relevant large animal model. Therefore, the present study established a standardized rotator cuff repair model using infraspinatus tendon insertion injury in beagle dogs. Adopting a self-controlled design and integrating CT/MRI imaging, quantitative histopathology, in vivo degradation analysis, and biomechanical testing within a single experimental framework, we performed a multi-dimensional, multi-time-point comprehensive evaluation (at 2, 4, 6, and 13 weeks) to systematically elucidate the safety, degradation kinetics, tissue integration pattern, and mechanical augmentation mechanism of the pADM patch.
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Materials and methods
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Experimental materials
The biological pADM patch was used a (BP-01, 50 mm × 50 mm, provided by BioPic (Chongqing) Biotechnology Co., Ltd., China). The patch was manufactured through a series of decellularization, purification, and sterilization procedures designed to preserve the native extracellular matrix architecture while minimising immunogenicity. The pADM patch was systematically characterized in vitro prior to in vivo implantation, including decellularization efficiency (residual DNA and total protein content), mechanical properties, cytocompatibility, and in vitro degradation kinetics. Suture anchors were used as control repair materials.
Twenty-four adult beagle dogs (ordinary grade, male:female = 1:1, body weight range 13–15 kg) were supplied by Hunan Hengxinwang Biotechnology Co., Ltd. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). This study was conducted in strict accordance with the ARRIVE 2.0 guidelines for the design, implementation, and reporting of animal experiments (Supplementary Table S1). Animal care and experimental procedures complied with relevant guidelines for laboratory animal care, and the protocol was approved by the Institutional Animal Care and Use Committee (EP-I-D-250205-00-01).
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Experimental design and grouping
A self-controlled design was employed. After a 7-day quarantine period, animals were randomly assigned to groups. The left shoulder joint of each dog was assigned to the test group (pADM patch combined with suture anchor repair), and the right shoulder joint served as the control group (suture anchor repair alone). Based on observation time points, animals were divided into 2W, 4W, 6W, and 13W groups (n = 6 per group). At all time points the examinations were performed under propofol anesthesia to ensure animal welfare.
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Animal model preparation and surgical procedure
Dogs were fasted for 8 h before surgery. Aanesthesia was induced with intravenous propofol (10 mg/mL, 1 mL/kg) and maintained with isoflurane (2.0%–2.5% in oxygen), with continuous monitoring of respiration and heart rate. Standard skin preparation, disinfection, and draping were performed. An incision was made along the inferior border of the scapular spine; the skin and subcutaneous tissue were incised sequentially, and the muscle layer was bluntly dissected to expose the insertion site of the infraspinatus tendon on the humeral greater tuberosity.
The infraspinatus tendon was transected at its insertion, and a segment of approximately 5 mm × 5 mm was excised to simulate a clinical moderate-sized tear (Figure 1). Soft tissue at the footprint area was debrided, bone tunnels were prepared using a tap, and one medial-row anchor (3.5 × 16 mm) and two lateral-row anchors (3.4 × 14 mm) were sequentially implanted. In the test group, the biological patch was rehydrated with normal saline, trimmed to a final size of approximately 18 mm × 10 mm (with its native thickness preserved), placed over the tendon-bone interface, and fixed to the lateral aspect of the greater tuberosity using a suture-bridge technique. In the control group, tendon suture and anchor fixation were performed using the same anchor types and suture-bridge technique, but without patch implantation. All surgical procedures were carried out by the same experienced surgeon. The incision was closed in layers. Animals were placed in a recovery room after surgery and returned to their housing after awakening. Postoperatively, meloxicam (0.2 mL/kg on day 1, followed by 0.1 mL/kg daily, subcutaneous) was administered for 5 days for analgesia, and ceftiofur sodium (2.2 mg/kg, intramuscular) for 5 days for antibiotic prophylaxis. At the designated time points (2, 4, 6, and 13 weeks postoperatively), animals were anesthetized by intramuscular injection of Zoletil at 2.5 mg/kg, followed by intravenous administration of propofol at 3 mg/kg. After confirming the absence of corneal and pedal withdrawal reflexes, euthanasia was performed under deep anesthesia by exsanguination. Briefly, a 10 cm transverse incision was made in the femoral triangle, and the femoral artery and vein were completely severed. A wet gauze was used to continuously wipe away blood and clots from the incision site, while running tap water was applied to maintain patency of the femoral arterial opening. Death occurred within 3–5 min as a result of acute hemorrhagic shock, and was further confirmed by the absence of a heartbeat on auscultation and pupillary dilation unresponsive to light.
[图：Surgical procedure of patch implantation for rotator cuff injury in a beagle dog, (a) Incision and tissue exposure, (b) ]
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Observation and evaluation parameters
Postoperative general health assessments were conducted daily and included evaluations of mental status, dietary intake, activity level, body weight, wound healing, and behavioral performance. Any abnormal findings or postoperative complications, including infection, hematoma, lameness, or other local and systemic adverse reactions, were recorded throughout the observation period.
Venous blood was collected preoperatively and at 2, 4, 6, and 13 weeks postoperatively for complete blood count, blood biochemistry, and coagulation function tests. To further evaluate the systemic inflammatory and immune responses to the implanted material, serum levels of immunoglobulin G (IgG), immunoglobulin M (IgM), and high-sensitivity C-reactive protein (CRP) were measured.
CT scans were performed preoperatively, immediately postoperatively, and at each time point to evaluate anchor position, bone tunnel status, bone resorption, and soft-tissue swelling. MRI was performed immediately postoperatively and at each time point to observe tendon continuity, signal intensity, soft-tissue repair, and joint cavity status.
Tendon thickness was measured on MRI images at three regions of interest (ROIs) preoperatively and at each time point to assess morphological recovery. The three ROIs were defined as follows: Rupture site measured perpendicular to the tendon fibers at the torn edge, 10 mm medial to the medial anchor; Fixation site measured at the patch–tendon interface corresponding to the central axis of the medial anchor; Fixation downstream site measured in the normal tendon region 15 mm lateral to the anchor edge, for comparison with the repaired site.
All measurements were obtained at standardized planes, avoiding tendon folding or stretching, and were performed by a single trained engineer with three repetitions per site; the average was used for analysis. After euthanasia, gross anatomy was performed to examine the morphology, size, color, and texture of major organs (heart, liver, spleen, lung, kidney) and to inspect the operative area, patch condition, and tendon-bone interface.
Tissue from the operative area was fixed, decalcified, dehydrated, and embedded for preparation of pathological sections. Hematoxylin-eosin and Masson staining were performed for evaluation of inflammatory and tissue reactions, semi-quantitative assessment of tendon repair quality using a previously established six-item scoring system (0-3) (Wang and He, 2022), and grading of in vivo patch degradation (De Jong et al., 2005).
Intact tendon-humerus specimens were subjected to tensile testing using a universal testing machine (Model E43.104; MTS Industrial Systems (China) Co., Ltd.) equipped with a 0–5 KN load cell. Both ends of each specimen were securely fixed in the grips along the direction of tendon loading, ensuring that the patch-tendon repair interface was precisely centered within the testing fixture without eccentric loading or compressive damage. The test was conducted at a constant loading rate of 30 mm/min (gauge length: 15 mm) for all specimens across all time points and groups. The maximum failure load (N) was determined as the peak point of the load-displacement curve: the ascending phase represents tendon stretching and elastic deformation, the peak represents the maximum load sustained by the repaired specimen, and the subsequent sharp drop indicates failure at the repair interface. All sample preparation and testing procedures were performed by the same operator to ensure consistency. For each time point and group, three parallel specimens were tested, and the average value was used for statistical analysis. The maximum failure load at the tendon-bone junction was recorded to evaluate the mechanical strength of the repaired site.
Blinding was applied as follows: imaging and biomechanical assessments were performed in a single-blind manner, and histopathological evaluations were performed in a double-blind manner (slides re-coded with unblinding after all readings).
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Statistical analysis
Data were analyzed with SPSS statistical software. Measurement data are presented as mean ± standard deviation (x̄ ± s). Comparisons between groups were performed using the paired Student’s t-test or the Wilcoxon signed-rank test. Comparisons among different time points were performed using One-Way Analysis of Variance (ANOVA). A P value <0.05 was considered statistically significant. A post hoc power analysis was conducted using G*Power 3.1 (α = 0.05) based on tendon thickness data at week 2, where data variability was greatest, representing the worst-case scenario. The achieved power values were 0.987 at the rupture site, 0.931 at the fixation site, and >0.999 at the downstream site, confirming that the sample size (n = 6) provides sufficient statistical power.
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Results
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General condition and safety
All dogs survived the postoperative period uneventfully and recovered smoothly. No abnormalities in mental status, diet, respiration, defecation, or spontaneous activity were detected during the observation period, and no adverse events (e.g., death, seizures, infection, hematoma, fat liquefaction, or lameness) occurred. Surgical incisions achieved primary healing without redness, exudation, or ulceration. Body weight showed normal growth trends at all time points, with no abnormal loss or fluctuation.
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Hematological and immunological findings
Complete blood count, blood biochemistry, and coagulation parameters remained within normal reference ranges at all postoperative time points (Supplementary Table S2, S3). Although some indices showed statistically significant variations, these were not physiologically significant and did not indicate organ injury, hematopoietic abnormalities, or coagulopathy.
Regarding immune-inflammatory markers (Figure 2), CRP exhibited a transient postoperative elevation, peaking at postoperative day 3 (23.546 ± 17.367 μg/mL) and week 2, then rapidly declining and returning to near-baseline levels by week 4 (5.550 ± 2.358 μg/mL), reflecting a typical acute inflammatory response following trauma. In contrast, IgG and IgM levels showed no abnormal fluctuations at any time point, suggesting that humoral immune function was not significantly affected and that the material did not elicit abnormal immune responses.
[图：Postoperative serum levels of CRP, IgG, and IgM measured at 3 days, 2, 4, 6 and 13W.]
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Imaging evaluation
CT examination showed regular morphology of the bone tunnels in the proximal humerus, stable anchor positions without displacement, loosening, or perforation, and no bone resorption or destruction around the bone tunnels (Figure 3). A small amount of gas and soft-tissue swelling was observed in the operative area immediately after surgery; swelling was reduced at 2 weeks, and soft-tissue morphology had largely normalized by 4, 6 and 13 weeks.
[图：Micro-CT analysis results of test and control group at pre-operative,intra-operative and 2, 4, 6 and 13 weeks. Red box i]
MRI examination revealed discontinuity of the infraspinatus tendon and structural disorganization in the operative area immediately after surgery (Figure 4; Supplementary Figure S1). At 2 weeks, tendon continuity was restored with intermediate signal intensity. At 4 and 6 weeks, tendon signal gradually decreased and structural regularity improved. At 13 weeks, tendon signal was low, and morphology and signal approached those of normal tissue; tendon-bone healing was satisfactory, and no obvious abnormal joint effusion was observed.
[图：T1 -weighted magnetic resonance imaging images. Red arrow: rupture site (10 mm medial to the medial anchor); yellow arro]
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Changes in tendon thickness
During the early postoperative period (2 and 4 weeks), tendon thickness decreased at all measured sites compared with preoperative values, followed by gradual recovery (Figure 5).
[图：The tendon thickness at the (a) rupture, (b) fixation and (c) fixation downstream site over 13 weeks of postoperatively.]
At the rupture site: Preoperative thickness was 2.2 ± 0.1 mm in the control group and 2.1 ± 0.1 mm in the test group. At 4 weeks, both groups reached their minimum values (control: 1.6 ± 0.1 mm; test: 1.8 ± 0.1 mm). Thereafter, thickness gradually increased, and by 13 weeks, both groups recovered to similar levels (control: 2.1 ± 0.6 mm; test: 2.1 ± 0.1 mm).
At the fixation site: The control group gradually decreased from 2.1 ± 0.1 mm to 1.7 ± 0.1 mm at 4 weeks; while the test group slowly decreased from 2.2 ± 0.1 mm to 1.9 ± 0.1 mm at 4 weeks. The decrease at this site was smaller than that at the stump, and thickness in the test group was slightly higher than in the control group.
Downstream of the fixation site: The control group decreased from 2.2 ± 0.2 mm to 1.8 ± 0.1 mm at 4 weeks; the test group decreased from 2.2 ± 0.2 mm to 1.9 ± 0.2 mm at 4 weeks. At 6 weeks, the test group had recovered to preoperative levels (no significant difference from preoperative values), whereas the control group remained below preoperative levels. At 13 weeks, both groups had recovered to normal levels, with no statistical difference between them.
Overall, tendon thickness in both groups was significantly reduced at 2 and 4 weeks postoperatively compared with preoperative values. At each corresponding time point, the infraspinatus tendon thickness in the test group was consistently greater than that in the control group. At 6 weeks postoperatively, tendon structural recovery in the test group was superior to that in the control group, with near-complete tissue reconstruction observed. By 13 weeks postoperatively, both groups showed restored structural integrity of the infraspinatus tendon.
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Gross anatomical observation
Gross anatomy at each time point showed that the major organs (heart, liver, spleen, lung, kidney) were normal in position, shape, color, and texture, with no necrosis, nodules, effusion, or abnormal adhesions. The operative area showed no obvious effusion, suppuration, or tissue necrosis. The patch adhered well to surrounding tissues with a clear interface, and no foreign-body granuloma or abnormal tissue hyperplasia was observed.
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Histopathological evaluation
Inflammatory and tissue reaction scores were similar between the test and control groups at all time points, with no statistically significant differences (Table 1; Supplementary Table S4). Both groups exhibited an increase in inflammation after surgery, peaking at 4 weeks (Weighted total score:13) and then gradually declining, which is consistent with the physiological inflammatory process of wound healing.
TABLE 1. Histopathological inflammatory reaction scores.
| Group | Statistical analysis of inflammation and tissue response | |||
|---|---|---|---|---|
| 2W M(P 25 ,P 75 ) | 4W M(P 25 ,P 75 ) | 6W M(P 25 ,P 75 ) | 13W M(P 25 ,P 75 ) | |
| Test group | 11.00 (11.00,11.50) | 13.00 (12.50,13.50) | 9.00 (9.00,9.50) | 7.00 (6.00,7.50) |
| Control group | 11.00 (11.00,12.00) | 13.00 (13.00,13.50) | 10.00 (9.50,10.50) | 7.00 (7.00,7.50) |
| Mean of test group | 11.33 | 13.00 | 9.33 | 6.67 |
| Mean of control group | 11.67 | 13.33 | 10.00 | 7.33 |
| Z | 0.258 | 0.471 | 0.943 | 0.471 |
| P | 0.796 | 0.637 | 0.346 | 0.637 |
As shown in Table 2 and Supplementary Table S5, semi-quantitative scores for tendon repair indicated that the test group had significantly better repair quality at each time point than the control group, as evidenced by more continuous fiber structure, more regular fiber arrangement, nuclear morphology resembling that of normal tendon, milder inflammatory cell infiltration, and more moderate neovascularization.
TABLE 2. Semi-quantitative scores for tendon repair.
| Group | Statistical analysis of semi-quantitative tendon repair scores | |||
|---|---|---|---|---|
| 2W M(P 25 ,P 75 ) | 4W M(P 25 ,P 75 ) | 6W M(P 25 ,P 75 ) | 13W M(P 25 ,P 75 ) | |
| Test group | 12.00 (11.50,12.00) | 8.00 (8.00,8.00) | 4.00 (4.00,4.00) | 2.00 (1.50,2.00) |
| Control group | 16.00 (15.50,16.50) | 10.00 (9.50,10.00) | 6.00 (5.50,6.50) | 4.00 (3.50,4.50) |
| Mean of test group | 11.67 | 8.00 | 4.00 | 1.67 |
| Mean of control group | 16.00 | 9.67 | 6.00 | 4.00 |
| Z | 1.993 | 2.121 | 2.087 | 1.993 |
| P | 0.046 | 0.034 | 0.037 | 0.046 |
Histological evaluation of the tendon-bone interface at serial time points revealed distinct repair patterns between the test and control groups (Figures 6, 7). At 2 weeks, the control group exhibited acute inflammation with abundant polymorphonuclear leukocytes (>30%) and extensive discontinuity of newly formed collagen fibers. The test group showed milder lymphocytic infiltration (inflammatory extent and neovascularization limited to 20–30%), with a structurally intact patch and only slight peripheral dissolution; collagen fibers maintained overall continuity with focal interruptions, and nascent fibers had begun merging with residual tendon-bone interface fibers.
[图：HE staining of control and test group at 2, 4, 6 and 13 weeks. Red arrow: implanted anchor area; black arrow: patch; gre]
[图：Masson staining of control and test group at 2, 4, 6 and 13 weeks. The green arrow: The interface between newly formed f]
At 4 weeks, the test group transitioned earlier to the proliferative/remodeling phase, with polymorphonuclear and lymphocytic infiltration reduced to minimal levels and macrophages present. Collagen continuity improved markedly, leaving only minor discontinuous areas, while the patch remained intact. The control group still retained some polymorphonuclear leukocytes and showed moderately loose fiber arrangement.
At 6 weeks, the test group demonstrated synchronized degradation-regeneration, with inflammatory infiltration and neovascularization decreasing to 10–19% and cell density normalizing first. The patch showed moderate degradation (cracks/fragments) with host collagen ingrowth. In the control group, although fibers became continuous and mildly loose, macrophage infiltration and neovascularization remained elevated (20–30%), indicating a more active remodeling state.
At 13 weeks, the patch had completely degraded in the test group, replaced by continuous autologous tissue with mature elongated, spindle-shaped nuclei; inflammatory infiltration was <10% and no anchor-site inflammation was observed. The control group displayed continuous fibers but with slightly elevated cell density and more rounded nuclei, suggesting delayed maturation. Extensive fiber-interface integration was evident in both groups.
In summary, the biological patch demonstrated three core advantages: attenuation of early inflammation, controlled degradation with orderly regeneration, and promotion of late physiological maturation. Complete, synchronous replacement by autologous tissue without residue was confirmed histologically, supporting its feasibility and superiority as a functional scaffold for tendon-bone healing.
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Biomechanical testing
At 2, 4, 6 and 13 weeks postoperatively, the maximum failure load at the tendon-bone junction was significantly higher in the test group than in the control group (Figure 8). The P-values for 2, 4, 6 and 13 weeks are 0.013, 0.049, 0.014, and 0.036, respectively.
[图：Mechanical testing data of maximum force to failure after 2, 4, 6 and 13W. Asterisks indicate significant differences us]
With increasing postoperative time, mechanical strength gradually improved in both groups, with a more pronounced enhancement in the test group, indicating that the biological patch effectively enhanced the mechanical stability of the repair site and supported early rehabilitation.
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Discussion
The high retear rate following repair of massive rotator cuff tears is attributed to multiple factors, including tendon degeneration, insufficient vascularity, limited biological healing capacity at the tendon-bone interface, and persistent complex mechanical loading (Mcelvany et al., 2015). Conventional suture-only repair restores anatomical continuity to some extent but fails to maintain long-term mechanical stability at the repair interface or effectively promote histological healing of the tendon-bone junction for large or severely degenerative rotator cuff tears (Mandaleson, 2021; Longo et al., 2021). Consequently, biological material-based augmentation has become a major research direction in rotator cuff repair. In the present study, using a beagle dog rotator cuff injury model and a 13-week multi-dimensional observation protocol, we systematically evaluated the safety, degradation behavior, and healing-promoting effects of a pADM patch for rotator cuff augmentation.
Safety is the primary prerequisite for clinical translation of biological patches. In this study, ADM patch implantation did not provoke abnormal inflammatory or immune responses; hematological, biochemical, and immunological indices remained within normal ranges, major organs were grossly intact, and histocompatibility was favorable. Local inflammatory responses in both groups showed an early increase followed by a late decrease, indicating that the ADM patch did not exacerbate the local inflammatory burden. This safety profile has important clinical significance because some commonly used biological materials, for example, SIS patches, despite being widely studied due to their ready availability have been shown to elicit rejection reactions and pronounced inflammation upon early transplantation (Cooper and Mirzayan, 2016; Walton et al., 2007; Toth et al., 2025). In contrast, the ADM patch used in this study demonstrated clear advantages in inflammatory control.
The core advantage of ADM lies in its retention of the three-dimensional ECM network, which provides a scaffold for adhesion, migration, and proliferation of fibroblasts and tenocytes (Brown and Badylak, 2014; Gadre et al., 2024). Compared with synthetic materials, ADM preserves key ECM components including type I and type III collagen, glycosaminoglycans (GAGs), elastin, and various matrix-bound proteins during decellularization (Brown and Badylak, 2014). These components not only maintain microstructural stability but also regulate cell behavior and local regeneration by releasing bioactive signals, thereby promoting orderly collagen deposition and tissue regeneration (Rijal, 2017; Liu et al., 2025). Native ECM materials also possess the ability to modulate the local immune microenvironment. Some studies have indicated that such materials can promote macrophage polarization from the M1 to the M2 phenotype, thereby reducing chronic inflammation and fibrosis risk and enhancing tissue regeneration capacity (Cornelison et al., 2018; Zhang et al., 2022). In the present study, the absence of persistent inflammation and the superior tissue healing observed in the test group indirectly support the hypothesis that the ADM patch may improve the repair environment through such immunomodulatory mechanisms.
During tendon-bone healing, the arrangement and density of collagen fibers are important morphological indicators of tissue maturity (Thomopoulos et al., 2015). Normal tendon tissue exhibits a highly ordered, dense collagen fiber structure aligned along stress directions, whereas early repair tissue typically presents as a disorganized, loose collagen network (Darrieutort-Laffite et al., 2024). As tissue remodeling progresses, collagen fibers gradually realign to form a more continuous, dense, and oriented mature structure (Kanazawa et al., 2016; Sohn and Cho, 2025). Masson staining in the present study showed that the test group had more continuous, dense, and oriented collagen fiber arrangement compared with the control group, suggesting that the ADM patch effectively promotes ECM remodeling and tissue maturation, bringing the repaired tissue closer to normal tendon architecture. A meta-analysis by Hurley et al. of five randomized controlled trials (307 patients) showed that the retear rate in the acellular collagen matrix patch augmentation group was 11% compared with 34% in the control group (Hurley et al., 2024). This clinical evidence quantitatively supports the histological basis of collagen remodeling promoted by ADM patches. These findings are consistent with previous studies, indicating that ADM not only provides passive mechanical support but also actively improves tendon-bone healing quality by regulating ECM reconstruction and the local microenvironment (Flury et al., 2018; Gillespie et al., 2016).
Angiogenesis is another important factor influencing tendon-bone healing (Brown and Badylak, 2014; Liu et al., 2025). Moderate neovascularization provides oxygen, nutrients, and cellular sources to the repair area, facilitating inflammation resolution and tissue regeneration (Zhang et al., 2022; Neishabouri et al., 2022). The native ECM structure retained by ADM serves as a scaffold for endothelial cell migration and microvessel ingrowth, and some matrix degradation products may also exert pro-angiogenic effects (Darrieutort-Laffite et al., 2024). Xu et al. (Xu et al., 2012) implanted a porcine, α-gal-reduced ADM patch in a primate rotator cuff repair model and observed abundant vascular structures at 3 months, with vessel density decreasing to levels similar to normal tendon by 6 months. That study also confirmed no significant inflammation by immunochemical and serum analyses, with histological remodeling into parallel collagen fiber alignment. These data are highly consistent with our observation of “moderate early neovascularization followed by gradual reduction and maturation” in the test group, indicating that ADM contributes to establishing a stable and orderly repair microenvironment.
The match between material degradation behavior and the tissue healing process is a core parameter for evaluating biological patch performance (Liu et al., 2025). An ideal patch should gradually degrade as new tissue matures while providing adequate mechanical support, achieving dynamic replacement. Through multi-time-point histological observation, the present study revealed a progressive degradation pattern of the ADM patch in the beagle rotator cuff repair model: “early structural integrity–midstage degradation with fibrous tissue ingrowth–late complete resorption,” with complete degradation occurring at approximately 13 weeks (Xu et al., 2012). This time course largely coincides with the critical remodeling phase of tendon-bone healing, indicating that the degradation kinetics of the patch are well matched to tissue repair demands. Ide et al. (Ide et al., 2009) in a rat ADM-reconstructed rotator cuff defect model, performed histological and biomechanical evaluations at 2, 6, and 12 weeks postoperatively and found that within 12 weeks the ADM patch had already been completely integrated histologically into a structure resembling normal controls, although collagen alignment remained somewhat irregular. Nicholson et al. (Nicholson et al., 2007) compared the integration behavior of cross-linked pADM patch (Zimmer Collagen Repair Patch) and porcine SIS patch in an ovine rotator cuff repair model. At 9 weeks, most of the SIS material had been resorbed, whereas the ADM patch still had macrophages attached on its surface; by 24 weeks, macrophages had disappeared from the ADM group, and the patch was completely integrated into the surrounding tissue with vascular and fibroblastic invasion (Nicholson et al., 2007). The complete resorption at 13 weeks in the present study may reflect species differences or the effect of patch cross-linking degree on degradation rate.
Biomechanical testing further confirmed that the test group had significantly higher tendon-bone junction strength at all time points, with a particularly prominent advantage in the early postoperative period. This directly reduces the risk of early retear and provides a mechanical basis for more aggressive early rehabilitation, which has clear clinical implications. Combined with histological observations, it can be concluded that the ADM patch achieves sustained improvement in repair performance by enhancing the mechanical integrity of the repair structure, promoting collagen maturation, and facilitating tendon-bone interface integration.
In clinical practice, for rotator cuff tears that are large, degenerative, or of poor soft-tissue quality, suture-only repair often yields unsatisfactory results. The ADM patch can be used as an augmentation layer covering the repair interface to reduce tension, improve the healing environment, and increase repair success rates. This study provides systematic preclinical evidence from a large animal model, comprehensively validating the safety and efficacy of the ADM patch for rotator cuff augmentation and supporting its further clinical translation.
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Conclusion
In this study, we systematically evaluated the safety and efficacy of a pADM patch for rotator cuff augmentation in a beagle dog rotator cuff injury model. The results demonstrated that the patch possesses good biocompatibility and in vivo safety, without inducing abnormal inflammatory responses, immune rejection, or major organ toxicity. Regarding tissue healing, the ADM patch significantly promoted tendon structure recovery, accelerated tendon-bone healing, and improved biomechanical strength at the repair site. Moreover, the patch exhibited stepwise degradation characteristics, and its degradation kinetics matched the host tissue remodeling process, with complete resorption by 13 weeks. In summary, the pADM patch demonstrated favorable safety and efficacy in this acute canine rotator cuff injury model, although further studies are warranted to validate its applicability in human chronic degenerative rotator cuff conditions.
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Limitations and future directions
This study is a large-animal self-controlled experiment with a limited sample size. The observation period was set to 13 weeks, which, although covering the main healing phase, still requires further investigation of longer-term in vivo reactions and long-term safety. The experiment was based on a beagle dog infraspinatus injury model, which has inherent differences from human rotator cuff injury in terms of pathological characteristics, mechanical environment, and healing capacity; thus, extrapolation of the results to clinical practice warrants caution and must be complemented by human studies.
In addition, several limitations of this study should be acknowledged. This study used a healthy, acute rotator cuff injury model, which differs fundamentally from chronic degenerative rotator cuff tears in humans characterised by tendon degeneration, fatty infiltration, muscle atrophy, and impaired intrinsic healing capacity and the present findings may not directly translate to this more clinically representative setting. Functional outcome assessments (e.g., joint range of motion, gait analysis) were not performed, which limits our ability to comprehensively evaluate the functional recovery after repair. The absence of molecular-level evaluations at the tendon-bone interface (e.g., growth factor expression, gene regulatory networks) restricts our understanding of the underlying mechanisms by which pADM promotes tendon-bone healing. Furthermore, this study was conducted in only a single animal species and did not include a head-to-head comparison with other commercially available biological patches, making it difficult to fully assess the superiority of pADM over existing products.
Future research may include multi-center, prospective clinical trials to further evaluate the safety and efficacy of the patch in humans. At the same time, imaging quantitative evaluation, molecular biological assays, and other means should be combined to deeply elucidate the molecular mechanisms by which the decellularized matrix regulates tendon-bone healing, providing a more precise theoretical basis for material optimization and clinical protocol improvement.