兽研引言
间充质干细胞（MSC）为基础的再生疗法已显示出对多种疾病的治疗潜力，包括癌症、心力衰竭、卒中、神经系统疾病、糖尿病、自身免疫性疾病和慢性炎症，这归因于其再生能力、免疫调节作用以及分泌促进组织修复和调节炎症的生物活性因子[1–5]。此外，MSCs可从多种组织来源中分离，包括骨髓、脂肪组织、胎盘、脐带、毛囊和牙齿组织，跨越多种动物物种，从而支持MSCs的开发和规模化生产[6–10]。MSCs的治疗效果部分由旁分泌机制介导，通过分泌细胞外囊泡（EVs），特别是外泌体，在细胞通讯中发挥关键作用，支持细胞功能[11, 12]。
外泌体是EVs的纳米级亚群，直径范围为30至150 nm，含有生物活性分子，包括核酸、脂质和蛋白质，以支持细胞功能[12, 13]。外泌体的生物学特性使其适合作为无细胞治疗剂，其临床潜力与细胞为基础的治疗相当，且免疫排斥、致瘤性、毒性和伦理问题的风险较低。这使其能够规模化储存和分发，因为外泌体不含活细胞且免疫原性表面分子的表达较低[12, 14]。基于外泌体的治疗相关的较低生物安全风险也使得跨物种异种给药成为可能[12, 15, 16]。先前的研究已报道人MSC来源外泌体在犬、猫和实验动物模型中的安全性和治疗疗效，表明外泌体介导的组织修复机制在跨物种屏障间相对保守[15, 17–21]。
多项临床前和临床研究已证明使用MSC来源外泌体的无细胞疗法在治疗神经退行性疾病、牙周组织再生、骨关节炎和其他退行性疾病中的安全性和治疗疗效[22–27]。先前的研究还表明，MSC-Exos在动物模型中加速伤口愈合和增强组织修复，人牙髓干细胞来源外泌体（huDPSC-Exos）被证明在伤口愈合过程中支持血管生成、炎症反应和免疫调节[6, 11, 13, 28]。这些再生特性突显了MSC-Exos在动物术后伤口愈合中的潜在应用，动物术后仍易发生手术并发症，如伤口裂开、持续性炎症、感染和败血症，从而延迟恢复并损害动物福利[29–32]。
犬牙髓干细胞（cDPSCs）因其微创、伦理上可接受的牙髓采集而成为兽医学再生治疗的有前景候选者，多项研究已基于国际细胞治疗学会（ISCT）建立的最低标准报道了其MSC样特征[33, 34]。然而，其外泌体（cDPSC-Exos）在很大程度上仍未得到探索，且尚无研究评估cDPSC-Exos在猫中的异种潜力。cDPSC-Exos的综合蛋白质组学表征也尚缺乏。
本初步研究的目标是表征cDPSCs和cDPSC-Exos，并通过蛋白质组学分析和体内异种应用于术后伤口愈合来评估其治疗潜力。这是首次将cDPSC表征、cDPSC-Exos表征和蛋白质组学分析与猫去势术后伤口的异种评估相结合的研究，作为标准化、临床相关且自然发生的手术模型。我们假设cDPSC-Exos将通过调节炎症、免疫调节和血管生成促进猫的术后伤口愈合。
材料与方法：
伦理批准
本研究按照公认的动物福利原则和ARRIVE 2.0指南进行。实验方案由印度尼西亚加查马达大学兽医学院伦理委员会审查并批准，批准号为101/EC-FKH/Int./2025。犬牙髓组织在适当的临床监督下从健康供体犬的常规拔牙过程中获取。猫的临床评估使用客户拥有的健康雄性猫在加查马达大学兽医学院Prof. Soeparwi动物医院进行择期去势手术，在获得主人书面知情同意后进行。
所有猫均接受标准化的围手术期护理，包括麻醉、镇痛、抗生素覆盖、术后监测和每日伤口评估。动物单独饲养，自由获取食物和水，所有程序由合格的兽医人员执行，以尽量减少疼痛、压力和不适。本研究采用阳性对照设计，因为不对术后伤口进行治疗被认为在伦理上不合适。研究期间未观察到不良反应、伤口裂开、感染或临床相关的全身并发症。
研究期间和地点
本研究于2025年1月至2026年1月在加查马达大学兽医学院综合实验室进行细胞分离与培养；在加查马达大学兽医学院生物化学与分子生物学实验室进行cDPSCs和cDPSC-Exos表征；在加查马达大学兽医学院Prof. Soeparwi动物医院进行体内观察。
研究设计
本研究采用体外与体内相结合的方法，包括cDPSC的分离与表征；cDPSC-Exos的分离、表征及蛋白质组学分析；以及一项随机对照预试验，用于异种评估去势后猫伤口愈合情况，如图1所示。
[图：犬牙髓干细胞（cDPSCs）分离与鉴定的示意图概述；cDPSC来源外泌体（cDPS]
细胞分离、培养与扩增
牙髓取自一只2.5岁健康家养雄性犬的单颗健康恒磨牙，该犬无全身性疾病或口腔病变史。采用组织块培养技术进行分离，方法改编自既往研究[7, 35]。将磨牙牙冠切开以采集牙髓。将牙髓剪碎至约0.5 cm，置于含Dulbecco改良Eagle培养基（Gibco™，Thermo Fisher Scientific，Waltham，MA，USA）的35 mm培养皿中，培养基补充10%胎牛血清（Gibco™）、1%抗生素-抗真菌剂（Gibco™）和1% GlutaMAX（Gibco™）；维持70%湿度；于37°C、5% CO₂条件下培养，每48小时更换培养基。当细胞达到约80%融合时，将其传代至三个含培养基的新35 mm培养皿中。根据ISCT对MSCs的最低要求，使用第3代cDPSCs进行表征。
集落形成实验
按照既往发表的方案[7, 36]，将分离的cDPSCs以每60 mm培养皿（TPP®，Trasadingen，Switzerland）500个细胞的浓度培养14天。14天后，用磷酸盐缓冲液（PBS，pH 7.4）洗涤集落，用冷甲醇固定20分钟，用结晶紫染色5分钟，计数含≥50个细胞的集落[7, 35]。集落形成效率（CFE）按以下公式计算：CFE（%）=（形成的集落数/接种的细胞数）× 100%。
增殖实验
在第1、5和7天，将cDPSCs在含5% alamarBlue™（Sigma-Aldrich，St. Louis，MO，USA）的培养基中孵育3小时，以评估增殖能力[7, 35]。在570 nm处记录吸光度，并根据产品指南将细胞活力量化为alamarBlue的还原百分比。
多向分化
采用既往发表的方案[7, 10, 13, 37, 38]，在体外评估cDPSCs的多向分化，包括成脂、成骨和成软骨分化。将cDPSCs接种于三个独立的24孔板中，并在如前所述配制的成骨、成软骨或成脂诱导培养基中培养[7, 38, 39]，随后进行Alizarin Red（Sigma-Aldrich）染色以检测成骨分化中的钙沉积，Alcian Blue（Sigma-Aldrich）染色以检测成软骨分化中的糖胺聚糖（GAG）积累，以及Oil Red O（Sigma-Aldrich）染色以根据细胞内脂滴形成评估成脂分化。
逆转录定量聚合酶链反应（RT-qPCR）
使用RT-qPCR作为流式细胞术的替代方法，评估MSC表面标志物（CD90、CD44、CD105、CD73和CD45）的基因表达。RT-qPCR还用于评估cDPSCs的干性相关标志物（NANOG、REX-1、OCT-4）、成骨分化标志物（RUNX-2、OSX、OCN）、成软骨分化标志物（COL-X、SOX-9）和成脂分化标志物（PPAR-γ、LPL）。本研究使用的特异性引物见表1。
Table 1. List of primer sequences used to characterize cDPSCs and cDPSC-Exos.
| Gene | Forward primer sequence (5’-3’) | Reverse primer sequence (5’-3’) |
|---|---|---|
| GAPDH | CCAACTGCTTGGCTCCTCTA | GTCTTCTGGGTGGCAGTGAT |
| CD90 | AGGACGAGGGGACATACACA | ATGCCCTCACACTTGACCAG |
| CD44 | CCCCATTACCAAAGACCACGA | TGGGATTTGAGGTTTCCGCA |
| CD105 | CGAGGAGTCTGTCACCGGAAA | GCGCCAAAGGTGATACCCAG |
| CD73 | GGCAACCTGATTTGTGATGCT | AGGTAATTGTGCCGTTGTTCC |
| CD45 | GTTTCCAGTTCTGTTTCCCCAG | CATTGGTCACAATTCACGGTATCA |
| NANOG | CAGCAGATGCAAGAACTTTCCA | AGCAGGTACCCCTGAGTCAC |
| REX-1 | AGGTTCTCACAGCAAGCTCA | CCAGCAAATTCTGCGCACTG |
| OCT-4 | AGGAGAAGCTGGAGCAAAACC | GTGATCCTCTTCTGCTTCAGGA |
| COL-X | AGCACCCCGAATCCATCTGAG | TGCCCGTAGGTGTTTGGTATC |
| SOX-9 | TCTGGAGGCTGCTGAACGA | TTCTTCACCGACTTCCTCCG |
| RUNX-2 | GGAAGAGGCAAGAGTTTCACC | GTGCTCACTTGCCAACAGAA |
| OSX | GCGTCCTCCCTGCTTGAG | GCTTTGCCCAGTGTCGTTG |
| OCN | GCCAGCCTATGGTCTCCTCTG | CCACCAGCTCCTTCTGTTCTCT |
| PPAR-γ | CCTCTTCCATGCTGTTATGGGT | TGGCATCTCTGTGTCAACCA |
| LPL | CTGGAGAGACTCAGAAAAAGGTAAT | TCCTTCTGTAGATTTGCTCAGGT |
| CD9 | ATTTCGTCTTCTGGCTTGCTGG | AGGGCACCAGCTCCAATCAG |
使用TRIzol®试剂（Invitrogen，美国加利福尼亚州卡尔斯巴德）和Direct-zol™ RNA小量制备试剂盒（Zymo Research，美国加利福尼亚州尔湾）提取总RNA；使用NanoDrop™（Thermo Fisher Scientific，美国马萨诸塞州沃尔瑟姆）进行RNA定量。使用QuantiNova®逆转录试剂盒（Qiagen，德国希尔登）进行逆转录，随后使用QuantiNova® SYBR® Green PCR试剂盒（Qiagen）在Rotor-Gene Q实时PCR循环仪（Qiagen）上进行qPCR扩增。对于干性和分化标志物，mRNA表达量以管家基因（GAPDH）进行归一化，使用公式ΔCt = Ct靶基因 - CtGAPDH计算；而表面标志物表达以百分比表示，使用公式2−ΔCt × 100%计算[7, 35, 40]。
外泌体分离与鉴定
使用Total Exosome Isolation Reagent（Invitrogen，货号4478359）按照产品说明书进行外泌体分离，方法如先前MSC来源外泌体研究中所报道[41, 42]。收集第3代cDPSC培养物的条件培养基（CM），于4 °C以2,000 × g离心30 min，获得无细胞和碎片的上清液。按条件培养基体积的25%向条件培养基中加入Total Exosome Isolation Reagent（Invitrogen），混合物于4 °C孵育过夜。孵育后，于4 °C以10,000 × g离心60 min收集外泌体沉淀，重悬于5 mL无菌PBS中，于−80 °C保存备用。通过RT-qPCR评估CD9转录本表达来鉴定cDPSC-Exos，同时使用纳米颗粒追踪分析（NTA）评估粒径分布。
外泌体蛋白质组学分析
使用Orbitrap Exploris 240高分辨率质谱仪（Thermo Fisher Scientific）对cDPSC-Exos进行蛋白质组学分析，方法遵循先前报道的MSC来源外泌体蛋白质组学工作流程[43, 44]。样品使用十二烷基硫酸钠裂解缓冲液裂解，经二硫苏糖醇还原，用质谱级胰蛋白酶消化，用1%三氟乙酸（TFA）酸化，并在分析前过滤。使用配备Acclaim PepMap 100 C18柱的Vanquish Horizon超高效液相色谱系统进行肽段分离，随后在Orbitrap Exploris 240高分辨率质谱仪（Thermo Fisher Scientific）上以正离子模式、Full MS/dd-MS²采集模式进行分析。原始数据使用Proteome Discoverer 2.5软件配合SequestHT搜索引擎对UniProt（Canis lupus familiaris）数据库进行处理。使用Reactome进行功能富集分析，使用STRING分析蛋白质-蛋白质相互作用（PPI）网络。由于C. l. familiaris数据库注释有限，与伤口愈合相关的功能和通路分析使用Homo sapiens数据库进行，以改善生物学解释。
蛋白质印迹分析
按照先前报道的工作流程[45]，使用iBlot™ 2和iBind™ Western Systems（Thermo Fisher Scientific）通过评估波形蛋白表达进行蛋白质印迹分析以验证蛋白质组学发现。使用BCA蛋白定量试剂盒（Thermo Fisher Scientific）测定蛋白浓度，在4-12% Bis-Tris凝胶上通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，并使用iBlot™ 2 Transfer System（Thermo Fisher Scientific）转移至聚偏二氟乙烯（PVDF）膜上。随后封闭膜并与小鼠抗波形蛋白单克隆抗体（V9）和兔抗β-肌动蛋白抗体（作为内参对照，Thermo Fisher Scientific）孵育，然后与辣根过氧化物酶标记的兔抗小鼠免疫球蛋白（Ig）G和山羊抗兔IgG二抗（Thermo Fisher Scientific）孵育。按照制造商说明书，使用SuperSignal™ West Pico PLUS化学发光底物（Thermo Fisher Scientific）显示蛋白条带。
猫术后伤口愈合的异种临床评价
在六只年龄≤5岁的临床健康雄性猫中进行了异种cDPSC-Exos治疗的临床评价，通过简单随机化将猫随机分配至两组：（1）cDPSC-Exos组，在去势后伤口局部涂抹cDPSC-Exos凝胶；（2）阳性对照组，局部涂抹Bioplacenton®。未设置阴性对照组是基于动物福利的伦理考虑，因为术后伤口不予处理可能导致不良并发症。
所有猫在主人知情同意后，于加查马达大学兽医学院Soeparwi教授动物医院接受去势手术。猫单独饲养，自由采食和饮水，并接受相同的麻醉、镇痛、抗生素和术后护理方案。术前给予曲马多（2-4 mg/kg IV）、雷尼替丁（1-2 mg/kg IV）和氨苄西林（20-30 mg/kg IV），局部镇痛采用睾丸内利多卡因（总剂量2 mg/kg）。使用阿法沙酮（2-5 mg/kg IV）诱导全身麻醉，并以异氟烷在氧气中维持。术后护理在Soeparwi教授动物医院住院病房进行，药物包括阿莫西林+克拉维酸（15 mg/kg，PO，q12h）连用5天，以及cDPSC-Exos凝胶或Bioplacenton®局部治疗每日一次连用5天。术后每日监测疼痛，术后3天给予曲马多（2-4 mg/kg，SC，q12h）。
采用血液学指标、宏观伤口评估和激光散斑对比成像（LSCI）组织灌注分析评估伤口愈合情况。伤口照片由独立研究者编码，所有结果评估均在不了解治疗分组的情况下以盲法进行。
cDPSC-Exos凝胶制备
本研究使用的基质凝胶含有2%羟丙基甲基纤维素（HPMC）胶凝剂、蒸馏水、2%对羟基苯甲酸甲酯、0.0018%对羟基苯甲酸丙酯和15%丙二醇[46]。将分离的cDPSC-Exos以30%浓度加入基质凝胶中，并符合药物检测标准，包括感官特性、铺展性、黏附性、黏度和pH的评估。
血液学指标
术前和术后第五天测量血液学指标，作为炎症和伤口愈合的全身性参数。观察参数包括白细胞（WBC）、白细胞（LEU）、中性粒细胞（NEU）、嗜酸性粒细胞（EOS）、单核细胞（MON）和血小板（PLT）。
伤口愈合评估
采用改编自术后缝合愈合演变量表[47]的评分系统评估伤口愈合过程。考虑到猫去势伤口的特定特征，该评分系统包括四个参数：（1）发红，（2）肿胀，（3）疼痛，（4）伤口闭合。每个参数按1至5分评分，分数越高表示伤口愈合结果越好，如表2所示。评估在术后立即进行，随后在术后第三天和第五天进行。
Table 2. Macroscopic wound-healing scoring.
| Parameter | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| Redness (Erythema) | Mild to no redness | Mild redness | Moderate redness, <50% of wound area | Clear redness, >50% of wound area | Severe and extensive redness |
| Swelling (Edema) | No swelling | Mild swelling | Moderate swelling, <50% of wound area | Marked swelling, >50% of wound area | Severe swelling, tense tissue |
| Pain | No pain response | Mild pain response | Mild–moderate pain response | Moderate pain response | Severe pain response upon palpation |
| Wound closure | Open wound <10% | Open wound <25% | Open wound <50% | Open wound >50% | Open wound consistent with incision length |
LSCI
LSCI是一种非侵入性技术，通过检测血流产生的激光散斑模式变化来评估组织灌注，从而实现微循环的近实时可视化[48, 49]。LSCI-ZW激光散斑对比成像系统（深圳瑞沃德生命科技有限公司，中国）的数字成像数据在去势后第5天记录。给予猫加巴喷丁（5 mg/kg）以便在LSCI记录期间进行操作。以约20-30 cm的工作距离获取图像，相机增益200，曝光时间20 ms，帧率1 fps，滤波常数15 s。通过在伤口区域及周围组织（距伤口1-2 cm）定义圆形感兴趣区（ROI）进行比较，进行灌注分析[49]。
统计分析
定性数据进行描述性分析，定量数据使用IBM SPSS Statistics for Mac版本29.0（IBM Corp.，Armonk，NY，USA）进行统计分析。伤口评分使用非参数Mann-Whitney检验分析，血液学和灌注率数据使用t检验分析，数据正态性经Shapiro-Wilk检验确认。统计显著性设定为p < 0.05。图表使用GraphPad Prism（版本10.6.1）生成，插图使用BioRender（biorender.com）创建。
结果
cDPSCs显示出MSC特性
采用组织块贴壁法成功从犬牙髓组织中分离出cDPSCs，并在第3代进行鉴定，结果显示其呈纺锤样形态（图2A）、贴壁生长，并通过第5天和第7天增殖率显著增加（p < 0.05）证实其增殖能力（图2D）。集落形成单位（CFU）结果显示为66.25 ± 4.11个集落，克隆形成效率为13.25 ± 0.82%（图2B和C）。NANOG（3.53 ± 0.17）、OCT-4（5.40 ± 0.42）和REX-1（3.46 ± 0.42）的mRNA表达证实了分离的cDPSCs具有自我更新能力并维持未分化状态（图2E）。RT-qPCR分析显示MSC表面标志物CD90（55.65 ± 21.97%）、CD44（64.67 ± 18.57%）、CD105（36.92 ± 8.25%）和CD73（34.85 ± 10.09%）阳性表达（图2F），而造血细胞表面标志物CD45不表达（0.02 ± 0.03%）（图2G）。
[图：cDPSCs的鉴定。(A) 第3代cDPSCs的代表性形态，显示纺锤状形状（100×；比例尺b]
本研究通过RT-qPCR从基因水平和多向分化染色从表型水平证实了cDPSCs的多能性（图3）。成骨分化表现为成骨分化标志物RUNX-2（4.43 ± 2.7）、OSX（5.45 ± 1.51）和OCN（3.79 ± 2.23）的显著上调（p < 0.05），茜素红染色进一步证实成骨诱导细胞中形成矿化基质，诱导细胞阳性染色面积（28.29 ± 14.86%）显著大于对照（0.02 ± 0.01%），对应增加1370.49倍（p < 0.05）（图3A和B）。SOX-9（67.34 ± 17.18）和COL-X（10.29 ± 0.74）的显著上调（p < 0.05），结合阿利新蓝染色证实的软骨诱导细胞GAG基质合成，阳性染色面积从对照的0.36 ± 0.28%显著增加至诱导细胞的49.59 ± 1.94%，增加136.82倍（p < 0.05），表明cDPSCs具有软骨分化潜能（图3D-E）。PPAR-γ（1.39 ± 0.17）和LPL（13.83 ± 1.49）的显著上调（p < 0.05）证实了成脂分化潜能，油红O染色进一步证实成脂诱导细胞内脂滴蓄积，阳性染色面积从对照的0.05 ± 0.03%显著增加至诱导细胞的2.67 ± 0.89%，对应增加59.18倍（p < 0.05）（图3G-I）。
cDPSC-Exos呈现外泌体样特征
采用梯度离心技术结合沉淀法分离试剂成功分离出cDPSC-Exos。粒径分布主要在30-150 nm范围内，平均粒径为111 ± 24 nm，浓度为1.1 × 10⁹颗粒/mL（图4A）。在分离组分中检测到CD9转录本表达（图4B），为外泌体样成分的存在提供了分子证据。
[图：cDPSCs的多向分化。(A) 通过茜素红染色评估的成骨分化（40×；比例尺ba]
[图：cDPSC来源外泌体（cDPSC-Exos）的鉴定。(A) 纳米颗粒追踪分析（NTA）显示颗粒]
cDPSC-Exos蛋白质组学图谱
这是首次对cDPSC-Exos进行蛋白质组学分析，共鉴定出来自Canis lupus familiaris的312种独特蛋白质，其中22种参与伤口愈合相关生物学过程（图5）。cDPSC-Exos中伤口愈合相关蛋白按丰度从高到低排列如下：磷脂酰肌醇3-激酶催化亚基3型（PIK3C3）、α-2-HS-糖蛋白（AHSG）、晚期内体/溶酶体衔接蛋白MAPK和mTOR激活因子2（LAMTOR2）、β-肌动蛋白（ACTB）、I型胶原α1（COL1A1）、磷脂酶D2（PLD2）、钙蛋白酶11（CAPN11）、膜联蛋白A1（ANXA1）、整合素α-6（ITGA6）、波形蛋白（VIM）、pellino E3泛素蛋白连接酶2（PELI2）、膜联蛋白A2（ANXA2）、胎球蛋白B（FETUB）、热休克蛋白家族B成员1（HSPB1）、膜联蛋白A6（ANXA6）、膜联蛋白A5（ANXA5）、白细胞介素-4受体（IL4R）、丙酮酸激酶M（PKM）、p21激活激酶6（PAK6）、微管蛋白α-1A（TUBA1A）、血小板反应蛋白-1（THBS1）和StAR相关脂质转移结构域蛋白13（STARD13）（图5A）。
根据功能分析，这些蛋白质分布于伤口愈合的所有阶段，包括止血期和炎症期中与免疫应答相关的通路，以及增殖期和重塑期中的血管生成、细胞迁移和ECM组织。某些蛋白质参与了与伤口愈合相关的多个生物学过程，如维恩图所示（图5C）。此外，PPI分析（图5D）揭示了cDPSC-Exos伤口愈合相关蛋白之间复杂的相互作用网络。ACTB和VIM显示出最高的相互作用，其次是THBS1，其与膜联蛋白家族成员表现出特异性相互作用。这些相互作用表明cDPSC-Exos的参与，特别是与增殖期和重塑期相关的蛋白质，COL1A1与多种细胞骨架和ECM相关蛋白的关联即为证据。具有高连接度的蛋白质，包括ACTB、VIM、COL1A1和THBS1，以及最丰富的蛋白质PIK3C3，参与了伤口愈合的几乎所有阶段。
蛋白质印迹分析显示cDPSC-Exos中波形蛋白（VIM）和β-肌动蛋白（ACTB）呈阳性表达（图5B）。这些发现与HRMS蛋白质组学结果一致，后者同样在伤口愈合相关蛋白中鉴定出ACTB和VIM。蛋白质印迹与蛋白质组学分析结果的一致性支持了HRMS数据的有效性，并表明蛋白质组学图谱中鉴定出的其他蛋白质也存在于cDPSC-Exos中。
[图：犬牙髓干细胞来源外泌体（cDPSC-Exos）的首次蛋白质组学分析。(A) 热图显示]
cDPSC-Exos凝胶制剂
局部凝胶以每克HPMC基质凝胶含3.3 × 10⁸个cDPSC-Exos颗粒配制，以达到30%的凝胶浓度。感官评价显示cDPSC-Exos凝胶澄清、透明、均匀且无气味。此外，药学评价证明了其递送cDPSC-Exos的安全性和有效性，凝胶pH为5，铺展性为7.05 ± 0.24 cm，滞留时间为5.32 ± 0.39分钟，粘度为5,782.75 ± 29.44 cP。该制剂在使用前于4°C储存，并通过感官评价监测其物理外观，包括澄清度、均匀性、颜色和气味。HPMC基质凝胶良好的药学特性表明，它可能作为一种有前景的递送系统，用于实验性伤口愈合研究和临床应用中cDPSC-Exos的局部给药。
伤口宏观评价
本研究中局部应用cDPSC-Exos凝胶支持了猫去势后伤口愈合，未出现任何临床并发症或不良事件，包括持续性炎症、伤口裂开、行为改变或感染迹象。第0、3和5天的伤口愈合宏观观察显示两组均有渐进性改善（图6A）。cDPSC-Exos组与阳性对照组之间的宏观伤口愈合评分在第3天的红肿和疼痛反应方面显示出显著差异（p < 0.05），以及第3天和第5天的肿胀方面亦有显著差异（图6B），而伤口闭合参数未显示显著差异（p > 0.05）。然而，cDPSC-Exos组的最终伤口闭合评分低于对照组，表明组织修复得到改善。
灌注率
第5天的LSCI结果显示，cDPSC-Exos组的灌注率（散斑血流指数；SFI）为1033.42 ± 274.61，低于对照组的1213.41 ± 313.22，但未观察到显著差异，这可能归因于样本量有限（图6D和6E）。
[图：去势后伤口愈合的宏观评估。(A) 对照组和cDPSC-Exo组的代表性伤口图像]
血液指标
cDPSC-Exos组与对照组之间WBC和PLT指标无显著差异，因为局部炎症很少引起全身性改变。然而，描述性地观察到了WBC和PLT动态的差异（图6C）。与术前血液学结果相比，cDPSC-Exos组在第5天观察到WBC、NEU和MON计数下降幅度更大，同时LYM和EOS计数升高。相反，对照组显示WBC、NEU和MON升高，而LYM和EOS降低。cDPSC-Exos组观察到PLT计数略有升高，而对照组显示PLT计数降低。
讨论
cDPSCs的表征
本研究中的cDPSCs采用组织块培养法分离，表现出与hDPSCs相当的克隆形成能力（约为9-12%[50]），并展现出与hDPSCs相似的生长模式的增殖能力，后者在培养3-7天内退出适应期并进入指数期[51]。与酶消化法相比，组织块培养法保留了天然的细胞微环境，细胞应激和损伤风险较低，更具成本效益，支持细胞贴壁和生长，并维持旁分泌信号机制，这可能有利于外泌体的收集[52, 53]。这种天然微环境也可能有助于维持cDPSCs的多能性和自我更新能力，这一点通过干性标志物的表达得到证实，反映了其向多个细胞谱系分化的能力[54]。此外，分离的cDPSCs表现出MSC特征，表现为MSC表面标志物CD90、CD105、CD73和CD44的表达以及造血细胞表面标志物CD45的不表达。尽管这些表面标志物是ISCT基于人MSCs定义的，但类似的表达模式已在动物来源的干细胞中报道，包括兔滑液[55]、马牙髓[7]、犬骨髓[10, 33, 34, 37, 56]以及既往研究中的犬牙髓[33, 34, 56]。尽管MSC鉴定通常按照ISCT标准使用流式细胞术进行，但本研究因方法学限制采用了RT-qPCR。既往研究报道RT-qPCR与流式细胞术分析之间CD90、CD105、CD73、CD44和CD45的表达模式一致[57, 58]，表明本研究结果足以代表分离的cDPSCs的MSC特征。
三系分化潜能，包括成软骨、成骨和成脂分化，是ISCT最低标准[59]中定义的MSC功能标志。分离的cDPSCs通过表型和转录水平评估表现出三系分化潜能。这种分化能力也已在既往犬来源干细胞研究中报道，包括来自骨髓、脂肪组织和牙髓的干细胞；然而，分化程度因组织来源而异[33, 34, 37, 60]。在本研究中，成骨分化在三系中最为显著，这与既往报道一致，即cDPSCs比犬骨髓MSCs（cBMSCs）具有更强的成骨潜能[37]，以及一项关于hDPSCs的研究报道其成骨和成软骨能力相对于其他MSC群体增强[61]。这表明cDPSCs的潜能不仅限于软组织，也适用于骨组织工程。
cDPSC-Exos的表征和蛋白质组学图谱
本研究成功分离了cDPSC来源的外泌体，证明使用梯度离心结合外泌体分离试剂可获得稳定且可测量的产量，这反映在cDPSC-Exos浓度处于外泌体分离的常见范围内，即10^9-10^10颗粒/mL[50, 62, 63]。CD9的阳性表达（图2B）作为支持cDPSCs外泌体生物发生的分子证据，与既往人类和动物的MSC-Exos研究一致[64–67]。然而，该表征仍属初步，因为MISEV 2023指南建议在蛋白质水平评估经典EV标志物（CD9、CD63、CD81、TSG101和Alix），并结合其他表征方法如超微结构成像和阴性标志物评估。
本研究首次提供了cDPSC-Exos的蛋白质组学图谱，揭示了伤口愈合相关蛋白。这些蛋白分布于组织修复的所有阶段，反映了如图7所示的协调而复杂的机制。值得注意的是，本研究中鉴定的若干蛋白，包括COL1A1、ANXA、VIM、THBS1、ITGA和ACTB，也已在牙髓及多种组织来源的MSC衍生外泌体蛋白质组学研究中报道[8, 37, 44, 68–70]。这一发现表明cDPSC-Exos与MSC衍生外泌体的再生和伤口愈合特性共享常见的分子组分。
[图：首次cDPSC-Exos蛋白质组学分析中鉴定的伤口愈合相关蛋白的示意图。蛋白]
基于功能分析，主要蛋白质参与调控止血期和炎症期的免疫反应，提示cDPSC-Exos可能在伤口愈合早期阶段主要发挥治疗作用，而该阶段对决定后续组织修复结局至关重要[71, 72]。此外，PPI分析揭示了伤口愈合相关蛋白之间的整合相互作用网络，其中ACTB和VIM等高连接度蛋白突显了细胞骨架组织和细胞运动在cDPSC-Exos生物学活性中的核心作用。与蛋白质组学结果一致，western blot分析证实了ACTB和VIM的表达（图3B），支持了其他已鉴定蛋白的有效性。这两种蛋白均与细胞迁移、结构稳定性和细胞外基质（ECM）重塑相关，而这些在伤口愈合的增殖期和重塑期至关重要[73, 74]。在伤口愈合过程中，VIM参与成纤维细胞迁移、增殖和分化，以及细胞骨架重塑和细胞外基质组织，以促进组织再生[75, 76]。ACTB通过细胞骨架重塑参与伤口愈合，驱动细胞迁移、上皮化和伤口收缩，特别是通过伤口边缘的肌动蛋白索形成，从而支持伤口闭合[77]。另一方面，VIM和ACTB揭示了外泌体的生物学完整性和纯度，因为这两种蛋白均与细胞骨架依赖性的EV生物发生相关。既往研究表明，VIM和ACTB通过调节囊泡运输、维持内体定位以及通过其在囊泡运输中的相互作用促进外泌体释放，参与外泌体生物发生；因此，丝状装配和解聚影响其分泌及参与EV途径[75, 78]。
基于PPI网络，THBS1与膜联蛋白家族成员的相互作用也支持血管生成和膜修复机制的调控，从而强化了cDPSC-Exos在组织再生中的作用[79, 80]。PIK3C3的高丰度可能反映其在外泌体生物发生和伤口愈合调控中的双重作用，因为它参与自噬和内吞作用，而这些对囊泡运输和细胞稳态至关重要[81]。由PIK3C3介导的自噬已涉及止血期的碎片清除、炎症期的炎症小体抑制和巨噬细胞极化，以及伤口修复后期的细胞迁移、血管生成和ECM重塑[81–83]。整合蛋白质组学分析提示，cDPSC-Exos所含蛋白不仅参与外泌体生物发生，还参与全面的伤口愈合机制，支持cDPSC-Exos促进组织修复的治疗潜力。
去势后伤口愈合的异种疗效
所鉴定的蛋白质可能通过在伤口愈合全过程中协调调控免疫反应、血管生成、细胞迁移和细胞外基质重塑，在机制上促进了本研究中观察到的炎症加速消退和伤口闭合。伤口愈合涉及众多组分，包括血细胞、细胞外基质、实质细胞和可溶性介质，通过四个主要阶段恢复组织完整性和功能：止血、炎症、增殖和重塑[47, 84]。炎症期的特征为发红，因血管舒张使血流增加至伤口部位；肿胀/水肿，因血管通透性增加使血浆渗漏至组织；以及疼痛，由前列腺素、细胞因子和趋化因子等介质释放引起[85]。cDPSC-Exos组第3天较低的发红、肿胀和疼痛评分表明其炎症反应比对照组更快。伤口闭合评分代表伤口愈合的晚期阶段，包括增殖和重塑，其特征为受损皮肤组织的再生和修复，而cDPSC-Exos组相对更快[47, 84]。LSCI结果与宏观表现一致，较低的灌注单位表明cDPSC-Exos组在第5天进入重塑期。伤口愈合过程早期发生活跃的血管生成，导致LSCI评估的SFI值较高，该值在重塑期逐渐下降，接近正常皮肤组织[86]。血管生成从现有血管系统形成新血管，从而维持对受损皮肤组织的氧气和营养供应，在组织再生和伤口愈合中发挥重要作用[87]。
血液指标与宏观观察及LSCI结果一致，表明cDPSC-Exos组伤口愈合加速。血小板在止血中起重要作用，cDPSC-Exos组血小板计数的轻微升高可能表明止血阶段结束，血小板计数恢复正常[84, 85]。在炎症期，中性粒细胞和巨噬细胞浸润受伤皮肤以对抗微生物、清除细胞碎片并释放炎症介质；因此，中性粒细胞和单核细胞计数的下降表明炎症期的消退[84]。中性粒细胞和单核细胞的趋势提示cDPSC-Exos组的炎症期持续时间相对短于对照组，这可能与cDPSC-Exos中丰富的免疫相关蛋白有关。淋巴细胞和嗜酸性粒细胞的增加可能表明伤口愈合后期的开始，因为它们在增殖期和重塑期发挥重要作用。EOS与凝血和纤溶系统相互作用，通过释放生长因子和蛋白酶促进ECM修饰和上皮修复，而淋巴细胞通过细胞因子介导的免疫调节调控组织再生和上皮屏障恢复[88, 89]。这些发现在组间具有可比性，提示cDPSC-Exos组与对照组相比伤口愈合有改善趋势，尽管未观察到统计学显著差异。伤口愈合的改善与先前关于MSC来源外泌体和基于干细胞的伴侣动物治疗研究一致，这些研究同样报道了组织修复的增强[5, 15, 17, 90]。
安全性、局限性与未来方向
异种cDPSC-Exos给药后未出现治疗相关的不良临床反应，这为跨物种无细胞再生方法的可行性提供了初步证据。先前的研究也报道了类似发现，证明MSC来源外泌体跨物种屏障的低免疫原性，包括hu-AdMSCs分泌组用于猫慢性龈口炎治疗、人细胞外泌体用于犬特应性皮炎治疗，以及大量人MSC-Exos研究揭示其在动物模型中加速伤口愈合[6, 14, 15, 18, 19]。尽管未观察到异种不良反应的证据，但本研究中cDPSC-Exos的免疫安全性仍需进一步研究。本研究采用的综合方法整合了蛋白质组学分析、血液学评估、组织灌注分析和临床伤口评价，为理解cDPSC-Exos的生物学效应提供了互补信息。
本初步研究存在若干局限性，特别是伤口愈合评价仅基于临床评估，缺乏组织学或分子学评估来表征组织再生和伤口愈合的细胞机制。此外，相对较小的样本量和有限的伤口大小可能限制对研究结果的解读。需要进一步研究，包括组织病理学、免疫组织化学和伤口组织的全面免疫学分析，以确认cDPSC-Exos异种应用的安全性。尽管存在这些局限性，本研究结果为cDPSC-Exos在猫伤口愈合中的潜力提供了初步证据。作为一种无细胞治疗方法，cDPSC-Exos可能在兽医学中具有转化潜力，但在更广泛的临床应用之前，还需要进一步的标准化、安全性评价和监管考量。
结论
cDPSC-Exos对猫术后伤口愈合显示出有前景的治疗潜力，表现为伤口愈合参数加速以及蛋白质组学分析鉴定的血管生成、抗炎和免疫调节蛋白的存在。本研究首次报道了cDPSC-Exos在猫手术伤口模型中的分离、表征、蛋白质组学分析和异种局部应用，并开创性地将伴侣动物牙髓作为异种无细胞产品的可持续来源，为临床兽医再生医学提供了实用途径。
蛋白质组学分析在cDPSC-Exos中鉴定出312种独特蛋白质，其中22种与伤口愈合全过程（止血、炎症、增殖和重塑）相关。在初步体内研究中，与Bioplacenton®相比，局部应用cDPSC-Exos凝胶（3.3 × 10⁸颗粒/g）显著改善了早期宏观伤口评分中的发红、肿胀和疼痛（p < 0.05），血液学参数和组织灌注（LSCI）呈支持性趋势，且未观察到不良反应。
这些发现支持cDPSC-Exos作为一种安全、异种、无细胞的再生疗法，来源于微创牙髓来源。该方法为伴侣动物术后伤口管理提供了一种可扩展的基于细胞治疗的替代方案，具有更广泛兽医应用的潜力。
该研究整合了全面的体外表征、详细的蛋白质组学分析以及在临床相关猫去势后模型中的随机对照初步体内评价，为分子机制和功能疗效提供了强有力的初步证据。
本初步研究存在若干局限性，特别是伤口愈合评价仅基于临床评估，缺乏组织学或分子学评估来表征组织再生和伤口愈合的细胞机制。此外，相对较小的样本量和有限的伤口尺寸可能限制了对研究结果的解读。
需要进一步研究，包括组织病理学、免疫组织化学和伤口组织的全面免疫学分析，以确认异种cDPSC-Exo应用的安全性。需要更大规模、把握度充分的随机对照试验、延长观察期、优化给药方案和制剂以及机制研究来验证疗效并支持临床转化。标准化、长期安全性评价和监管考量在更广泛应用之前也至关重要。
尽管存在这些局限性，本研究结果为cDPSC-Exos在猫伤口愈合中的潜力提供了初步证据。作为一种无细胞治疗方法，cDPSC-Exos可能在兽医学中具有转化潜力。鉴于本研究的初步性质、有限的样本量以及缺乏组织学和分子学伤口评估，需要进一步开展把握度充分的研究，结合全面的外泌体表征、组织病理学评价和机制研究，以验证这些初步发现并支持cDPSC-Exos作为兽医学无细胞再生疗法的未来临床转化。
数据可用性
支持本研究结果的数据可在本文及其补充材料中获取，包括原始NTA数据、多向分化染色定量、全长western blot图像、蛋白质组学数据集、详细动物信息、原始伤口愈合数据和ARRIVE 2.0清单。补充材料和其他数据可向通讯作者合理索取。
生成式人工智能声明
生成式人工智能工具仅用于改善语言、语法和可读性，未被列为作者。所有科学内容、分析、解释和结论均由作者开发并验证，作者对工作的准确性和完整性承担全部责任。
作者贡献
MP：构思并设计研究。YSD和MP：进行动物实验、解读数据、进行统计分析并起草手稿。DA：实施去势手术。AH和TB：参与干细胞表征和数据解读。CS和SN：参与外泌体表征、批判性审阅和编辑手稿。MP、YSD、AH、TB、CS和SN：修订并批准最终手稿。所有作者均已阅读并批准最终手稿。
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INTRODUCTION
Mesenchymal stem cell (MSC)-based regenerative therapy has demonstrated therapeutic potential for various diseases, including cancer, heart failure, stroke, neurological disorders, diabetes mellitus, autoimmune diseases, and chronic inflammation, owing to their regenerative capacity, immunomodulatory effects, and secretion of bioactive factors that promote tissue repair and modulate inflammation [1–5]. Furthermore, MSCs can be isolated from various tissue sources, including bone marrow, adipose tissue, placenta, umbilical cord, hair follicles, and dental tissue, across various animal species, thereby supporting the development and large-scale production of MSCs [6–10]. The therapeutic effects of MSCs are partly mediated by paracrine mechanisms through the secretion of extracellular vesicles (EVs), particularly exosomes, which play a crucial role in cellular communication, supporting cellular functions [11, 12].
Exosomes are a nanoscale subpopulation of EVs with diameters ranging from 30 to 150 nm that contain bioactive molecules, including nucleic acids, lipids, and proteins, to support cellular functions [12, 13]. The biological properties of exosomes make them suitable as cell-free therapy agents, with clinical potential comparable to cell-based therapy and a lower risk of immune rejection, tumorigenesis, toxicity, and ethical concerns. This enables scalable storage and distribution, as exosomes do not contain viable cells and exhibit lower expression of immunogenic surface molecules [12, 14]. The lower biosafety risks associated with exosome-based therapy also enable xenogeneic administration across species [12, 15, 16]. Previous studies have reported the safety and therapeutic efficacy of human MSC-derived exosomes in canine, feline, and laboratory animal models, suggesting a relatively conserved mechanism of exosome-mediated tissue repair across species barriers [15, 17–21].
Several preclinical and clinical studies have demonstrated the safety and therapeutic efficacy of cell-free therapy using MSC-derived exosomes in the treatment of neurodegenerative diseases, periodontal tissue regeneration, osteoarthritis, and other degenerative disorders [22–27]. Previous studies also suggest that MSC-Exos accelerate wound-healing and enhance tissue repair in animal models, with human dental pulp stem cell-derived exosomes (huDPSC-Exos) shown to support angiogenesis, inflammatory responses, and immunomodulation during the wound-healing process [6, 11, 13, 28]. These regenerative properties highlight the potential application of MSC-Exos for postoperative wound-healing in animals, which remain susceptible to post-surgical complications such as wound dehiscence, prolonged inflammation, infection, and septicemia, thereby delaying recovery and compromising animal welfare [29–32].
Canine dental pulp stem cells (cDPSCs) are promising candidates for regenerative therapy in veterinary medicine because of the minimally invasive, ethically favorable dental pulp collection, and various studies have reported their MSC-like characteristics based on the minimum criteria established by the International Society for Cellular Therapy (ISCT) [33, 34]. However, their exosomes (cDPSC-Exos) remain largely unexplored, and no studies have evaluated the xenogeneic potential of cDPSC-Exos in cats. Comprehensive proteomic characterization of cDPSC-Exos is also lacking.
The objective of this pilot study was to characterize cDPSCs and cDPSC-Exos and to evaluate their therapeutic potential through proteomic profiling and in vivo xenogeneic application for postoperative wound-healing. This is the first study to combine cDPSC characterization, cDPSC-Exos characterization and proteomic profiling, with xenogeneic evaluation in cats post-castration wounds as a standardized, clinically relevant, and naturally occurring surgical model. We hypothesized that cDPSC-Exos would promote postoperative wound-healing in cats through regulation of inflammation, immunomodulation, and angiogenesis.
MATERIALS AND METHODS:
Ethical approval
This study was conducted in accordance with accepted animal welfare principles and the ARRIVE 2.0 guidelines. The experimental protocol was reviewed and approved by the Ethics Committee, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia, under approval number 101/EC-FKH/Int./2025. Canine dental pulp tissue was obtained from a healthy donor dog during routine dental extraction with appropriate clinical oversight. The feline clinical evaluation was conducted using client-owned healthy male cats undergoing elective castration at Prof. Soeparwi Animal Hospital, Faculty of Veterinary Medicine, Universitas Gadjah Mada, after obtaining written informed consent from the owners.
All cats received standardized perioperative care, including anesthesia, analgesia, antibiotic coverage, postoperative monitoring, and daily wound assessment. Animals were housed individually with free access to food and water, and all procedures were performed by qualified veterinary personnel to minimize pain, stress, and discomfort. The study used a positive-control design because leaving post-surgical wounds untreated was considered ethically inappropriate. No adverse reactions, wound dehiscence, infection, or clinically relevant systemic complications were observed during the study period.
Study period and location
This study was conducted from January 2025 to January 2026 at the Integrated Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University for cell isolation and culture; the Laboratory of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Universitas Gadjah Mada for cDPSCs and cDPSC-Exos characterization; and Prof. Soeparwi Animal Hospital, Faculty of Veterinary Medicine, Universitas Gadjah Mada for in vivo observation.
Study design
This study employed a combined in vitro and in vivo approach consisting of cDPSC isolation and characterization; cDPSC-Exos isolation, characterization, and proteomic profiling; and a randomized controlled pilot study for xenogeneic evaluation of post-castration wound-healing in cats, as demonstrated in Figure 1.
[图：Schematic overview of canine dental pulp stem cells (cDPSCs) isolation and characterization; cDPSC-derived exosome (cDPS]
Cell isolation, culture, and expansion
Dental pulp was obtained from a single healthy permanent molar of a 2.5-year-old domestic male dog with no history of systemic disease or oral pathology. Isolation involving tissue explant technique was adapted from previous work [7, 35]. The crown of the molar tooth was sectioned to collect the dental pulp. The dental pulp was minced to ±0.5 cm and placed into a 35 mm culture plate containing Dulbecco’s modified Eagle medium (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (Gibco™), 1% antibiotic-antimycotic (Gibco™), and 1% GlutaMAX (Gibco™); maintained in 70% humidity; and incubated at 37°C in 5% CO₂ with medium replacement every 48 h. When the cells reached approximately 80% confluence, they were subcultured into three new 35 mm culture plates containing medium. Passage 3 of the cDPSCs was used for characterization based on MSCs’ minimal requirements by ISCT.
Colony-forming assay
Isolated cDPSCs were cultured at a concentration of 500 cells per 60 mm culture dish (TPP®, Trasadingen, Switzerland) for 14 days, following the previously published protocol [7, 36]. After 14 days, colonies were washed with Phosphate-buffered saline (PBS, pH 7.4), fixed with cold methanol for 20 min, stained with crystal violet for 5 min, and colonies containing ≥50 cells were counted [7, 35]. Colony-forming efficiency (CFE) was calculated using the formula CFE(%) = (number of colonies formed/number of cells seeded) × 100%.
Proliferation assay
cDPSCs were incubated in culture medium with 5% alamarBlue™ (Sigma-Aldrich, St. Louis, MO, USA) for 3 hours to assess the proliferation capability on days 1, 5, and 7 [7, 35]. The absorbance was recorded at 570 nm, and cell viability was quantified as the percentage reduction of alamarBlue according to the product’s guidelines.
Multilineage differentiation
The cDPSCs multilineage differentiation, including adipogenesis, osteogenesis, and chondrogenesis, was assessed in vitro using a previously published protocol [7, 10, 13, 37, 38]. cDPSCs were seeded into three separate 24-well plates and cultured in osteogenic, chondrogenic, or adipogenic induction media formulated as previously described [7, 38, 39], followed by Alizarin Red (Sigma-Aldrich) staining to detect calcium deposition in osteogenic differentiation, Alcian Blue (Sigma-Aldrich) staining to detect glycosaminoglycan (GAG) accumulation in chondrogenic differentiation, and Oil Red O (Sigma-Aldrich) staining to assess adipogenic differentiation based on intracellular lipid droplet formation.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The gene expression of MSC surface markers (CD90, CD44, CD105, CD73, and CD45) was assessed using RT-qPCR as an alternative approach to flow cytometry. RT-qPCR was also used to assess stemness-related markers (NANOG, REX-1, OCT-4), osteogenic differentiation markers (RUNX-2, OSX, OCN), chondrogenic differentiation markers (COL-X, SOX-9), and adipogenic differentiation markers (PPAR-γ, LPL) of cDPSCs. Specific primers used in this study are presented in Table 1.
Table 1. List of primer sequences used to characterize cDPSCs and cDPSC-Exos.
| Gene | Forward primer sequence (5’-3’) | Reverse primer sequence (5’-3’) |
|---|---|---|
| GAPDH | CCAACTGCTTGGCTCCTCTA | GTCTTCTGGGTGGCAGTGAT |
| CD90 | AGGACGAGGGGACATACACA | ATGCCCTCACACTTGACCAG |
| CD44 | CCCCATTACCAAAGACCACGA | TGGGATTTGAGGTTTCCGCA |
| CD105 | CGAGGAGTCTGTCACCGGAAA | GCGCCAAAGGTGATACCCAG |
| CD73 | GGCAACCTGATTTGTGATGCT | AGGTAATTGTGCCGTTGTTCC |
| CD45 | GTTTCCAGTTCTGTTTCCCCAG | CATTGGTCACAATTCACGGTATCA |
| NANOG | CAGCAGATGCAAGAACTTTCCA | AGCAGGTACCCCTGAGTCAC |
| REX-1 | AGGTTCTCACAGCAAGCTCA | CCAGCAAATTCTGCGCACTG |
| OCT-4 | AGGAGAAGCTGGAGCAAAACC | GTGATCCTCTTCTGCTTCAGGA |
| COL-X | AGCACCCCGAATCCATCTGAG | TGCCCGTAGGTGTTTGGTATC |
| SOX-9 | TCTGGAGGCTGCTGAACGA | TTCTTCACCGACTTCCTCCG |
| RUNX-2 | GGAAGAGGCAAGAGTTTCACC | GTGCTCACTTGCCAACAGAA |
| OSX | GCGTCCTCCCTGCTTGAG | GCTTTGCCCAGTGTCGTTG |
| OCN | GCCAGCCTATGGTCTCCTCTG | CCACCAGCTCCTTCTGTTCTCT |
| PPAR-γ | CCTCTTCCATGCTGTTATGGGT | TGGCATCTCTGTGTCAACCA |
| LPL | CTGGAGAGACTCAGAAAAAGGTAAT | TCCTTCTGTAGATTTGCTCAGGT |
| CD9 | ATTTCGTCTTCTGGCTTGCTGG | AGGGCACCAGCTCCAATCAG |
Total RNA was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and Direct-zol™ RNA Miniprep kit (Zymo Research, Irvine, CA, USA); with RNA quantification performed using NanoDrop™ (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using QuantiNova® Reverse Transcription kit (Qiagen, Hilden, Germany), followed by qPCR amplification using QuantiNova® SYBR® Green PCR kit (Qiagen) on Rotor-Gene Q real-time PCR cycler (Qiagen). The mRNA expression was normalized to the housekeeping gene (GAPDH) using the formula ΔCt = Ct target gene - Ct GAPDH for stemness and differentiation markers, while surface marker expression was presented as a percentage calculated using the formula 2−ΔCt × 100% [7, 35, 40].
Exosome isolation and characterization
Exosome isolation was performed using Total Exosome Isolation Reagent (Invitrogen, catalog no. 4478359) following the product’s protocol as previously reported in MSC-derived exosome studies [41, 42]. Conditioned medium (CM) from passage 3 cDPSC cultures was collected and centrifuged at 2,000 × g for 30 min at 4 °C to obtain a cell- and debris-free supernatant. Total Exosome Isolation Reagent (Invitrogen) was added to the conditioned medium at 25% of the conditioned medium volume, and the mixture was incubated overnight at 4 °C. Following incubation, centrifugation at 10,000 × g for 60 min at 4 °C was performed to collect the exosomal pellet, which was resuspended in 5 mL of sterile PBS and stored at −80 °C until further use. Characterization of cDPSC-Exos was performed by evaluating CD9 transcript expression using RT-qPCR, while particle size distribution was assessed using nanoparticle tracking analysis (NTA).
Exosomes proteomic profiling
Proteomic analysis of cDPSC-Exos was performed using an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific) following previously reported MSC-derived exosome proteomic workflows [43, 44]. Samples were lysed using Sodium dodecyl sulfate-based lysis buffer, reduced with dithiothreitol, digested with MS-grade trypsin, acidified with 1% trifluoroacetic acid (TFA), and filtered prior to analysis. Peptide separation was carried out using a Vanquish Horizon Ultra-high-performance liquid chromatography system equipped with an Acclaim PepMap 100 C18 column, followed by analysis on an Orbitrap Exploris 240 high-resolution mass spectrometer (Thermo Fisher Scientific) operating in positive ion mode with Full MS/dd-MS² acquisition. Raw data were processed using Proteome Discoverer 2.5 with the SequestHT search engine against the UniProt (Canis lupus familiaris) database. Functional enrichment analysis was performed using Reactome, and protein-protein interaction (PPI) networks were analyzed using STRING. Due to the limited annotation available in the C. l. familiaris database, functional and pathway analyses related to wound-healing were conducted using the Homo sapiens database to improve biological interpretation.
Western blot analysis
Western blot analysis was performed to validate the proteomic findings by assessing vimentin expression using the iBlot™ 2 and iBind™ Western Systems (Thermo Fisher Scientific) following a previously reported workflow [45]. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific), separated using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4-12% Bis-Tris gels, and transferred onto polyvinylidene difluoride (PVDF) membranes using the iBlot™ 2 Transfer System (Thermo Fisher Scientific). Membranes were then blocked and incubated with mouse anti-vimentin monoclonal antibody (V9) and rabbit anti-β-actin antibody as the internal loading control (Thermo Fisher Scientific), followed by Horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (Ig)G and goat anti-rabbit IgG secondary antibodies (Thermo Fisher Scientific). Protein bands were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions.
Xenogeneic clinical evaluation of postoperative wound-healing in cats
Clinical evaluation of xenogeneic cDPSC-Exos therapy was conducted in six clinically healthy male cats aged ≤5 years, which were randomly assigned by simple randomization into two groups: (1) the cDPSC-Exos group, receiving topical cDPSC-Exos gel on post-castration wounds, and (2) the positive control group, receiving topical Bioplacenton®. The absence of a negative control group was based on ethical considerations regarding animal welfare, since leaving post-surgical wounds untreated could potentially lead to adverse complications.
All cats underwent castration at Prof. Soeparwi Animal Hospital, Faculty of Veterinary Medicine, Gadjah Mada University, following owner-informed consent. Cats were housed individually with ad libitum access to food and water and received the same anesthesia, analgesia, antibiotic, and postoperative care protocols. Tramadol (2-4 mg/kg IV), ranitidine (1-2 mg/kg IV), and ampicillin (20-30 mg/kg IV) were administered preoperatively, while local analgesia was provided by intratesticular lidocaine (2 mg/kg total dose). General anesthesia was induced with alfaxalone (2–5 mg/kg IV) and maintained with isoflurane in oxygen. Postoperative care was carried out in the inpatient ward of Prof. Soeparwi Animal Hospital, with medications including amoxicillin + clavulanic acid (15 mg/kg, PO, q12h) for 5 days and topical therapy with cDPSC-Exos gel or Bioplacenton® once daily for 5 days. Postoperative pain was monitored daily, with tramadol (2-4 mg/kg, SC, q12h) administered for 3 days postoperatively.
Wound healing was evaluated using hematological profiles, macroscopic wound assessment, and tissue perfusion analysis with laser speckle contrast imaging (LSCI). Wound photographs were coded by an independent investigator, and all outcome assessments were performed in a blinded manner without knowledge of treatment allocation.
cDPSC-Exos gel preparation
The base gel used in this study contained 2% hydroxypropyl methylcellulose (HPMC) gelling agent, distilled water, 2% methyl paraben, 0.0018% propyl paraben, and 15% propylene glycol [46]. The isolated cDPSC-Exos were added to the base gel at a concentration of 30%, and conformed to pharmaceutical testing standards, including assessments of organoleptic properties, spreadability, adhesiveness, viscosity, and pH.
Hematology profile
Hematological profiles were measured preoperatively and on the fifth postoperative day as systemic parameters for inflammation and wound-healing. The parameters observed included white blood cells (WBC), leukocytes (LEU), neutrophils (NEU), eosinophils (EOS), monocytes (MON), and platelets (PLT).
Wound-healing assessment
The wound healing process was evaluated using a scoring system adapted from the postoperative suture-healing evolution scale [47]. Considering the specific characteristics of castration wounds in cats, the scoring system includes four parameters: (1) redness, (2) swelling, (3) pain, and (4) wound closure. Each parameter is rated on a scale of 1 to 5, with higher scores indicating better wound-healing outcomes, as shown in Table 2. Assessments were conducted immediately after surgery, and subsequently on the third and fifth days post-operation.
Table 2. Macroscopic wound-healing scoring.
| Parameter | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| Redness (Erythema) | Mild to no redness | Mild redness | Moderate redness, <50% of wound area | Clear redness, >50% of wound area | Severe and extensive redness |
| Swelling (Edema) | No swelling | Mild swelling | Moderate swelling, <50% of wound area | Marked swelling, >50% of wound area | Severe swelling, tense tissue |
| Pain | No pain response | Mild pain response | Mild–moderate pain response | Moderate pain response | Severe pain response upon palpation |
| Wound closure | Open wound <10% | Open wound <25% | Open wound <50% | Open wound >50% | Open wound consistent with incision length |
LSCI
LSCI is a non-invasive technique used to assess tissue perfusion by detecting changes in laser speckle patterns generated by blood flow, thereby enabling near-real-time visualization of microcirculation [48, 49]. Digital imaging data from the LSCI-ZW Laser Speckle Contrast Imaging System (Shenzhen RWD Life Science Co., Ltd., China) were recorded on the 5th day after castration. Cats were administered gabapentin (5 mg/kg) to facilitate handling during LSCI recording. Images were acquired at a working distance of approximately 20-30 cm using a camera gain of 200, an exposure time of 20 ms, a frame rate of 1 fps, and a filter constant of 15 s. Perfusion analysis was conducted by defining circular regions of interest (ROIs) over the wound area and the surrounding tissue (1-2 cm from the wound) for comparison [49].
Statistical analysis
Qualitative data were analyzed descriptively, while quantitative data were analyzed statistically using IBM SPSS Statistics for Mac, version 29.0 (IBM Corp., Armonk, NY, USA). Wound scores were analyzed using the nonparametric Mann-Whitney test, while hematology and perfusion rate data were analyzed using a t-test, as the normality of the data was confirmed using the Shapiro-Wilk test. Statistical significance was set at p < 0.05. Graphs and plots were generated using GraphPad Prism (version 10.6.1), and illustrations were created using BioRender (biorender.com).
RESULTS
cDPSCs revealed MSC properties
The cDPSCs were successfully isolated from canine dental pulp tissue using the tissue explantation technique and characterized at passage 3, which revealed spindle-like morphology (Figure 2A), adherence to the culture dish, and showed proliferation capacity confirmed by a significant increase of proliferation rate on the 5th and 7th day (p < 0.05) (Figure 2D). The colony-forming unit (CFU) result showed 66.25 ± 4.11 colonies and 13.25 ± 0.82% clonogenic efficiency (Figures 2B and C). The self-renewal and maintenance of the non-differentiation state of isolated cDPSCs were confirmed by the mRNA expression of NANOG (3.53 ± 0.17), OCT-4 (5.40 ± 0.42), and REX-1 (3.46 ± 0.42) (Figure 2E). The RT-qPCR analysis indicated positive expression of MSC surface markers CD90 (55.65 ± 21.97%), CD44 (64.67 ± 18.57%), CD105 (36.92 ± 8.25%), and CD73 (34.85 ± 10.09%) (Figure 2F), and lack of hematopoietic cell surface marker CD45 expression (0.02 ± 0.03%) (Figure 2G).
[图：Characterization of cDPSCs . (A) Representative morphology of passage 3 cDPSCs showing spindle-like shape (100×; scale b]
The multipotency of cDPSCs in the present study was confirmed both genetically through RT-qPCR and phenotypically through multilineage staining (Figure 3). Osteogenic differentiation demonstrated by the significant upregulation (p < 0.05) of osteogenic differentiation markers RUNX-2 (4.43 ± 2.7), OSX (5.45 ± 1.51) and OCN (3.79 ± 2.23), further confirmed by Alizarin Red staining showing mineralized matrix formation in osteogenically induced cells, with a significantly greater positive staining area in induced cells (28.29 ± 14.86%) compared with controls (0.02 ± 0.01%), corresponding to a 1370.49-fold increase (p < 0.05) (Figures 3A and B). The significant upregulation (p < 0.05) of SOX-9 (67.34 ± 17.18) and COL-X (10.29 ± 0.74), supported by the Alcian Blue staining demonstrated GAG matrix synthesis in chondrogenic induced cells, with a significant increase of positive staining area from 0.36 ± 0.28% in controls to 49.59 ± 1.94% in induced cells, representing a 136.82-fold increase (p < 0.05) represent the chondrogenic differentiation potential of cDPSCs (Figures 3D-E). The adipogenic differentiation potential was confirmed by the significant upregulation (p < 0.05) of PPAR-γ (1.39 ± 0.17) and LPL (13.83 ± 1.49), further confirmed by the Oil Red O staining demonstrated intracellular lipid droplet accumulation in adipogenic induced cells, with a significant increase in positive staining area from 0.05 ± 0.03% in controls to 2.67 ± 0.89% in induced cells, corresponding to a 59.18-fold increase (p < 0.05) (Figures 3G–I).
cDPSC-Exos revealed exosome-like characteristics
The cDPSC-Exos were successfully isolated using a gradual centrifugation technique combined with a precipitation-based isolation reagent. Particle size distribution predominantly within the 30-150 nm range, with an average particle size of 111 ± 24 nm and a concentration of 1.1 × 10⁹ particles/mL (Figure 4A). CD9 transcript expression was detected in the isolated fraction (Figure 4B), providing molecular evidence supporting the presence of exosome-like components.
[图：Multilineage differentiation of cDPSCs . (A) Osteogenic differentiation assessed by Alizarin Red staining (40×; scale ba]
[图：Characterization of cDPSC -derived exosomes ( cDPSC -Exos ). (A) Nanoparticle tracking analysis (NTA) showing the partic]
cDPSC-Exos proteomic profile
This is the first cDPSC-Exos proteomic analysis, which identifies 312 unique proteins from Canis lupus familiaris, including 22 involved in wound-healing-related biological processes (Figure 5). The wound-healing-related proteins of cDPSC-Exos were ranked from the highest abundance as follows: phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), alpha-2-HS-glycoprotein (AHSG), late endosomal/lysosomal adaptor MAPK and mTOR activator 2 (LAMTOR2), actin beta (ACTB), collagen type I alpha 1 (COL1A1), phospholipase D2 (PLD2), calpain 11 (CAPN11), annexin A1 (ANXA1), integrin alpha-6 (ITGA6), vimentin (VIM), pellino E3 ubiquitin protein ligase 2 (PELI2), annexin A2 (ANXA2), fetuin-B (FETUB), heat shock protein family B member 1 (HSPB1), annexin A6 (ANXA6), annexin A5 (ANXA5), interleukin-4 receptor (IL4R), pyruvate kinase M (PKM), p21-activated kinase 6 (PAK6), tubulin alpha-1A (TUBA1A), thrombospondin-1 (THBS1), and StAR-related lipid transfer domain protein 13 (STARD13) (Figure 5A).
According to the functional analysis, these proteins were distributed across all phases of wound-healing, including immune response-related pathways in the hemostasis and inflammation phases; as well as angiogenesis, cell migration, and ECM organization during the proliferation and remodeling phases. Certain proteins were involved in multiple biological processes associated with wound-healing, as illustrated in the Venn diagram (Figure 5C). Additionally, PPI analysis (Figure 5D) revealed a complex interaction network among the wound healing-related proteins of cDPSC-Exos. ACTB and VIM showed the highest interactions, followed by THBS1, which showed specific interactions with members of the annexin family. These interactions indicate the involvement of cDPSC-Exos, particularly proteins implicated in the proliferation and remodeling phases, as evidenced by the association of COL1A1 with several cytoskeletal and ECM-related proteins. Proteins with high connectivity, including ACTB, VIM, COL1A1, and THBS1, as well as the most abundant protein PIK3C3, were involved in nearly all phases of wound-healing.
The western blot analysis revealed positive expression of vimentin (VIM) and β-actin (ACTB) in the cDPSC-Exos (Figure 5B). These findings were consistent with the HRMS proteomic results, which also identified ACTB and VIM among the wound-healing-related proteins. The aligned western blot and proteomic analyses support the validity of the HRMS data and suggest that the other proteins identified in the proteomic profile are also present in the cDPSC-Exos.
[图：The first proteomic analysis of canine dental pulp stem cell-derived exosome ( cDPSC -Exos ). (A) Heatmap showing the re]
cDPSC-Exos gel formulation
The topical gel was formulated with 3.3 × 10⁸ particles of cDPSC-Exos per gram of HPMC base gel to achieve a 30% gel concentration. Organoleptic evaluation showed that the cDPSC-Exos gel was clear, transparent, homogeneous, and odorless. Furthermore, pharmaceutical evaluation demonstrated its safety and effectiveness in delivering cDPSC-Exos, with a gel pH of 5, spreadability of 7.05 ± 0.24 cm, residence time of 5.32 ± 0.39 minutes, and viscosity of 5,782.75 ± 29.44 cP. The formulation was stored at 4°C prior to use, and its physical appearance was monitored through organoleptic evaluation, including clarity, homogeneity, color, and odor. The favorable pharmaceutical characteristics of the HPMC-based gel suggest it may serve as a promising delivery system for the topical administration of cDPSC-Exos in experimental wound-healing studies and clinical applications.
Wound macroscopic evaluation
Topical application of cDPSC-Exos gel in this study supported post-castration wound-healing in cats without any clinical complications or adverse events, including prolonged inflammation, wound dehiscence, behavioral changes, or signs of infection. Macroscopic observation of wound-healing on days 0, 3, and 5 showed progressive improvement in both groups (Figure 6A). Macroscopic wound-healing scores between the cDPSC-Exos group and the positive control group showed significant differences (p < 0.05) in the redness and pain response on day 3, as well as in the swelling on days 3 and 5 (Figure 6B), while the wound closure parameters did not show significant differences (p > 0.05). However, the final wound closure score in the cDPSC-Exos group was lower than that in the control group, indicating improved tissue repair.
Perfusion rate
The LSCI results on the 5th day showed a lower perfusion rate (Speckle flow index; SFI) in the cDPSC-Exos group (1033.42 ± 274.61) compared to the control group (1213.41 ± 313.22), though no significant difference was observed, which may be attributed to the limited sample size (Figures 6D and 6E).
[图：Macroscopic evaluation of post-castration wound-healing. (A) Representative wound images from the control and cDPSC -Exo]
Blood profile
There were no significant differences in the WBC and PLT profiles between the cDPSC-Exos and control groups, as local inflammation rarely causes systemic alterations. Nevertheless, differences in the WBC and PLT dynamics were observed descriptively (Figure 6C). Compared to the pre-operative hematology result, a greater decrease in WBC, NEU, and MON counts, along with increased LYM and EOS counts, was observed in the cDPSC-Exos group on the 5th day. In contrast, the control group showed increases in WBC, NEU, and MON, while LYM and EOS decreased. A slight increase in PLT counts was observed in the cDPSC-Exos group, while the control group showed decreased PLT counts.
DISCUSSION
Characterization of cDPSCs
cDPSCs in the present study were isolated using the tissue explantation method, exhibited clonogenic capacity comparable to hDPSCs, which ranges between 9-12% [50], and demonstrated a proliferation capacity with a growth pattern similar to hDPSCs, which exit the adaptive phase and enter the exponential phase within 3-7 days of culture [51]. Compared to the enzymatic digestion method, the tissue explant method preserves the native cellular microenvironment, has a lower risk of cellular stress and damage, is more cost-effective, supports cell attachment and outgrowth, and maintains paracrine signaling mechanisms, which may be beneficial for exosome collection [52, 53]. This native microenvironment may also contribute to the maintenance of multipotency and self-renewal in cDPSCs, as evidenced by the expression of stemness markers, reflecting their ability to differentiate into multiple cell lineages [54]. Furthermore, the isolated cDPSCs exhibited MSC characteristics, as indicated by the expression of MSC surface markers CD90, CD105, CD73, and CD44 and the lack expression of the hematopoietic cell surface marker CD45. Although these surface markers are defined by ISCT based on human MSCs, similar expression patterns have been reported in animal-derived stem cells, including those from rabbit synovial fluid [55], equine dental pulp [7], canine bone marrow [10, 33, 34, 37, 56], as well as canine dental pulp in previous studies [33, 34, 56]. Although MSC characterization is conventionally performed using flow cytometry according to ISCT criteria, RT-qPCR was employed in the present study due to methodological limitations. Previous studies have reported consistent expression patterns of CD90, CD105, CD73, CD44, and CD45 between RT-qPCR and flow cytometric analyses [57, 58], indicating the present findings sufficiently represent the MSC characteristics of the isolated cDPSCs.
The trilineage differentiation potential, including chondrogenesis, osteogenesis, and adipogenesis, serves as a functional hallmark of MSCs, as defined in the ISCT minimal requirements [59]. The isolated cDPSCs demons-trated trilineage differentiation potential, as assessed through phenotypic and transcriptional approaches. This differentiation capacity has also been reported in previous studies of canine-derived stem cells, including those from bone marrow, adipose tissue, and dental pulp; however, the extent of differentiation varies by tissue source [33, 34, 37, 60]. In the present study, osteogenic differentiation was the most pronounced among the three lineages, consistent with previous reports demonstrating that cDPSCs possess greater osteogenic potential than canine bone marrow MSCs (cBMSCs) [37], as well as a study on hDPSCs reporting enhanced osteogenic and chondrogenic capacities relative to other MSC populations [61]. This indicates that cDPSCs' potential is not limited to soft tissue but also to bone tissue engineering.
Characterization and proteomic profile of cDPSC-Exos
The successful isolation of cDPSC-derived exosomes in the present study demonstrates a stable and measurable yield using gradual centrifugation combined with exosome isolation reagent, as reflected by the cDPSC-Exos concentration within the common range of exosome isolation, 109-1010 particle/mL [50, 62, 63]. The positive expression of CD9 (Figure 2B) serves as molecular evidence to support exosome biogenesis from cDPSCs; in line with previous MSC-Exos studies in humans and animals [64–67]. However, this characterization remains preliminary, as the MISEV 2023 guidelines recommend the assessment of canonical EV markers at the protein level (CD9, CD63, CD81, TSG101, and Alix) along with additional characterization approaches such as ultrastructural imaging and evaluation of negative markers.
This study provides the first proteomic profiling of cDPSC-Exos, which revealed wound healing-related proteins. These proteins were distributed across all phases of tissue repair, reflecting a coordinated and complex mechanism as illustrated in Figure 7. Notably, several proteins identified in the present study, including COL1A1, ANXA, VIM, THBS1, ITGA, and ACTB, have also been reported in proteomic studies of MSC-derived exosomes from dental pulp and various tissue sources [8, 37, 44, 68–70]. This finding suggests that cDPSC-Exos share molecular components commonly associated with the regenerative and wound-healing properties of MSC-derived exosomes.
[图：Schematic illustration of wound-healing-related proteins identified in the first cDPSC -Exos proteomic analysis. Protein]
Based on the functional analysis, the predominant proteins are involved in regulating the immune response during the hemostasis and inflammation phases, suggesting that cDPSC-Exos may primarily exert their therapeutic effects during the early stages of wound-healing, which are critical for determining subsequent tissue repair outcomes [71, 72]. Additionally, PPI analysis revealed an integrated interaction network among wound-healing-related proteins, with highly connected proteins such as ACTB and VIM highlighting the central role of cytoskeletal organization and cell motility in the biological activity of cDPSC-Exos. Consistent with the proteomic results, western blot analysis confirmed the expression of ACTB and VIM (Figure 3B), supporting the validity of other identified proteins. Both proteins are associated with cell migration, structural stability, and extracellular matrix (ECM) remodeling, which are essential during the proliferation and remodeling phases of wound-healing [73, 74]. During wound-healing, VIM is involved in fibroblast migration, proliferation, and differentiation, as well as in cytoskeletal remodeling and extracellular matrix organization, to promote tissue regeneration [75, 76]. ACTB is involved in wound-healing through cytoskeletal remodeling, which drives cell migration, epithelialization, and wound contraction, particularly via actin cable formation at the wound edge, thereby supporting wound closure [77]. On the other hand, VIM and ACTB revealed the biological integrity and purity of exosomes, as both proteins are associated with cytoskeleton-dependent EV biogenesis. Previous studies have demonstrated that VIM and ACTB are involved in exosome biogenesis by regulating vesicle transport, maintaining endosomal positioning, and facilitating exosome release through their interaction in vesicular transport; thus, filament assembly and disassembly influence their secretion and involvement in EV pathways [75, 78].
Based on the PPI network, THBS1 interactions with annexin family members also support the regulation of angiogenesis and membrane repair mechanisms, thereby strengthening the role of cDPSC-Exos in tissue regeneration [79, 80]. The high abundance of PIK3C3 may reflect its dual roles in exosome biogenesis and wound-healing regulation, as it is involved in autophagy and endocytosis, which are essential for vesicle trafficking and cellular homeostasis [81]. Autophagy mediated by PIK3C3 has been implicated in debris clearance during hemostasis, inflammasome inhibition, and macrophage polarization during inflammation, as well as cell migration, angiogenesis, and ECM remodeling during the late phases of wound repair [81–83]. The integrated proteomic analysis suggests that cDPSC-Exos contain proteins involved not only in exosome biogenesis but also in the comprehensive wound-healing mechanisms, supporting the therapeutic potential of cDPSC-Exos in promoting tissue repair.
Xenogeneic efficacy in post-castration wound-healing
The identified proteins may mechanistically contribute to the accelerated resolution of inflammation and wound closure observed in the present study by coordinately regulating immune responses, angiogenesis, cell migration, and extracellular matrix remodeling throughout the wound-healing process. Wound-healing involves numerous components, including blood cells, extracellular matrix, parenchymal cells, and soluble mediators, that restore tissue integrity and function through four main phases: hemostasis, inflammation, proliferation, and remodeling [47, 84]. The inflammation phase is characterized by redness, as vasodilatation increases blood flow to the wound site; swelling/edema, as increased vascular permeability allows plasma to leak into the tissue; and pain, caused by the release of mediators such as prostaglandins, cytokines, and chemokines [85]. The lower redness, swelling, and pain scores in the cDPSC-Exos group on day 3 indicate a faster inflammatory response than in the control group. The wound closure score represents the late stages of wound-healing, including proliferation and remodeling, characterized by the regeneration and repair of damaged skin tissue, which are relatively faster in the cDPSC-Exos group [47, 84]. The LSCI results were consistent with the macroscopic findings, as a lower perfusion unit indicates that the cDPSC-Exos group entered the remodeling phase on day 5. Active angiogenesis occurs in the early stage of the wound-healing process, resulting in a higher SFI value as assessed by LSCI, which gradually decreases during the remodeling phase, approaching that of normal skin tissue [86]. Angiogenesis forms new blood vessels from the existing vasculature, thereby maintaining oxygen and nutrient supply to damaged skin tissue and playing an important role in tissue regeneration and wound-healing [87].
The blood profile was consistent with the macroscopic and LSCI results, indicating accelerated wound-healing in the cDPSC-Exos group. Platelets play an important role in hemostasis, and the slight increase in platelet counts in the cDPSC-Exos group may indicate the end of hemostasis as platelet counts return to normal [84, 85]. During the inflammatory phase, neutrophils and macrophages infiltrate the wounded skin to fight microorganisms, clear cellular debris, and release inflammatory mediators; therefore, a decrease in neutrophil and monocyte counts indicates resolution of the inflammatory phase [84]. The neutrophil and monocyte trends suggest that the inflammatory phase in the cDPSC-Exos group occurred over a relatively shorter period than in the control group, which may be associated with the abundance of immune-related proteins in cDPSC-Exos. Increased lymphocytes and eosinophils may indicate the onset of the late stage of wound-healing, as they play important roles in the proliferative and remodeling phases. EOS interact with the coagulation and fibrinolytic systems, promoting ECM modification and epithelial repair by releasing growth factors and proteases, while lymphocytes regulate tissue regeneration and epithelial barrier restoration through cytokine-mediated immune modulation [88, 89]. These findings were comparable between groups and suggest a trend toward improved wound-healing in the cDPSC-Exos group compared with the control group, although no statistically significant difference was observed. The improvement in wound-healing is consistent with previous studies of MSC-derived exosomes and stem cell-based therapies in companion animals, which have similarly reported enhanced tissue repair [5, 15, 17, 90].
Safety, limitations, and future directions
The absence of treatment-related adverse clinical reactions following xenogeneic cDPSC-Exos administration provides preliminary evidence supporting the feasibility of cross-species cell-free regenerative approaches. Similar findings have been reported in previous studies demonstrating the low immunogenicity of MSC-derived exosomes across species barriers, including hu-AdMSCs secretome in feline chronic gingivostomatitis therapy, human cell exosomes in canine atopic dermatitis treatment, and numerous human MSC-Exos studies revealed acceleration in animal model wound-healing [6, 14, 15, 18, 19]. Although no evidence of xenogeneic adverse reactions was observed, the immunological safety of cDPSC-Exos in this study warrants further investigation. The comprehensive approach adopted in the present study, integrating proteomic profiling, hematological assessment, tissue perfusion analysis, and clinical wound evaluation, provides complementary information to understand the biological effects of cDPSC-Exos.
This pilot study has several limitations, particularly that the wound healing evaluation was based solely on clinical assessments, without histological or molecular assessments to characterize tissue regeneration and the cellular mechanisms of wound-healing. In addition, the relatively small sample size and limited wound size may limit the interpretation of the findings. Further studies including histopathology, immunohistochemistry, and comprehensive immunological analyses of wound tissue are needed to confirm the safety of cDPSC-Exos xenogeneic application. Despite these limitations, the present findings provide preliminary evidence of the potential of cDPSC-Exos in feline wound-healing. As a cell-free therapeutic approach, cDPSC-Exos may have translational potential in veterinary medicine, although further standardization, safety evaluation, and regulatory considerations are needed before broader clinical application.
CONCLUSION
cDPSC-Exos demonstrated promising therapeutic potential for postoperative wound-healing in cats, as indicated by accelerated wound healing parameters and the presence of angiogenic, anti-inflammatory, and immunomodulatory proteins identified by proteomic analysis. This study represents the first report describing the isolation, characterization, proteomic profiling, and xenogeneic topical application of cDPSC-Exos in a feline surgical wound model, and pioneers the use of companion-animal dental pulp as a sustainable source for xenogeneic cell-free products, offering a practical pathway for clinical veterinary regenerative medicine.
Proteomic analysis identified 312 unique proteins in cDPSC-Exos, including 22 associated with wound healing processes across all phases (hemostasis, inflammation, proliferation, and remodeling). In the pilot in vivo study, topical cDPSC-Exos gel (3.3 × 10⁸ particles/g) significantly improved early macroscopic wound scores for redness, swelling, and pain (p < 0.05) compared with Bioplacenton®, with supportive trends in hematological parameters and tissue perfusion (LSCI), and no adverse reactions observed.
These findings support cDPSC-Exos as a safe, xenogeneic, cell-free regenerative therapy derived from a minimally invasive dental pulp source. This approach offers a scalable alternative to cell-based therapies for post-surgical wound management in companion animals, with potential for broader veterinary applications.
The study integrates comprehensive in vitro characterization, detailed proteomic profiling, and a randomized controlled pilot in vivo evaluation in a clinically relevant feline post-castration model, providing strong preliminary evidence of both molecular mechanisms and functional efficacy.
This pilot study has several limitations, particularly that the wound healing evaluation was based solely on clinical assessments, without histological or molecular assessments to characterize tissue regeneration and the cellular mechanisms of wound-healing. In addition, the relatively small sample size and limited wound size may limit the interpretation of the findings.
Further studies, including histopathology, immunohistochemistry, and comprehensive immunological analyses of wound tissue, are needed to confirm the safety of the application of xenogeneic cDPSC-Exo. Larger well-powered randomized controlled trials, extended observation periods, optimized dosing and formulation, and mechanistic investigations are required to validate efficacy and support clinical translation. Standardization, long-term safety evaluation, and regulatory considerations will also be essential before broader application.
Despite these limitations, the present findings provide preliminary evidence of cDPSC-Exos' potential for feline wound healing. As a cell-free therapeutic approach, cDPSC-Exos may have translational potential in veterinary medicine. Given the pilot nature of the study, the limited sample size, and the absence of histological and molecular wound assessments, further well-powered studies with comprehensive exosome characterization, histopathological evaluation, and mechanistic investigations are required to validate these preliminary findings and support future clinical translation of cDPSC-Exos as a cell-free regenerative therapy in veterinary medicine.
DATA AVAILABILITY
The data supporting the findings of this study are available in this article and its Supplementary Materials, including the raw NTA data, quantification of multilineage differentiation staining, full-length western blot images, the proteomic dataset, detailed animal information, raw wound healing data, and the ARRIVE 2.0 checklist. Supplementary materials and additional data are available from the corresponding author upon reasonable request.
GENERATIVE AI DECLARATION
Generative AI tools were used solely to improve language, grammar, and readability, and were not listed as authors. All scientific content, analysis, interpretation, and conclusions were developed and verified by the authors, who take full responsibility for the accuracy and integrity of the work.
AUTHORS’ CONTRIBUTIONS
MP: Conceptualized and designed the study. YSD and MP: Conducted the animal experiments, interpreted the data, performed the statistical analysis, and drafted the manuscript. DA: Performed the castration surgeries. AH and TB: Contributed to stem cell characterization and data interpretation. CS and SN: Contributed to exosome characterization, critically reviewed, and edited the manuscript. MP, YSD, AH, TB, CS, and SN: Revised and approved the final manuscript. All authors have read and approved the final manuscript.