Three-dimensional gelatin microspheres loaded human umbilical cord mesenchymal stem cells for chronic tendinopathy repair

doi:10.12307/2025.019

Abstract

Abstract: BACKGROUND: The absence of blood vessels in tendon tissue makes tendon repair challenging. Therefore, improving tendon healing and raising the efficacy of stem cell and other therapeutic cell transplantation after tendon damage have become hotspots for research in both clinical and scientific contexts.
OBJECTIVE: The stem cells and gelatin microcarrier scaffold were joined to form tissue engineered stem cells. Human umbilical cord mesenchymal stem cells cultured in gelatin microcarriers were used to investigate the therapeutic impact and mode of action on tendinopathy healing in rats in vitro and In vivo.
METHODS: (1) In vitro cell experiments: After seeding human umbilical cord mesenchymal stem cells with three-dimensional gelatin microcarriers, the cell vitality and survival were assessed. Human umbilical cord mesenchymal stem cells conventionally cultured were cultured as controls. (2) In vivo experiment: Adult SD rats were randomly assigned to normal group, tendinopathy group, 2D group (tendinopathy + conventional culture of human umbilical cord mesenchymal stem cells), and 3D group (tendinopathy + gelatin microcarrier three-dimensional culture of human umbilical cord mesenchymal stem cells), with 6 rats in each group. Four weeks after therapy, animal behavior tests and histopathologic morphology of the Achilles tendon was examined.
RESULTS AND CONCLUSION: (1) In vitro cell experiments: the seeded human umbilical cord mesenchymal stem cells on gelatin microcarriers showed high viability and as time went on, the stem cell proliferation level grew. Compared with the control group, 3D stem cell culture preserved cell viability. (2) In vivo experiment: Following a 4-week treatment, the 3D stem cell culture group showed a significant improvement in both functional recovery of the lower limbs and histopathological scores when compared to the tendinopathy group. The 2D stem cell culture group also showed improvement in tendinopathy injury, but its effect is not as much as the 3D stem cell culture group. (3) The outcomes demonstrate that human umbilical cord mesenchymal stem cells cultured with three-dimensional gelatin microcarrier can promote the repair and regeneration of tendon injury tissue, and the repair effect is better than that of conventional human umbilical cord mesenchymal stem cells.

Key words: tendinopathy, tendon injury, human umbilical cord mesenchymal stem cell, tissue-engineered stem cell, microcarrier, three-dimensional culture

CLC Number:

Li Dijun, Jiu Jingwei, Liu Haifeng, Yan Lei, Li Songyan, Wang Bin. Three-dimensional gelatin microspheres loaded human umbilical cord mesenchymal stem cells for chronic tendinopathy repair[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(7): 1356-1362.

Figures/Tables 1

2.1 明胶微载体的形态特征实验所使用明胶微载体支架由多达数万个微载体压缩而制成，最终呈多层多个状态(图2A)。将200 μL 人脐带间充质干细胞悬液接种于明胶微载体上，整片微载体吸水膨胀而不散开(图2B)，便于细胞贴壁；接种2 h后随着细胞的贴壁及营养物质消耗，微载体颜色变黄但仍呈圆形膨胀状态(图2C)，添加培养基后微载体能均匀分布于培养皿中(图2D)。此外，使用扫描电镜观察明胶微载体的显微结构呈现疏松多孔蜂窝状，具有较高的孔隙率(大于90%)，这均为细胞的贴壁吸附提供了条件(图2E，F)。 2.2 明胶微载体培养对人脐带间充质干细胞活性的影响人脐带间充质干细胞接种于明胶微载体1，3，5 d后，对明胶微载体三维培养的人脐带间充质干细胞进行死活染色以观察接种后细胞存活情况和增殖状态(图3A)，可见大多数细胞存活(85%以上)，仅少量细胞死亡；随着接种时间的推移活细胞数量明显增加，显示出良好的增殖状态(图3B，C)。同时探索了明胶微载体三维培养对人脐带间充质干细胞活力的影响，CCK-8检测发现随着时间的增加，细胞活性不受影响(P > 0.05)(图3D)，由此可见：三维培养环境可维持人脐带间充质干细胞的活力。该结果进一步说明明胶微载体支架具有良好的生物相容性。 2.3 实验动物数量分析实验选用24只SD大鼠全部存活良好，并纳入最终结果分析。 2.4 动物行为学治疗4周后使用CatWalk进行大鼠步态分析，大鼠自由通过固定跑道时能清晰记录四肢印记(图4A，B)。正常组大鼠节律整齐地通过固定跑道；肌腱病组大鼠在通过跑道时节律紊乱；与肌腱病组及2D组相比，3D组大鼠下肢步态节律更齐(图4C)，恢复效果更好。进一步分析发现肌腱病形成后局部痛阈减小，大鼠触地面积以及站立的时间减少(图4D)，3D组治疗后大鼠功能显著恢复，同时2D组也显示出一定治疗效果(P < 0.05)，但疗效不及3D组。 2.5 组织病理学分析苏木精-伊红染色显示，正常组细胞和细胞外基质良好；肌腱病组结构不完整，纤维排列结构混乱，伴有大量炎性细胞浸润及新生血管生成；3D组纤维排列对齐，无明显炎症细胞浸润(图5A)。组织学评分(纤维结构、纤维排列、核圆程度、炎症细胞、血管数量、细胞数量)也显示，与肌腱病组相比，3D干细胞治疗后组织学评分更低恢复更佳(图5B)。同时行Masson染色进一步观察各组纤维分布排列情况，结果显示正常组为排列整齐的肌纤维与胶原纤维，而肌腱病组胶原纤维明显水肿(蓝色区域)，3D组的胶原纤维水肿程度被逆转(图5A)；同时，胶原容积分数显著低于肌腱病组和2D组(P < 0.001)(图5C) 。 2.6 组织炎症因子分泌结果通过免疫荧光染色法观察组织中炎症因子分泌情况，结果显示：正常组具有少量炎症因子(白细胞介素6及肿瘤坏死因子α)表达，肌腱病组炎症因子明显增多，而3D组白细胞介素6及肿瘤坏死因子α的表达明显降低(图6，7)。"

References

[1] MILLAR NL, SILBERNAGEL KG, THORBORG K, et al. Tendinopathy. Nat Rev Dis Primers. 2021;7(1):1.
[2] LUI PP, CHAN LS, FU SC, et al. Expression of sensory neuropeptides in tendon is associated with failed healing and activity-related tendon pain in collagenase-induced tendon injury. Am J Sports Med. 2010; 38(4):757-764.
[3] DOCHEVA D, MÜLLER SA, MAJEWSKI M, et al. Biologics for tendon repair. Adv Drug Deliv Rev. 2015;84:222-239.
[4] CHILDRESS MA, BEUTLER A. Management of chronic tendon injuries. Am Fam Physician. 2013;87(7):486-490.
[5] VOLETI PB, BUCKLEY MR, SOSLOWSKY LJ. Tendon healing: repair and regeneration. Annu Rev Biomed Eng. 2012;14:47-71.
[6] AICALE R, BISACCIA RD, OLIVIERO A, et al. Current pharmacological approaches to the treatment of tendinopathy. Expert Opin Pharmacother. 2020;21(12):1467-1477.
[7] MAZZOCCA AD, CHOWANIEC D, MCCARTHY MB, et al. In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers. Knee Surg Sports Traumatol Arthrosc. 2012;20(9):1666-1672.
[8] KOKUBU S, INAKI R, HOSHI K, et al. Adipose-derived stem cells improve tendon repair and prevent ectopic ossification in tendinopathy by inhibiting inflammation and inducing neovascularization in the early stage of tendon healing. Regen Ther. 2020;14:103-110.
[9] TANG Y, CHEN C, LIU F, et al. Structure and ingredient-based biomimetic scaffolds combining with autologous bone marrow-derived mesenchymal stem cell sheets for bone-tendon healing. Biomaterials. 2020;241:119837.
[10] TSUCHIYA A, TAKEUCHI S, WATANABE T, et al. Mesenchymal stem cell therapies for liver cirrhosis: MSCs as “conducting cells” for improvement of liver fibrosis and regeneration. Inflamm Regen. 2019; 39:18.
[11] TROYER DL, WEISS ML. Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells. 2008;26(3):591-599.
[12] ROURA S, FARRÉ J, SOLER-BOTIJA C, et al. Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells. Eur J Heart Fail. 2006;8(6):555-563.
[13] WANG B, LIU W, LI JJ, et al. A low dose cell therapy system for treating osteoarthritis: In vivo study and in vitro mechanistic investigations. Bioact Mater. 2021;7:478-490.
[14] LIU H, YAN X, JIU J, et al. Self-assembly of gelatin microcarrier-based MSC microtissues for spinal cord injury repair. Chem Eng J. 2023;451: 138806.
[15] YANG Z, WANG B, LIU W, et al. In situ self-assembled organoid for osteochondral tissue regeneration with dual functional units. Bioact Mater. 2023;27:200-215.
[16] ZHOU T, YUAN Z, WENG J, et al. Challenges and advances in clinical applications of mesenchymal stromal cells. J Hematol Oncol. 2021; 14(1):24.
[17] DORON G, WOOD LB, GULDBERG RE, et al. Poly(ethylene glycol)-Based Hydrogel Microcarriers Alter Secretory Activity of Genetically Modified Mesenchymal Stromal Cells. ACS Biomater Sci Eng. 2023;9(11): 6282-6292.
[18] CHEN AK, REUVENY S, OH SK. Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction. Biotechnol Adv. 2013;31(7): 1032-1046.
[19] LOUBIÈRE C, SION C, DE ISLA N, et al. Impact of the type of microcarrier and agitation modes on the expansion performances of mesenchymal stem cells derived from umbilical cord. Biotechnol Prog. 2019;35(6):e2887.
[20] YAN X, ZHANG K, YANG Y, et al. Dispersible and Dissolvable Porous Microcarrier Tablets Enable Efficient Large-Scale Human Mesenchymal Stem Cell Expansion. Tissue Eng Part C Methods. 2020;26(5):263-275.
[21] 李帝均,王桂杉,刘海峰,等.肌腱病动物模型的研究进展[J].中国实验动物学报,2022,30(2):260-266.
[22] ZHANG H, CHEN Y, FAN C, et al. Cell-subpopulation alteration and FGF7 activation regulate the function of tendon stem/progenitor cells in 3D microenvironment revealed by single-cell analysis. Biomaterials. 2022;280:121238.
[23] MILLAR NL, MURRELL GA, MCINNES IB. Inflammatory mechanisms in tendinopathy - towards translation. Nat Rev Rheumatol. 2017;13(2): 110-122.
[24] OREFF GL, FENU M, VOGL C, et al. Species variations in tenocytes’ response to inflammation require careful selection of animal models for tendon research. Sci Rep. 2021;11(1):12451.
[25] JIAO X, ZHANG Y, LI W, et al. HIF-1α inhibition attenuates severity of Achilles tendinopathy by blocking NF-κB and MAPK pathways. Int Immunopharmacol. 2022;106:108543.
[26] ACKERMAN JE, BEST KT, MUSCAT SN, et al. Metabolic Regulation of Tendon Inflammation and Healing Following Injury. Curr Rheumatol Rep. 2021;23(3):15.
[27] LEE SY, KWON B, LEE K, et al. Therapeutic Mechanisms of Human Adipose-Derived Mesenchymal Stem Cells in a Rat Tendon Injury Model. Am J Sports Med. 2017;45(6):1429-1439.
[28] RODAS G, SOLER-RICH R, RIUS-TARRUELLA J, et al. Effect of Autologous Expanded Bone Marrow Mesenchymal Stem Cells or Leukocyte-Poor Platelet-Rich Plasma in Chronic Patellar Tendinopathy (With Gap >3 mm): Preliminary Outcomes After 6 Months of a Double-Blind, Randomized, Prospective Study. Am J Sports Med. 2021;49(6): 1492-1504.
[29] XUE Y, KIM HJ, LEE J, et al. Co-Electrospun Silk Fibroin and Gelatin Methacryloyl Sheet Seeded with Mesenchymal Stem Cells for Tendon Regeneration. Small. 2022;18(21):e2107714.
[30] CHEN G, ZHANG W, ZHANG K, et al. Hypoxia-Induced Mesenchymal Stem Cells Exhibit Stronger Tenogenic Differentiation Capacities and Promote Patellar Tendon Repair in Rabbits. Stem Cells Int. 2020; 2020:8822609.