Probing the effects of polysaccharide hydrogel composition on the viability and pro-angiogenic function of human adipose-derived stromal cells.

Fiona E Serack, Kaylee A Fennell, Christina Iliopoulos, John T Walker, John A Ronald, Brian G Amsden, David A Hess, Lauren E Flynn
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Abstract

Cell therapies harnessing the pro-vascular regenerative capacities of mesenchymal stromal cell (MSC) populations, including human adipose-derived stromal cells (hASCs), have generated considerable interest as an emerging treatment strategy for peripheral arterial disease (PAD) and its progression to critical limb ischemia (CLI). There is evidence to support that polysaccharide hydrogels can enhance therapeutic efficacy when applied as minimally-invasive delivery systems to support MSC survival and retention within ischemic tissues. However, there has been limited research to date on the effects of hydrogel composition on the phenotype and function of encapsulated cell populations. Recognizing this knowledge gap, this study compared the pro-angiogenic function of hASCs encapsulated in distinct but similarly-modified natural polysaccharide hydrogels composed of methacrylated glycol chitosan (MGC) and methacrylated hyaluronic acid (MHA). Initial in vitro studies confirmed high viability (>85%) of the hASCs following encapsulation and culture in the MGC and MHA hydrogels over 14 days, with a decrease in the cell density observed over time. Moreover, higher levels of a variety of secreted pro-angiogenic and immunomodulatory factors were detected in conditioned media samples collected from the hASCs encapsulated in the MGC-based hydrogels compared to the MHA hydrogels. Subsequent testing focused on comparing hASC delivery within the MGC and MHA hydrogels to saline controls in a femoral artery ligation-induced CLI (FAL-CLI) model in athymic nu/nu mice over 28 days. For the in vivo studies, the hASCs were engineered to express tdTomato and firefly luciferase to quantitatively compare the efficacy of the two platforms in supporting the localized retention of viable hASCs through longitudinal cell tracking with bioluminescence imaging (BLI). Interestingly, hASC retention was significantly enhanced when the cells were delivered in the MHA hydrogels as compared to the MGC hydrogels or saline. However, laser Doppler perfusion imaging (LDPI) indicated that the restoration of hindlimb perfusion was similar between the treatment groups and controls. These findings were corroborated by endpoint immunofluorescence (IF) staining showing similar levels of CD31+ cells in the ligated limbs at 28 days in all groups. Overall, this study demonstrates that enhanced MSC retention may be insufficient to augment vascular regeneration, emphasizing the complexity of designing biomaterials platforms for MSC delivery for therapeutic angiogenesis. In addition, the data points to a potential challenge in approaches that seek to harness the paracrine functionality of MSCs, as strategies that increase the secretion of immunomodulatory factors that can aid in regeneration may also lead to more rapid MSC clearance in vivo.

探究多糖水凝胶成分对人脂肪源性基质细胞活力和促血管生成功能的影响。
利用间充质基质细胞(MSC)群(包括人脂肪源性基质细胞(hASCs))促进血管再生能力的细胞疗法作为治疗外周动脉疾病(PAD)及其发展为严重肢体缺血(CLI)的一种新兴治疗策略,引起了人们的极大兴趣。有证据表明,当多糖水凝胶作为微创递送系统用于支持间充质干细胞在缺血组织中存活和保留时,可以提高治疗效果。然而,迄今为止,有关水凝胶成分对包裹细胞群表型和功能影响的研究还很有限。认识到这一知识空白,本研究比较了由甲基丙烯酸化乙二醇壳聚糖(MGC)和甲基丙烯酸化透明质酸(MHA)组成的不同但类似改性的天然多糖水凝胶中包裹的 hASCs 的促血管生成功能。初步体外研究证实,在 MGC 和 MHA 水凝胶中封装和培养 14 天后,hASCs 的存活率很高(>85%),但随着时间的推移,细胞密度有所下降。此外,与 MHA 水凝胶相比,从封装在 MGC 水凝胶中的 hASCs 收集的条件培养基样本中检测到了更高水平的各种促血管生成和免疫调节分泌因子。随后的测试重点是在无胸腺 nu/nu 小鼠股动脉结扎诱导的 CLI(FAL-CLI)模型中,比较 MGC 和 MHA 水凝胶中的 hASC 输送与生理盐水对照组在 28 天内的输送情况。在体内研究中,hASCs被设计为表达tdTomato和萤火虫荧光素酶,以便通过生物发光成像(BLI)进行纵向细胞追踪,定量比较两种平台在支持有活力的hASCs局部保留方面的功效。有趣的是,与 MGC 水凝胶或生理盐水相比,当细胞在 MHA 水凝胶中输送时,hASC 的保留率明显提高。然而,激光多普勒灌注成像(LDPI)显示,治疗组和对照组的后肢灌注恢复情况相似。终点免疫荧光(IF)染色也证实了这些发现,28 天时,所有组结扎肢体中的 CD31+ 细胞水平相似。总之,这项研究表明,间充质干细胞保留能力的增强可能不足以促进血管再生,从而强调了为治疗性血管生成而设计间充质干细胞递送生物材料平台的复杂性。此外,这些数据还指出了试图利用间充质干细胞旁分泌功能的方法所面临的潜在挑战,因为增加有助于再生的免疫调节因子分泌的策略也可能导致间充质干细胞在体内更快地被清除。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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