Research Centre for Experimental Orthopaedics, Orthopaedic University Hospital, Heidelberg University Hospital, Schlierbacher Landstrasse 200a, Heidelberg, 69118, Germany.
Orthopaedic University Hospital, Heidelberg University Hospital, Heidelberg, Germany.
Stem Cell Res Ther. 2024 Apr 5;15(1):98. doi: 10.1186/s13287-024-03710-7.
In vitro chondrogenesis of mesenchymal stromal cells (MSCs) driven by the essential chondro-inducer transforming growth factor (TGF)-β is instable and yields undesired hypertrophic cartilage predisposed to bone formation in vivo. TGF-β can non-canonically activate bone morphogenetic protein-associated ALK1/2/3 receptors. These have been accused of driving hypertrophic MSC misdifferentiation, but data remained conflicting. We here tested the antihypertrophic capacity of two highly specific ALK1/2/3 inhibitors - compound A (CompA) and LDN-212854 (LDN21) - in order to reveal potential prohypertrophic contributions of these BMP/non-canonical TGF-β receptors during MSC in vitro chondrogenesis.
Standard chondrogenic pellet cultures of human bone marrow-derived MSCs were treated with TGF-β and CompA (500 nM) or LDN21 (500 nM). Daily 6-hour pulses of parathyroid hormone-related peptide (PTHrP[1-34], 2.5 nM, from day 7) served as potent antihypertrophic control treatment. Day 28 samples were subcutaneously implanted into immunodeficient mice.
All groups underwent strong chondrogenesis, but GAG/DNA deposition and ACAN expression were slightly but significantly reduced by ALK inhibition compared to solvent controls along with a mild decrease of the hypertrophy markers IHH-, SPP1-mRNA, and Alkaline phosphatase (ALP) activity. When corrected for the degree of chondrogenesis (COL2A1 expression), only pulsed PTHrP but not ALK1/2/3 inhibition qualified as antihypertrophic treatment. In vivo, all subcutaneous cartilaginous implants mineralized within 8 weeks, but PTHrP pretreated samples formed less bone and attracted significantly less haematopoietic marrow than ALK1/2/3 inhibitor groups.
Overall, our data show that BMP-ALK1/2/3 inhibition cannot program mesenchymal stromal cells toward stable chondrogenesis. BMP-ALK1/2/3 signalling is no driver of hypertrophic MSC misdifferentiation and BMP receptor induction is not an adverse prohypertrophic side effect of TGF-β that leads to endochondral MSC misdifferentiation. Instead, the prohypertrophic network comprises misregulated PTHrP/hedgehog signalling and WNT activity, and a potential contribution of TGF-β-ALK4/5-mediated SMAD1/5/9 signalling should be further investigated to decide about its postulated prohypertrophic activity. This will help to successfully engineer cartilage replacement tissues from MSCs in vitro and translate these into clinical cartilage regenerative therapies.
在体外，间充质基质细胞（MSCs）在必需的软骨诱导转化生长因子（TGF）-β的作用下进行软骨生成，这种软骨生成不稳定，并且产生体内不期望的易形成骨的肥大软骨。TGF-β可以非经典地激活骨形态发生蛋白相关的ALK1/2/3 受体。这些受体被指控驱动肥大 MSC 错分化，但数据仍然存在冲突。我们在这里测试了两种高度特异性的 ALK1/2/3 抑制剂 - 化合物 A（CompA）和 LDN-212854（LDN21） - 的抗肥大能力，以揭示这些 BMP/非经典 TGF-β 受体在 MSC 体外软骨生成过程中的潜在促肥大作用。
用 TGF-β和 CompA（500 nM）或 LDN21（500 nM）处理人骨髓来源的 MSC 的标准软骨生成微球培养物。从第 7 天开始，甲状旁腺激素相关肽（PTHrP[1-34]，2.5 nM，每日 6 小时脉冲）作为有效的抗肥大对照处理。第 28 天的样本皮下植入免疫缺陷小鼠。
所有组都经历了强烈的软骨生成，但与溶剂对照相比，ALK 抑制使 GAG/DNA 沉积和 ACAN 表达略有但显著降低，同时肥大标志物 IHH-、SPP1-mRNA 和碱性磷酸酶（ALP）活性也略有降低。当校正软骨生成程度（COL2A1 表达）时，只有脉冲 PTHrP 而不是 ALK1/2/3 抑制可作为抗肥大治疗。在体内，所有皮下软骨植入物在 8 周内都发生了矿化，但与 ALK1/2/3 抑制剂组相比，经 PTHrP 预处理的样本形成的骨较少，吸引的造血骨髓明显较少。
总的来说，我们的数据表明，BMP-ALK1/2/3 抑制不能将间充质基质细胞编程为稳定的软骨生成。BMP-ALK1/2/3 信号不是肥大 MSC 错分化的驱动因素，BMP 受体诱导不是导致软骨内 MSC 错分化的 TGF-β 的不利促肥大副作用。相反，促肥大网络包括调节异常的 PTHrP/ hedgehog 信号和 WNT 活性，并且 TGF-β-ALK4/5 介导的 SMAD1/5/9 信号的潜在贡献需要进一步研究，以决定其假定的促肥大活性。这将有助于成功地从 MSC 体外工程软骨替代组织，并将其转化为临床软骨再生治疗。
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