Supplementary Material for: Distinct Gene Expression Patterns Defining Human Osteoblasts' Response to BMP2 Treatment: Is the Therapeutic Success All a Matter of Timing?

<i>Background:</i> Bone morphogenetic proteins (BMPs) play a key role in bone formation. Local application of BMP2 (Dibotermin alfa) supports bone formation when applied to complex fractures. However, up to 33% of patients do not respond to this therapy.<i>Purpose:</i> Aiming to investigate whether inter-individual responses to BMP2 treatment can be predicted by gene expression patterns, we investigated the effect of BMP2 on primary human osteoblasts and THP-1 cell-derived osteoclasts from 110 donors. <i>Methods:</i> Osteoblasts were obtained by collagenase digestion of spongy bone tissues. Osteoclasts were differentiated from THP-1 cells using the conditioned media of the osteoblasts. Viability was determined by resazurin conversion. As functional characteristics AP and Trap5B activity were measured. Gene expression levels were determined by RT-PCR in 21 of the 110 evaluated donors and visualized by electrophoresis. <i>Results:</i> Based on our data, we could classify three response groups: (i) In 51.8% of all donors, BMP2 treatment induced osteoblast function. These donors strongly expressed the BMP2 inhibitor Noggin <i>(NOG)</i>, the alternative BMP2 receptors repulsive guidance molecule B <i>(RGMb)</i> and activin receptor-like kinase 6<i>(Alk6)</i>, as well as the Wnt inhibitor sclerostin <i>(SOST)</i>. (ii) In 17.3% of all donors, BMP2 treatment induced viability. In these donors, the initial high <i>SOST</i> expression significantly dropped with BMP2 treatment. (iii) 30.9% of all donors were not directly affected by BMP2 treatment. These donors expressed high levels of the pseudoreceptor BMP and activin membrane-bound inhibitor <i>(BAMBI)</i> and lacked <i>SOST</i>expression. In all donors, <i>SOST</i> expression correlated directly with receptor activator of NF-κB ligand <i>(RANKL)</i> expression, defining the cells' potential to stimulate osteoclastogenesis. <i>Conclusions:</i> Our data identified three donor groups profiting from BMP2 treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclastogenesis, depending on their expression of <i>BAMBI</i>, <i>SOST</i>, <i>NOG</i>, and <i>RANKL</i>. On the basis of patients' respective expression profiles, the clinical application of BMP2 as well as its timing might be modified in order to better fit the patients' needs to promote bone formation or to inhibit bone resorption.

背景：骨形态发生蛋白（Bone morphogenetic proteins，BMPs）在骨形成过程中发挥关键作用。将BMP2（地特莫肽α，Dibotermin alfa）局部应用于复杂性骨折时，可促进骨形成。然而，多达33%的患者对该治疗无应答。
目的：为探究个体间对BMP2治疗的应答差异是否可通过基因表达模式预测，我们针对110名供者的原代人成骨细胞及THP-1细胞源性破骨细胞，研究了BMP2对其的作用效果。
方法：通过胶原酶消化海绵骨组织获取成骨细胞。利用成骨细胞的条件培养基，将THP-1细胞诱导分化为破骨细胞。采用刃天青还原法检测细胞活力。以碱性磷酸酶（alkaline phosphatase，AP）和抗酒石酸酸性磷酸酶5B（Tartrate-resistant acid phosphatase 5B，Trap5B）活性作为功能学检测指标。对110名供者中的21名进行逆转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）检测基因表达水平，并通过电泳进行可视化分析。
结果：基于本研究数据，我们可将供者分为三类应答组：(i) 51.8%的供者经BMP2治疗后成骨细胞功能被激活。此类供者高表达BMP2抑制剂Noggin（NOG）、替代性BMP2受体排斥性引导分子B（RGMb）及激活素受体样激酶6（Alk6），同时高表达Wnt抑制剂骨硬化蛋白（SOST）。(ii) 17.3%的供者经BMP2治疗后细胞活力提升。此类供者初始高表达的SOST在BMP2治疗后显著下调。(iii) 30.9%的供者未受BMP2治疗的直接影响。此类供者高表达假受体BMP与激活素膜结合抑制剂（BAMBI），且无SOST表达。在所有供者中，SOST的表达与核因子κB受体活化因子配体（receptor activator of NF-κB ligand，RANKL）的表达呈正相关，这决定了细胞刺激破骨细胞生成的能力。
结论：本研究数据确定了三类可从BMP2治疗中获益的供者群体：一类通过直接激活成骨细胞功能或提升细胞活力获益，另一类则通过抑制破骨细胞生成间接获益，这取决于供者的BAMBI、SOST、NOG及RANKL表达谱。基于患者各自的表达特征，可对BMP2的临床应用方案及给药时机进行调整，以更贴合患者需求，从而更好地促进骨形成或抑制骨吸收。