Lu Fangzhou, Groven Rald V M, Meesters Dennis M, Bitorina Albert, Poeze Martijn, Tang Shan, van Griensven Martijn, Blokhuis Taco J, Shiri-Sverdlov Ronit
Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht 6229 ER, the Netherlands; Division of Trauma Surgery, Department of Surgery, Maastricht University Medical Center, Maastricht 6229 HX, the Netherlands.
Department of Cell Biology-Inspired Tissue Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht 6229 ER, the Netherlands; Department of Orthopaedic, Trauma- and Reconstructive Surgery, RWTH Aachen University Hospital, Aachen, Germany.
Bone. 2025 Oct;199:117574. doi: 10.1016/j.bone.2025.117574. Epub 2025 Jun 26.
The role and involvement of immune cells in the fracture healing cascade, specifically neutrophils, is not yet fully understood. During tissue regeneration, cathepsin (CTS) D and cathepsin G have been identified as being involved in various cellular processes and associated with neutrophil function. This study aimed to determine the expression of these 2 cathepsins in fracture hematoma. Firstly, these two cathepsins were identified and scrutinized using a bioinformatics approach. Secondly, these two cathepsins were investigated for their expression in the human fracture hematoma (FH) as well as in N1 (pro-inflammatory) and N2 (regenerative) neutrophil phenotypes.
To review the latest research on CTSD and G gene expressions in fracture healing, bioinformatics analysis was firstly performed. Subsequently, to identify CTSD and G genes, the expression of them was assessed in human FH samples throughout different phases of the fracture healing cascade, and potential correlations with patient characteristics were explored. To confirm that gene expression translated to protein production, their corresponding protein levels in FH were evaluated via immunofluorescence. Finally, human neutrophils were harvested and polarized into N0, N1, or N2 phenotypes, after which their expression of CTSD and CTSG was analyzed.
Bioinformatics analysis revealed distinct expression patterns of CTSD and CTSG in the fracture healing cascade in one earlier rodent study. In human, 58 FHs (0-19 days post-trauma) were harvested. The expression of CTSD significantly increased over fracture healing time, while the expression of CTSG remained constant throughout the early phases of fracture healing. Both proteins were found to be expressed throughout the FH. In neutrophils from five human donors, the expression of CTSD was higher in N2 neutrophils compared to N1, while CTSG was expressed more in N1 compared to N2 neutrophils.
This study was the first to investigate the association of CTSD and CTSG in the fracture healing cascade. It was shown that the expression of CTSD enzyme was associated with early fracture healing phases, as well as with specific neutrophil phenotypes (N1 or N2). Furthermore, these expression dynamics of CTSD and CTSG support the increasing N2/N1 phenotype ratio over time during fracture healing in humans, reflecting a shift from inflammation to regeneration.
This study determined to investigate the cathepsin D and G expression in human fracture healing after reviewing the latest research progress. Along with characterizing the dynamics of these cathepsins in fracture hematoma, we demonstrate their association with two distinct neutrophil phenotypes, N1 and N2. These findings enhance the understanding of cathepsins and the roles of N1 and N2 neutrophils in fracture healing, providing a theoretical foundation for developing future therapies and biomaterials.
免疫细胞，尤其是中性粒细胞，在骨折愈合级联反应中的作用和参与情况尚未完全明确。在组织再生过程中，组织蛋白酶D（CTS D）和组织蛋白酶G已被确定参与各种细胞过程并与中性粒细胞功能相关。本研究旨在确定这两种组织蛋白酶在骨折血肿中的表达情况。首先，采用生物信息学方法对这两种组织蛋白酶进行鉴定和分析。其次，研究这两种组织蛋白酶在人骨折血肿（FH）以及N1（促炎）和N2（再生）中性粒细胞表型中的表达情况。
为了回顾关于CTSD和G基因在骨折愈合中表达的最新研究，首先进行了生物信息学分析。随后，为了鉴定CTSD和G基因，评估它们在骨折愈合级联反应不同阶段的人FH样本中的表达情况，并探讨与患者特征的潜在相关性。为了确认基因表达转化为蛋白质产生，通过免疫荧光评估它们在FH中的相应蛋白质水平。最后，收集人中性粒细胞并将其极化为N0、N1或N2表型，然后分析它们的CTSD和CTSG表达情况。
生物信息学分析揭示了在一项早期啮齿动物研究中CTSD和CTSG在骨折愈合级联反应中的不同表达模式。在人类中，收集了58例FH（创伤后0 - 19天）。CTSD的表达在骨折愈合过程中显著增加，而CTSG的表达在骨折愈合的早期阶段保持恒定。两种蛋白质在整个FH中均有表达。在来自五名人类供体的中性粒细胞中，与N1相比，N2中性粒细胞中CTSD的表达更高，而与N2中性粒细胞相比，N1中CTSG的表达更多。
本研究首次探讨了CTSD和CTSG在骨折愈合级联反应中的关联。结果表明，CTSD酶的表达与骨折愈合的早期阶段以及特定的中性粒细胞表型（N1或N2）相关。此外，CTSD和CTSG的这些表达动态支持了人类骨折愈合过程中N2/N1表型比例随时间增加，反映了从炎症到再生的转变。
本研究在回顾最新研究进展后，决定研究组织蛋白酶D和G在人类骨折愈合中的表达。除了表征这些组织蛋白酶在骨折血肿中的动态变化外，我们还证明了它们与两种不同的中性粒细胞表型N1和N2的关联。这些发现加深了对组织蛋白酶以及N1和N2中性粒细胞在骨折愈合中作用的理解，为未来开发治疗方法和生物材料提供了理论基础。
