Mechanisms of Integrin α2β1 driven cell-matrix interaction in the course of bone regeneration and fracture healing

• Applicant: Professor Dr. Richard Stange
• Subject Area: Orthopaedics, Traumatology, Reconstructive Surgery
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437825722

• Final Report Year: 2024

Final Report Abstract

The project aimed to characterize the role of integrin α2β1 and its influence on cell-matrix interactions and matrix formation during fracture healing. It investigated the mechanism behind the increased collagen formation and deposition, as well as the expression of pro-osteoblastic genes when integrin-α2 expression is suppressed. Finally, the potential of inhibiting integrin α2 to improve fracture healing was explored. Characterization of the Role of Integrin α2: Studies on mice at different stages of fracture healing (7, 14, and 21 days) showed that integrin-α2-deficient animals exhibited earlier mineralization of the fracture callus. Histological analyses also revealed that both the formation and degradation of the cartilaginous callus occurred earlier in integrin-α2-deficient animals, indicating accelerated fracture healing. Mechanism of Collagen Formation and Gene Expression: It was demonstrated that the early activation of the BMP (Bone Morphogenetic Protein) signaling pathway in integrin-α2- deficient animals leads to increased extracellular matrix formation and osteoblast differentiation. These findings were reproduced in cell culture experiments. Primary osteoblasts from integrin-α2-deficient animals exhibited enhanced mineralization competence, mediated by a secreted factor identified as BMP-2, similar to that observed during fracture healing. Potential of Integrin α2 Inhibition: Attempts to inhibit integrin α2β1 using the specific snake venom rhodocetin were unsuccessful. MC3T3 cells were found to be unsuitable due to low integrin α2 expression and lack of significant BMP-2 activation. Alternatives, such as the integrin-α2-blocking antibody AB1950Z, resulted in off-target effects. The investigation of different carrier substances (agarose, collagen, Matrigel, blood coagulate) for drug delivery showed that none achieved sufficiently delayed release kinetics. A planned osteotomy could not be carried out due to organizational and personnel constraints. Results and Discussion: The project confirmed the hypothesis that integrin-α2 deficiency promotes fracture healing. Further research is required to fully understand the mechanistic relationship, which could be achieved through unbiased analytical approaches such as proteomics or single-cell sequencing. siRNA technology for the inhibition of integrin α2 showed promising results and will be pursued as a potential therapeutic approach. A clinical study on the role of integrin α2 in human fracture healing has begun, aiming to determine whether integrin α2 can serve as a prognostic marker for poor fracture healing.

Publications

• Einfluss von kollagenbindenden Integrinen auf den Frakturheilungsprozess an der α2-defizienten Maus (2019) Fachbereich Medizin, Universität Münster
  Louisa Wendler
  
• MuSkTYR. Integrin α2β1 affects fracture healing by elevated collagen expression and impaired bone remodeling. (2019)
  Melanie Brand, Daniel Kronenberg, Jens Everding, Louisa Wendler, Beate Eckes & Richard Stange
  
• Bone Regeneration: A Novel Osteoinductive Function of Spongostan by the Interplay between Its Nano- and Microtopography. Cells, 9(3), 654.
  Vordemvenne, Thomas; Wähnert, Dirk; Koettnitz, Julian; Merten, Madlen; Fokin, Nadine; Becker, Andreas; Büker, Björn; Vogel, Asaria; Kronenberg, Daniel; Stange, Richard; Wittenberg, Günther; Greiner, Johannes FW; Hütten, Andreas; Kaltschmidt, Christian & Kaltschmidt, Barbara
  
• Europaen calcified tissue society congress 2020. The integrin ⍺2β1-dependent collagen upregulation is linked to the TGF-β superfamily
  Melanie Brand, Daniel Kronenberg, Jens Everding, Beate Eckes & Richard Stange
  
• MuSkTYR. Integrin α2β1 influences bone remodeling by regulating osteoblastogenesis via BMP signaling. (2021)
  Melanie Brand, Daniel Kronenberg, Jens Everding, Louisa Wendler & Richard Stange
  
• MuSkTYR. Mechanical stress leads to morphology change of cells (2021)
  Lena Schemmelmann, Melanie Brand, Daniel Kronenberg & Richard Stange
  
• Spongostan™ Leads to Increased Regeneration of a Rat Calvarial Critical Size Defect Compared to NanoBone® and Actifuse. Materials, 14(8), 1961.
  Wähnert, Dirk; Koettnitz, Julian; Merten, Madlen; Kronenberg, Daniel; Stange, Richard; Greiner, Johannes F. W.; Kaltschmidt, Christian; Vordemvenne, Thomas & Kaltschmidt, Barbara
  
• The small-molecule protein ligand interface stabiliser E7820 induces differential cell line specific responses of integrin α2 expression. BMC Cancer, 21(1).
  Hülskamp, Michael David; Kronenberg, Daniel & Stange, Richard
  
• The small-molecule protein ligand interface stabilizer E7820 induces differential cell line specific responses of integrin a2 expression (2021) Fachbereich Medizin, Universität Münster
  Michael D. Hülskamp
  
• Deutscher Kongress für Orthopädie und Unfallchirurgie 2022. Targeting integrin α2 expression in osteoblastic cells to improve fracture healing
  Michael D. Hülskamp, Daniel Kronenberg & Richard Stange
  
• Human Sex Matters: Y-Linked Lysine Demethylase 5D Drives Accelerated Male Craniofacial Osteogenic Differentiation. Cells, 11(5), 823.
  Merten, Madlen; Greiner, Johannes F. W.; Niemann, Tarek; Grosse, Venhaus Meike; Kronenberg, Daniel; Stange, Richard; Wähnert, Dirk; Kaltschmidt, Christian; Vordemvenne, Thomas & Kaltschmidt, Barbara
  
• The role of the collagen-binding integrin α2β1 in bone metabolism and fracture healing (2022) Fachbereich Biologie, Universität Münster
  Melanie Brand
  
• Integrin α2β1 deficiency enhances osteogenesis via BMP-2 signaling for accelerated fracture repair. Bone, 190, 117318.
  Kronenberg, Daniel; Brand, Melanie; Everding, Jens; Wendler, Louisa; Kieselhorst, Eric; Timmen, Melanie; Hülskamp, Michael D. & Stange, Richard