The masquelet technique, also called an induced membrane technique (IMT), was first introduced to treat critical size bone defect up to 25 cm in 1986 by Pr. Alain Charles Masquelet et al. IMT is a two-stage surgical operation that recently provides a more popular option for the treatment of critical size bone defect as well as fracture nonunion. Basically, the first stage of IMT comprises the implantation of a polymethylmethacrylate (PMMA) cement spacer loaded with antibiotics into defect site. During 6 to 8 weeks, this spacer induces the formation and maturation of a thin layer called induced membrane surrounding it via a foreign body immune response. In stage II, the spacer is carefully removed while keeping induced membrane at the fracture site, autologous bone graft is then performed to provoke bone healing. IMT endows several beneficial effects including the relatively simple management versus vascularized fibular grafts and no significant difference in healing time among various defect sizes. Of note, multiple surgeries and long recovery time are the difficulties still needed to be conquered in patients treated with IMT. In view of the fact that induced membrane has been considered as an indispensable component for successful union, a more detailed and comprehensive analysis of it will improve the process of IMT to ameliorate those difficulties.

The clinical study design will follow the description in Chang Gung Medical Foundation Institutional Review Board, which will be begun in the first year until reaching 15 patients for each group. Briefly, patients of open fracture will be randomly divided into two groups to reduce selection bios, comprising spacer (with induced membrane) and without spacer groups (without induced membrane). In a without spacer group (No operation of stage I), soft tissues near fracture sites with a size of 1 cm2 will be collected to be used as an appropriate control of induced membrane (1 cm2) from the spacer group (Stage I + II) at the time point of stage II of IMT. Tissues will be dissected into three parts for proteomics, metabolomics, and paraffinized section, stored at minus 80 degrees prior to experiments. The section is imperative in this project to characterize type H vessel formation and the capacity of bone healing. For this purpose, immunohistochemistry (IHC) or Immunofluorescence (IF) will be performed to measure identified proteins from proteomics results in both of soft tissues and induced membrane. The characterized markers comprising cluster of differentiation 31 (CD31) and Endomucin (Emcn) will be used to detect type H vessels. Colorimetric methods including Masson's trichrome, Alcian blue, Alizarin red S, H\&E and Safranin O-Fast green can also be performed to unveil histological insights of induced membrane. In the multi-omics, tissues will be used to unveil novel targets, which will then be compared to the levels of vascular endothelial growth factor (VEGF) and osteocalcin. Furthermore, western blotting and real-time reverse transcriptase-Polymerase Chain Reaction (RT-PCR) will be performed to validate downstream regulation of the identified metabolites.

The venous blood (3ml in vacutainers with heparin) will be collected from both groups at the Department of Orthopedic Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan. The protocol was based on a report identifying VEGF as a potential biomarker of the union in IMT. After drawing, blood will be centrifuged (1000RPM, 10min at room temperature) for 30 minutes and stored at minus 80 degrees until analysis. Blood samples from the time point of collecting induced membrane are prepared for multi-omics analysis to identify systemic markers for type H vessel or induced membrane, and VEGF and osteocalcin will be used as serum indicators for bone mass. Blood samples from all time points will be used to identify a novel biomarker for type H vessels and bone healing. As for metabolite targets, targeted methods of metabolomics will be employed to verify them in blood. The time points for collecting blood samples of stage I will be the day of surgery, the next day of surgery, and 1, 2, and 4 weeks after surgery. As for stage II, the time points will be the day of surgery, the next day of surgery, and 1, 2, 12, and 14 weeks after surgery.