Effect of changes in serum levels of endogenous hydrogen sulfide on fracture healing

Abstract Background: Fracture is a common disease; many factors affect fracture healing. Recent studies have confirmed that hydrogen sulfide (H2S) plays an essential role in bone formation, but most of these studies are drawing conclusions based on animal experiment; whether H2S could promote fracture healing in patients is still unclear. We aim to investigate the change of serum H2S in fracture patients, and analyze its effort on fracture healing. Methods: This is a single-center, prospective cohort study. Patients with spinal or limb fracture will be recruited. Patient's serum and urine will be collected at baseline for examination (serum H2S, β-CTX, OC, PINP, 25-OH-VitD3, S-CTX, urinary calcium, and urinary creatinine). All patients will be followed-up for 24 months in outpatients settings, the image of X-ray or CT will be reviewed and fracture healing will be judged by 2 experienced orthopedic physicians. The difference in serum parameters especially H2S will be compared between patients with fracture healed within 9 months and those with fracture unhealed at 9 months. Discussion: Results of the trial could provide insight into influence of H2S on fracture healing. Ethics and dissemination: The study was approved by the ethics committee of School of Medicine UESTC & Sichuan Provincial People's Hospital Ethics Committee. All the participants will be asked to provide written informed consent before data collection. The findings of the study will be published in peer-reviewed journals and will be presented at national or international conferences.


Introduction
Fracture is common in clinical practice, healing without deformity and restoring limb function as soon as possible is the goal of fracture treatment. To date, the detailed pathophysi-ological mechanism of fracture healing has not been fully clarified. Previous research elucidated that the process mainly depends on the action of osteoblasts and osteoclasts, and is regulated by many cytokines, such as bone morphogenetic protein-2 (BMP-2), transforming growth factor b (TGF-b) and fibroblast growth factor (FGF). [1,2] Hydrogen sulfide (H 2 S) is a gastrotransmitter and plays important regulatory roles in cardiovascular, gastrointestinal, and neurological diseases. [3,4] Cystathionine b synthase (CBS), cystathionine c lyase (CSE) or 3-mercaptopyruvate sulfurtransferase are key enzymes generating H 2 S as L-cysteine as a substrate. [5] In recent years, many studies have confirmed H 2 S plays an essential role in bone metabolism. H 2 S decreases osteoclast differentiation induced by nicotine or lipopolysaccharide [6] and osteoclast progenitor differentiation, [7] which decreases necrotic bone absorption during bone healing, but benefits osteoporosis. As well, H 2 S decreases matrix metalloproteinase activity in bone matrix, [8] thereby accelerating matrix mineralization. Finally, H 2 S promotes bone marrow mesenchymal stem cells mineralization by Wnt signals. [9] and reduces MC3T3-E1 preosteoblast cell injury induced by H 2 O 2.
[10] Our work found that CSE-H 2 S sulfhydrated Runtrelated transcript factor 2 (RUNX2) enhanced its transactivation and increased osteoblast differentiation and maturation, thereby promoting rat bone healing. [11] However, the above studies are based on animals or cultured cells, whether H 2 S plays similar role in patients with fracture is still unclear.

Trial registration number: ChiCTR1900026045
This work was supported by the Science and Technology Plan of Sichuan Province (no. 19ZDYF2615). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors report no conflicts of interest.

Study Protocol Clinical Trial
Medicine ®

OPEN
In this study, we aimed to investigate the change of serum H 2 S in fracture patients, and analyze its effort on fracture healing.

Study design
This is a single center, prospective, cohort study at the Sichuan Provincial People's Hospital, which is a referral hospital for trauma and has case volume necessary for the study. Trial is registered at Chinese Clinical Trial Registry (ChiCTR1900026045). This study included fracture group and control group, and is a 24-month study, which started in July 2019 and will be ended in June 2021.
The primary outcome is concentrations of serum H 2 S. The secondary outcomes are bone turnover markers [12,13] (including serum b-C-terminal telopeptide of type I collagen (b-CTX), procollagen type I N-terminal propeptide (PINP), osteocalcin (OC), serum C-terminal telopeptide of type I collagen (S-CTX),25-OH-VitD 3 and urinary calcium/creatinine) and time between surgery and healing of the fracture. These indexes will be measured at specific time points, as described in Figure 1.

Participants 2.2.1. Inclusion criteria.
Patients with spinal or limb fracture will be recruited. Patients with fracture occurred within 3 weeks will be included, and the fracture should be confirmed by X-ray or CT. All patients (1000 patients) will be asked to provide written informed consent.

Exclusion criteria.
Patients with wound infection or serious injury requiring amputation will be excluded. Patients with a predicted survival time of less than 9 months because of other serious illness will also be excluded. Patients with pregnancy, psychological disturbance that affects outcome evaluation, or cognitive impairment will also be excluded.

Recruitment
Participants will be recruited from inpatient or outpatient settings in Sichuan Provincial People's Hospital. We will also recruit patients through advertise posters or local media. Patients who meet the inclusion criteria will be asked to provide written inform consent, and baseline evaluation including basic information collection, measurement of bone metabolic markers, measurement of serum H 2 S will be performed subsequently.
2.4. Data collection 2.4.1. Basic information. Basic information such as patient age, gender, and fracture site will be extracted from the hospital medical record system. We will record the basic information of the included participants, which include age, gender, location of facture, smoking, frequency and mode of exercise, accompanied disease, long-term medication (name of medication, dose, and frequency of administration), and other conditions that related to bone fracture.

Measurement of serum H 2 S.
We will use modified methylene blue method for measurement of H 2 S level. [14] We will add Zn 2+ to serum sample to deposit H 2 S, HS À , S 2À , and plasma protein; NaOH will then be used to re-dissolve plasma protein.

Fracture group
Control group Measurement of serum H 2 S and biochemical markers of bone turnover.
Measurement of serum H 2 S and biochemical markers of bone turnover, review X-ray or CT in the orthopedic clinic every month aŌer operaƟon unƟl fracture heal. ZnS deposition was re-dissolved by the addition of N, Ndimethyl-p-phenylenediamine, and the remnant protein was deposited by trichloroacetic acid. After centrifugation, ferriammonium sulfate was added to the supernatant fluid to generate methylene blue, which was analyzed by spectrophotometer at 665 nm.

Outcomes
Standard for fracture healing: (1) No pain in the fracture part and no pain along the longitudinal axis. (2) Raise the limb without discomfort.
(3) Use proper force to reverse the affected limb and no abnormal activity in the fracture. (4) The injured limb can meet the following requirements to lift 1 kg weight to 1 minute; The lower extremity can walk 3 minutes without holding on the ground, not less than 30 steps. Continuous observation of 2 weeks of fracture without deformation. (5) X-ray shows the fracture line blurred, with continuous callus through the fracture line. [15,16] Fracture healing was defined as being up to the all above standards.
Fracture healing is judged by 2 experienced orthopedic physicians, which usually takes 4 to 8 months after reduction and fixation, and if not heal for more than 9 months, it is considered delayed or non-union. [17] 2.6. Planned statistical analysis The analysis will be conducted by Statistical Package for the Social Sciences (SPSS) Statistics software, version 19.0 (IBM Corporation, Armonk, NY). Continuous data will be presented as the mean ± standard deviation (X±S), while categorical data will be presented as raw numbers and frequencies. Kolmogorov-Smirnov and Shapiro-Wilk tests will be used to test whether data distribution is normal. Data with normal distribution will be analyzed by Student t test. Non-parametric tests Mann-Whitney U test will be used to assess the difference between patients with healed fracture and those without when data distribution is skewed. Logistic regression model will be developed with whether fracture is healed at 9 months as dependent variable, with level of H 2 S as independent variable, and with age, gender, baseline characteristics, and other parameters as covariates. P < .05 is considered statistical significance.

Discussion and practical implications
Increasing data suggest that H 2 S plays an essential role in bone metabolism. Our previous study found that overexpressed CSE promoted bone fracture healing through increasing collagen secretion, promoting endochondral ossification, and building a full bridge between fracture sites in rat femur. [11] However, whether endogenous H 2 S has the same effect on patients with fracture is still unclear. Therefore, we design an observational study to investigate the relationship between serum H 2 S concentration and bone fracture healing.
The study is an important and innovative prospective cohort study that will provide insight into influence of H 2 S on fracture healing, which, to the best of our knowledge, is the first study in human. The findings may have important implications for therapeutic strategies targeted at promoting healing of fractures.

Ethics and dissemination
The study was approved by the ethics committee of School of Medicine UESTC & Sichuan Provincial People's Hospital Ethics Committee. All the participants gave written informed consent before data collection. The findings of the study will be published in peer-reviewed journals and will be presented at national or international conferences.