Noninvasive assessment of the repair process in diabetic wound healing using multimodal imaging techniques

Noninvasive assessment of the repair process in diabetic wound healing using multimodal imaging techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Noninvasive assessment of the repair process in diabetic wound healing using multimodal imaging techniques Yaling Li, Hongjiu Zhang, Yongliang Ren, Heng Wang, Genmao Cao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8616724/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract Background The wound healing process in diabetic patients is complex and often accompanied by risks of infection and ischemia. Currently, clinical assessment mainly relies on visual examination and surface measurement, which have limitations such as strong subjectivity and the inability to conduct in-depth evaluations. In this study, we established an integrated multimodal non-invasive imaging platform to accurately evaluate the healing process of diabetic wounds and quantitatively assess the therapeutic potential of Zn²⁺-based treatment methods in promoting microcirculation reconstruction. Methods We implemented a novel technical platform combining laser speckle contrast imaging (LSCI), second near-infrared region (NIR-II) imaging, and optical coherence tomography angiography (OCTA) to conduct longitudinal, high-resolution imaging of full-thickness skin wound healing in both normal and db/db diabetic mouse models. The platform enabled dynamic monitoring of vascular and structural changes throughout the healing process. Results The multimodal imaging approach successfully provided comprehensive quantitative data: LSCI revealed real-time dynamic changes in cutaneous blood perfusion, NIR-II imaging delineated the spatial-temporal evolution of vascular network structures, while OCTA offered detailed characterization of internal wound microarchitecture and microvascular patterns. This integrated methodology permitted both qualitative and quantitative assessment of wound repair capacity with unprecedented resolution. Conclusions The developed imaging platform represents a significant advancement for predicting wound healing outcomes and evaluating treatment strategies for diabetic wounds. It provides a powerful tool for assessing the efficacy of novel drugs and shows potential for guiding optimal intervention timing in clinical settings. wound healing diabetes mellitus laser speckle contrast imaging (LSCI) second near-infrared region (NIR-II) imaging optical coherence tomography angiography (OCTA) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Skin wounds from burns, surgery, and chronic ulcers can be life-threatening. Diabetes mellitus (DM), characterized by hyperglycemia, leads to skin wounds in 19%-34% of patients[ 1 ]. Hyperglycemia-induced pathological changes, such as peripheral vascular disease and neuropathy, significantly impair wound healing[ 2 ], resulting in slow repair and increased infection risk[ 3 ]. Therefore, timely and accurate assessment of skin wounds is crucial for formulating effective and targeted treatment strategies. Wound healing involves hemostasis, inflammation, proliferation, and remodeling[ 4 , 5 ]. Notably, the cutaneous vasculature plays a crucial regulatory role in these phases, influencing both the process and outcome of wound healing[ 6 ]. Therefore, identifying and quantifying changes in cutaneous blood vessels is fundamental for assessing the status of wound healing and optimizing wound care management. Currently, the assessment of wound healing mainly relies on clinical techniques, including visual inspection and surface measurement, such as monitoring wound size, color, odor, exudate characteristics, and scab formation[ 7 ]. However, this method is limited to the skin surface and is largely influenced by the subjective experience of medical staff[ 8 ]. Although histological analysis of biopsy tissue is the gold standard for assessing wound healing, it is an invasive procedure that causes secondary damage and increases the risk of infection[ 9 ]. Therefore, researchers have developed a variety of non-invasive detection techniques to assist in assessing wound healing, including magnetic resonance imaging (MRI), ultrasound imaging, polarization-sensitive optical coherence tomography (PS-OCT), and laser Doppler flowmetry (LDF)[ 10 – 13 ]. The above non-invasive techniques have limitations in terms of cost, operator dependence, and imaging depth. There is an urgent need to develop better non-invasive assessment solutions. In vivo tissue imaging is crucial for directly observing the dynamic changes in the complex wound repair process (such as skin angiogenesis). Laser speckle contrast imaging (LSCI) is an innovative optical imaging technique that can quantitatively and non-invasively evaluate the movement of red blood cells in the dermal microvasculature[ 14 , 15 ]. LSCI has the advantages of short imaging time, high resolution, wide imaging range, non-invasiveness, and non-contact operation. It can be used to evaluate blood flow and perfusion dynamics, which helps predict the healing rate[ 16 ]. Near-infrared region (NIR) fluorescence imaging has the advantages of being non-radioactive and non-invasive, and has become a promising technique for monitoring blood perfusion. In the second near-infrared region (1000-1700nm, NIR-II), due to the significantly improved contrast between the pathological area and the background, spatial resolution, and penetration depth, non-invasive imaging of angiogenesis can be achieved[ 17 ]. Continuous monitoring and evaluation of wound healing through NIR-II fluorescence imaging may provide valuable information for patients' prognosis and care. Optical coherence tomography angiography (OCTA) is a functional extension of the OCT imaging protocol[ 18 ]. It utilizes the hemodynamic changes caused by the movement of red blood cells to generate contrast, with the advantages of high resolution, no need for labeling, and non-invasiveness. Currently, it is widely used in clinical settings to examine vascular changes in retinopathy and the wound-healing process[ 19 , 20 ]. This method can perform three-dimensional imaging of the epidermal tissue structure and the vascular system, thus contributing to the monitoring of pathophysiological changes in skin wounds[ 21 ]. Existing studies have clearly demonstrated that zinc ions (Zn 2+ ) play a crucial role in skin wound healing. Zn²⁺ stimulates angiogenesis through vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), degrades the matrix through matrix metalloproteinases (MMPs), promotes collagen expression, and exerts anti-inflammatory effects[ 22 – 24 ]. This study constructed a multimodal imaging platform integrating LSCI, NIR-II, and OCTA. Longitudinal imaging of full-thickness skin wounds in C57BL/6 and db/db mice enabled simultaneous dynamic evaluation of vascular structure and perfusion during diabetic wound repair. It quantified vascular remodeling and perfusion evolution, providing a novel quantitative tool for efficacy evaluation and prognosis prediction with clinical translation value. 2. Materials and Methods 2.1 Animal Surgery All animal procedures were approved by the Animal Care and Use Committee of the Second Hospital of Shanxi Medical University (DW2023053). Ten 8-week-old male C57BL/6 mice and ten 8-week-old male db/db mice (Nanjing Junke Bioengineering Co., Ltd.) were anesthetized via inhalation of isoflurane (3.8%). Following confirmation of sedation levels using the toe pinch test, the dorsal hair was shaved off. All surgical instruments were sterilized, and the surgical area was scrubbed with povidone-iodine. A 6mm biopsy punch was used to create a circular wound with a diameter of 6mm in the disinfected skin of the mouse's back[ 2 ]. Post-surgery, the mice were placed on a heating pad to recover to their normal state. Upon the conclusion of the experiments, all animals were anesthetized with isoflurane (2–5%) and then euthanized by cervical dislocation, ensuring that the animals were in a painless state throughout the process. Moreover, animal care and all experimental procedures adhered to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. 2.2 In Vivo Imaging Starting from the day of surgery (Day 0), in vivo imaging of the wound area was performed using LSCI, NIR-II, and OCTA every other day for a duration of 7 days. During the imaging process, each mouse was anesthetized with 3.8% isoflurane and subsequently immobilized on a heating pad maintained at 37℃, with continuous anesthesia administered to minimize bodily movements during imaging. The imaging was conducted under controlled room temperature (23–24℃) to maintain stable blood flow in the wound area of the mice. We utilized a laser speckle contrast imager (RFLSI ZW, RWD Life Science CO, China) to quantify cutaneous blood flow perfusion. This system employs a laser wavelength of 785nm, with the laser head positioned 15cm above the skin, resulting in an image size of 2.8×2.1cm and a resolution of 3.9 µm/pixel. The image acquisition time was set at 10 seconds. After anesthesia, the mice were placed on the imaging platform and allowed to acclimatize for 5 minutes before initiating the recording of skin blood flow. In vivo wound blood flow imaging of mice was performed using the RWD laser speckle imaging system coupled with LSCI v4.0 software (RWD Life Science). We employed an NIR-II imager (NIR-II, Beijing Digital Precision Medical Technology Co., China) to assess cutaneous vascular architecture and blood flow perfusion. NIR-II fluorescence was captured by an InGaAs short-wave infrared camera (Xenics Cheetah-640CL TE3), which was equipped with a filter wheel (1000nm, 1100nm, 1200nm, 1300nm, 1400nm, 1500nm, Thorlabs, USA) positioned in front of the camera, allowing only the passage of NIR-II fluorescence. The excitation laser wavelength was set at 808nm with a power output of 5000mW, and the image acquisition time was 2 minutes. After anesthesia, the mice were placed in a darkroom and injected with 200 µg of ICG (0.1mg/ml, Dandong Pharmaceutical Factory, Liaoning, China) via the tail vein, simultaneously initiating the fluorescence imaging process. In vivo wound vascular structure and perfusion of mice were acquired using NIR-II coupled DPM-IVFM-NIR-OF software for 2 minutes. We used an optical coherence tomography angiographer (Micro-VCC, Optoprobe, UK) to measure the microcirculation structure and blood flow within the skin. This system utilizes a swept-source laser with a central wavelength of 1060 nm, featuring an A-scan acquisition rate of 200 kHz and a scanning area of 8mm×8mm. After anesthesia, the mice were placed on the imaging platform, and physiological saline was applied to the skin defect area to ensure close contact between the probe and the skin, thereby preventing the formation of air bubbles. In vivo imaging of wound microcirculation structure and blood flow was performed in mice using OCTA coupled OPTO-III scanning software. 2.3 Image processing The blood flow perfusion data obtained from LSCI were analyzed by calculating the mean perfusion units (PU) within the region of interest (ROI). We used the RFLSI-Analysis software to calculate the mean perfusion in the skin defect area. Specifically, the mean blood flow perfusion within the designated ROI (region over the skin defect) was extracted and recorded at three distinct time points, and the mean of these measurements was calculated to avoid potential temporal variability introduced by the equipment. The blood flow signals in the NIR-II window were analyzed by calculating the mean signal-to-background ratio (SBR) within the region of interest (ROI). We used the DPM-IVFM-NIR-OF software to calculate the mean SBR in the skin defect area. The mean blood flow signals within the designated ROI (region over the skin defect) were extracted and recorded at three distinct time points, and the mean of these measurements was calculated to mitigate potential temporal variability introduced by the equipment. The vascular-related parameters obtained from OCTA were analyzed by calculating the pixels within the region of interest (ROI), including: A = pixels occupied by the vascular area, S = pixels deemed as the length of the vessels, P = pixels within the perimeter of the vessel, and X = total pixels within the vessel map. Vascular parameters, including density, diameter, branching complexity, and skeleton density, were quantified from OCTA data using PyOct 8.0 software. Averag_Area = A/X -- the ratio of the total image area occupied by the vascular system with the vessel area map to the total image area, indicating the vascular density within the analyzed region. Average_Skeleton_Density = S/X -- the ratio of vessel length as represented in the skeletonized vessel map to the total image area, providing insights into the distribution and compactness of the vascular network. Average_Diameter = A/S -- the ratio of the vessel area map to the skeletonized vessel map, providing the average vessel caliber Average_Complexity = P 2 /(4π*A) -- calculated using the circumference and vessel area maps, providing the degree of branching and intricacy within the vascular structure. 2.4 Hematoxylin and eosin staining To examine alterations in the skin tissue architecture, paraffin-embedded skin tissue sections (5-µm-thick) were stained with hematoxylin and eosin (H&E). Pathologic images were obtained using an optical microscope (Pannoramic MIDI, 3DHISTECH Ltd., Hungary). 2.5 Statistical analysis Twenty mice were subjected to wound healing experiments. They were all euthanized at the study endpoint (i.e., Day 7), with in vivo data utilized for statistical analysis of vascular structural and functional parameters. Quantitative data were expressed as mean ± standard deviation (SD). Statistical comparisons between groups were conducted using either one-way ANOVA or a t-test. GraphPad Prism 10.1.0 software was utilized for statistical analysis and graph generation. A significance level of p < 0.05 was applied to determine statistical significance. 3. Results We used adult wild-type (C57BL/6) mice and type 2 diabetes (db/db) model mice to compare the healing process of their full-thickness skin wounds. A total of 20 mice were included in the experiment (10 in the C57BL/6 group and 10 in the db/db group). During the experiment, one db/db mouse that did not receive drug intervention unexpectedly died on Day 7. Therefore, we set the imaging endpoint on Day 7 after surgery. 3.1 Administration of Zn 2+ accelerates the repair process of wound healing Firstly, we established models of full-thickness skin defects (diameter: 6mm) in both C57BL/6 and db/db mice. Since the wound microenvironment is acidic and it has been demonstrated that Zn 2+ has an important role in promoting vascular angiogenesis and accelerating wound healing[ 25 , 26 ]. The treatment group received a reagent capable of releasing Zn 2+ under acidic conditions, administered every other day at a dose of 50µl (1mg/ml) post-induction of skin wounds. The structure of the Zn 2+ reagent is shown in the transmission electron microscopy (TEM) image (Figure S1 ) . Figure 1 a depicts the healing process of skin wounds from Day 1 to Day 7 in C57BL/6 and db/db mice, respectively. Visually, skin wound healing in db/db mice was significantly impaired compared to C57BL/6 mice. At day 7, the wound healing area of db/db mice was significantly smaller than that of C57BL/6 mice. After Zn 2+ administration, both C57BL/6 and db/db mice exhibited accelerated wound healing rates ( Fig. 1 b ) . Further assessment of wound healing rates based on wound size revealed that the Zn 2+ -treated groups had significantly higher healing rates compared to their respective controls ( Fig. 1 c ) . Specifically, the wound closure rates on Day 7 were 86% and 54% in the Zn 2+ -treated C57BL/6 and db/db mice, respectively, compared to 72% and 39% in their untreated counterparts (P < 0.0001, P < 0.0001), suggesting that Zn 2+ formulations can promote skin wound healing. 3.2 LSCI reveals blood flow perfusion during wound healing LSCI provides two-dimensional dynamic maps of blood flow perfusion based on fluctuations in speckle patterns induced by red blood cell motion, enabling the monitoring of hemodynamic changes throughout the wound healing process. Firstly, in vivo imaging was performed on normal C57BL/6 mice and db/db mice to obtain baseline information (Figure S2) . Interestingly, in C57BL/6 mice, LSCI not only allowed for the visualization of overall skin microcirculatory blood flow perfusion but also revealed the structure of larger vessels. In contrast, due to alterations in skin structure in db/db diabetic mice, partial reflection of laser light occurred, leading to deviations in imaging results. Therefore, for db/db mice, LSCI imaging was focused on localized wound areas. Figure 2 a shows a sequential series of LSCI images captured at days 0, 1, 3, 5, and 7 during the cutaneous wound healing process in both C57BL/6 mice and db/db mice, respectively. The type, severity, and stage of wound healing significantly impact the level of blood flow perfusion. A lower blood flow perfusion in the wound area compared to the surrounding tissues indicates the presence of eschar formation or an open epidermal injury. Conversely, a higher blood flow perfusion in the wound area compared to the surrounding tissues indicates the occurrence of wound healing. As shown in Fig. 2 a, C57BL/6 mice exhibited a notable difference in blood flow perfusion between the wound and adjacent tissues, and Zn 2+ treatment increased perfusion within the wound area. Similarly, Zn 2+ administration augments blood flow perfusion in the wound region of db/db mice. Upon further quantification of the LSCI data, it was found that the wound region of C57BL/6 mice maintained a high level of blood flow perfusion from day 0 to 3, gradually declining from day 5. However, Zn 2+ treatment sustained this elevated perfusion level until day 7 in C57BL/6 mice. The same results were observed in the wound model of db/db mice ( Fig. 2 b ) . 3.3 NIR-II imaging reveals changes in vascular structure during wound healing ICG was injected into C57BL/6 mice and db/db mice via the tail vein for NIR-II fluorescence imaging. Firstly, NIR-II imaging was performed on normal C57BL/6 mice and db/db mice to obtain baseline information (Figure S3) . NIR-II clearly showed the vascular morphology of the dorsal skin in C57BL/6 mice; db/db mice were less well imaged due to interference from adipose tissue and other factors, but the dorsal skin vascular structures could still be visualized. Figure 3 a shows a series of NIR-II pseudo-color images of the wound healing process in C57BL/6 mice and db/db mice on days 0, 1, 3, 5, and 7, respectively. During the initial 0–3 days, the vascular signal intensity in the wound area was elevated in C57BL/6 mice, with a downward trend observed in db/db mice. Compared with the control group, following Zn 2+ treatment, the vascular signal intensity in the wound area of both C57BL/6 mice and db/db mice continued to rise until day 5 and began to show a decreasing trend on day 7 ( Fig. 3 b ) . Interestingly, the NIR-II imaging revealed that wounds with higher signal intensities healed at a faster rate than those with lower signals, which is consistent with the pathologic features of wound healing. 3.4 OCTA shows structural changes in microcirculation during the wound healing process OCTA is an emerging ophthalmic imaging technology that allows rapid and non-invasive reconstruction of the three-dimensional structure of retinal and choroidal vessels. By utilizing OCTA technology, we monitored model mice with wounds to obtain the perfusion of microcirculatory structures in the wound area. Firstly, OCTA imaging was performed on the dorsal skin of normal C57BL/6 and db/db mice to obtain baseline information. Based on the skin tissue structure, the OCTA volume was segmented into three layers using the semi-automatic segmentation software integrated with the OCTA instrument, with depths divided into layer 1 (0–20µm), layer 2 (20–30µm), and layer 3 (30–100µm), respectively (Figure S4a) . Among them, layer2 had the highest vascular density and best reflected the microcirculatory structure and blood flow changes during wound healing. Therefore, layer 2 was chosen for quantitative analysis. Figure S4b shows sequential mask maps on days 0, 1, 3, 5, and 7 during skin wound healing in C57BL/6 mice and db/db mice, which provides basic information on various vascular parameters. Based on this basic information, the OCTA analysis software can analyze vascular parameters, including vascular complexity, vascular diameter, vascular skeleton density, and vascular blood flow velocity. These vascular parameters are able to quantify the vascular responses, providing a comprehensive assessment of the wound healing process. Figure 4 a is a pseudo-color map of the vascular area based on mask maps. In contrast to the uniform vascular distribution presented in the skin of normal mice, vasodilatation is evident around the wound site. As healing progresses, angiogenesis gradually migrates into the wound, forming neovascular sprouts. Compared with C57BL/6 mice, db/db mice had a significantly delayed establishment of microcirculation than C57BL/6 mice due to their hyperglycemic factors (P = 0.0012). However, the rate of angiogenesis was accelerated after Zn 2+ administration ( Fig. 4 b ) . During the healing process, vascular complexity, vessel diameter, and vascular skeleton density all increased over time, indicating enhanced microcirculation augmented blood perfusion within the wound area (Figure S5) . 3.5 Zn 2+ promotes angiogenesis and tissue repair As mentioned above, previous studies have clearly demonstrated that Zn 2+ plays a crucial role in skin wound healing. We selected the wound-area tissues of C57BL/6 mice and db/db mice from the control group and the Zn 2+ -treated group on Day 7 for hematoxylin-eosin (H&E) staining to evaluate relevant histological changes, including angiogenesis, collagen deposition, and epithelialization ( Fig. 5 ) . Compared with the control group, the Zn 2+ -treated group showed increased angiogenesis in the wound area, higher levels of collagen deposition, and epithelialization. The collagen fiber bundles in the Zn 2+ -treated group were arranged more tightly and compactly, with abundant angiogenesis and continuous epithelialization. The same results were observed in both C57BL/6 mice and db/db mice. Compared with C57BL/6 mice, db/db mice had reduced angiogenesis, collagen deposition, and epithelialization in the wound area, leading to a delayed wound healing process. 4. Discussion The wound healing process represents the body's inherent ability to restore tissue integrity after injury[ 27 , 28 ]. Wound healing in diabetic patients is usually complex, often accompanied by infection and ischemia[ 29 ]. Long-term elevated blood glucose can lead to vascular damage, insufficient blood perfusion, and prolonged wound healing time[ 30 ]. Therefore, it is particularly important to monitor vascular structure and blood perfusion around the wound site, along with wound interventions. In this study, we used LSCI, NIR-II, and OCTA multimodal imaging techniques to monitor skin wound healing in C57BL/6 mice and db/db mice, and combined with histopathologic examinations to evaluate the efficacy of these three imaging techniques for assessing the wound healing process. Through continuous monitoring of skin wounds in both C57BL/6 and db/db mice over 7 days, we observed that the wound healing rate of db/db mice significantly lagged behind that of C57BL/6 mice. Furthermore, Zn 2+ -treatment promoted angiogenesis and accelerated the wound healing process. Laser speckle contrast imaging (LSCI) is a high-resolution and high-contrast optical imaging technique commonly employed to characterize hemodynamic variations in short-term physiological experiments[ 31 ]. LSCI is commonly used in neurophysiological studies to dynamically image alterations in cerebral blood flow[ 32 ]. In recent years, there have also been related studies using LSCI to study skin and wound perfusion[ 33 , 34 ]. LSCI can provide noninvasive real-time feedback on changes in perfusion and can monitor the microcirculation in the outer layer of the skin[ 35 ]. In this study, we used an RFLSI ZW laser speckle blood flow imaging system designed based on LSCI technology with higher resolution to monitor and present the microcirculation blood flow perfusion distributions within living tissues in real time. Our findings showed that LSCI was able to monitor hemodynamic changes during wound healing, with quantification revealing the perfusion information of the wound area’s microcirculation. Notably, blood flow perfusion in the wound area increased from day 0 to day 3, and Zn 2+ treatment was observed to prolong this period of elevated perfusion, thereby facilitating wound healing. The second near-infrared region (NIR-II, 900–1800 nm) imaging has emerged as a novel optical imaging modality with promising clinical application prospects in recent years[ 2 ]. It enables the visualization of clinically occult lesions and surrounding important structures with higher sensitivity and resolution, and has already achieved applications in some clinical fields, such as hepatectomy and pancreatic tumor resection, etc[ 36 – 38 ]. Indocyanine green (ICG), the only cyanine dye approved by the Food and Drug Administration (FDA) in the United States, has demonstrated robust NIR-II imaging outcomes in both patients and small animals[ 39 ]. By injecting ICG via the tail vein in mice and performing NIR-II imaging, we were able to comprehensively visualize the vascular structural changes within the wound area, thus reflecting the ability of wound healing. Surprisingly, we found that NIR-II signal values correlated with healing outcomes in our mouse model, with high signals suggesting good healing outcomes. This discovery has the potential to provide valuable insights for selecting optimal intervention timings in clinical wound management. Optical coherence tomography angiography (OCTA) is a relatively new, non-invasive technique that provides high-resolution images of retinal and choroidal vascularization[ 15 , 40 ]. It has been reported in healthy subjects, in patients with different ocular and systemic diseases, as well as in diverse animal models[ 21 , 41 ]. OCTA, which operates without the need for contrast agents, can show perfusion of microcirculatory tissues, which is useful for studying diseases in which microvascular morphology and perfusion change over time[ 42 , 43 ]. By monitoring changes in microcirculatory structure and perfusion during wound healing using OCTA, we found that neovascular sprouts gradually migrated inward over time. Furthermore, OCTA provides many vascular parameters during wound healing, including vascular area density, vascular diameter, and vascular complexity, suggesting its potential as a future indicator for wound assessment. In this experiment, it was originally planned to conduct longer-term observations and multimodal imaging. However, a db/db mouse that did not receive drug intervention unexpectedly died on Day 7, resulting in the premature termination of the observation of this individual. This unexpected event limited the data integrity of this study. In subsequent studies, a longer observation period and more comprehensive imaging strategies will be employed to address this deficiency. Zn 2+ plays a central role in enzymatic reactions, collagen synthesis, and antibacterial activities, and its toxicity is reduced, and the healing effect is remarkable after being compounded with polymers[ 44 ]. It should be noted that, as it was not the research objective of this study, the molecular mechanism by which Zn 2+ promotes wound healing was not explored. Future research can focus on the regulatory role of Zn 2+ in the diabetic wound microenvironment. In addition, the operating procedures of the multimodal imaging techniques involved in this study need to be further standardized, which is crucial for the translation of research findings into clinical applications. The multimodal imaging system integrating LSCI, NIR-II, and OCTA enables non-destructive visualization of tissue microstructure and microvasculature in murine skin, facilitating longitudinal monitoring of anatomical and vascular dynamics during wound healing. By synergistically complementing information across modalities, this approach provides a comprehensive perspective on healing progression and deepens mechanistic understanding of the process. We anticipate that this integrated platform will significantly advance wound intervention strategies and accelerate the development of novel therapeutics. 5. Conclusion The developed imaging platform represents a significant advancement for predicting wound healing outcomes and evaluating treatment strategies for diabetic wounds. It provides a powerful tool for assessing the efficacy of novel drugs and shows potential for guiding optimal intervention timing in clinical settings. Abbreviations DM: Diabetes Mellitus LSCI: Laser Speckle Contrast Imaging NIR-II: Second Near-infrared Region Imaging OCTA: Optical Coherence Tomography Angiography MRI: Magnetic Resonance Imaging PS-OCT: Polarization-Sensitive Optical Coherence Tomography LDF: Laser Doppler Flowmetry ICG: Indocyanine Green Declarations Ethics approval and consent to participate All animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH) and approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (Approval No. DW2023053). All measures were aimed at minimizing animal suffering and discomfort, including the use of appropriate anesthesia and analgesia during surgery, as well as providing a standard-compliant feeding environment and postoperative care. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work received the support from the Regional Cooperation Program of Shanxi Province, China (Grant No. 202204041101038); The Leading Talent Team Building Program of Shanxi Province, China (Grant No. 202204051002010); Construction and Demonstration of Molecular Diagnosis and Treatment Platform for Vascular Diseases in Shanxi Province, China (Grant No. SCP-2023-17); Translational Medicine Engineering Research Center for Vascular Diseases of Shanxi Province, China (Grant No. 2022017); Central government-guided local project, China (Grant No. YDZJSX2021C026); General Project of National Natural Science Foundation of China (Grant No. 81770695 and 81870354); Graduate Practical Innovation Project of the Education Department of Shanxi Province, China (Grant No.2023SJ145); Graduate Student Academic Innovation Project of the Education Department of Shanxi Province, China (Grant No. 2025XS309). Authors' contributions Y.L., H.Z., and Y.R. contributed equally to this study. Y.L., H.Z., and Y.R. were responsible for the conception and design of the study, the acquisition, analysis, and interpretation of data, and the drafting of the manuscript. H.W. and G.C. contributed significantly to the acquisition and analysis of data. K.F. and C.L. provided overall guidance on the project, including the design and execution of the experiments. Y.Z. and J.H. were responsible for the statistical analysis of the data and for ensuring the accuracy and consistency of the results presented in the manuscript. H.D. oversaw the entire project, including the design, execution, and analysis of the study. He was also responsible for the final approval of the version. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable. References Lei, H. and D. 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Bioact Mater, 2024. 37 : p. 14-29. Tang, X., et al., Metallic elements combine with herbal compounds upload in microneedles to promote wound healing: a review. Front Bioeng Biotechnol, 2023. 11 : p. 1283771. Yang, F., et al., Sustained release of magnesium and zinc ions synergistically accelerates wound healing. Bioact Mater, 2023. 26 : p. 88-101. Han, Z., et al., Zn(2+)-Loaded adhesive bacterial cellulose hydrogel with angiogenic and antibacterial abilities for accelerating wound healing. Burns Trauma, 2023. 11 : p. tkac048. Kirsner, R.S. and W.H. Eaglstein, The wound healing process. Dermatol Clin, 1993. 11 (4): p. 629-40. Rodrigues, M., et al., Wound Healing: A Cellular Perspective. Physiol Rev, 2019. 99 (1): p. 665-706. Bai, Q., et al., Potential Applications of Nanomaterials and Technology for Diabetic Wound Healing. Int J Nanomedicine, 2020. 15 : p. 9717-9743. Jiang, P., et al., Current status and progress in research on dressing management for diabetic foot ulcer. Front Endocrinol (Lausanne), 2023. 14 : p. 1221705. Rege, A., et al., In vivo laser speckle imaging reveals microvascular remodeling and hemodynamic changes during wound healing angiogenesis. Angiogenesis, 2012. 15 (1): p. 87-98. Dunn, A.K., et al., Dynamic imaging of cerebral blood flow using laser speckle. J Cereb Blood Flow Metab, 2001. 21 (3): p. 195-201. Couturier, A., et al., Reproducibility of high-resolution laser speckle contrast imaging to assess cutaneous microcirculation for wound healing monitoring in mice. Microvasc Res, 2022. 141 : p. 104319. Hsieh, M.C., et al., Improvement of clinical wound microcirculation diagnosis using an object tracking-based laser speckle contrast imaging system. APL Bioeng, 2024. 8 (1): p. 016105. Mennes, O.A., et al., The Association between Foot and Ulcer Microcirculation Measured with Laser Speckle Contrast Imaging and Healing of Diabetic Foot Ulcers. J Clin Med, 2021. 10 (17). Chen, K., et al., Rising sun or strangled in the cradle ？A narrative review of near-infrared fluorescence imaging-guided surgery for pancreatic tumors. Int J Surg, 2024. Zhang, Z., et al., NIR-II fluorescence image-guided surgery prolongs the relapse-free survival of hepatocellular carcinoma patients. HPB (Oxford), 2024. 26 (7): p. 963-966. Cai, Z., et al., NIR-II fluorescence microscopic imaging of cortical vasculature in non-human primates. Theranostics, 2020. 10 (9): p. 4265-4276. Reinhart, M.B., et al., Indocyanine Green: Historical Context, Current Applications, and Future Considerations. Surg Innov, 2016. 23 (2): p. 166-75. Alnawaiseh, M., et al., Feasibility of optical coherence tomography angiography to assess changes in retinal microcirculation in ovine haemorrhagic shock. Crit Care, 2018. 22 (1): p. 138. Gao, Y., et al., Assessment of alterations in the retina and vitreous in pre- and post-COVID-19 patients using swept-source optical coherence tomography and angiography: A comparative study. J Med Virol, 2023. 95 (10): p. e29168. Deegan, A.J., et al., Optical coherence tomography angiography monitors human cutaneous wound healing over time. Quant Imaging Med Surg, 2018. 8 (2): p. 135-150. Yousefi, S., et al., Assessment of microcirculation dynamics during cutaneous wound healing phases in vivo using optical microangiography. J Biomed Opt, 2014. 19 (7): p. 76015. Sun, X., et al., Construction of pH-Sensitive Multifunctional Hydrogel with Synergistic Anti-Inflammatory Effect for Treatment of Diabetic Wounds. Pharmaceutics, 2025. 17 (5). Additional Declarations No competing interests reported. Supplementary Files supplementarymaterials.docx Supplementary description Supplementary Figure 1. The transmission electron microscopy (TEM) image of Zn 2+ reagent. (Scale bar: 50nm) Supplementary Figure 2. Gray-scale images and pseudo-color images of LSCI in normal C57BL/6 mice and db/db mice. Supplementary Figure 3. Pseudo-color images of NIR-II imaging in normal C57BL/6 mice and db/db mice. Supplementary Figure 4. (a) Representative all layers, layer1 (0-20μm), layer2 (20-30μm), and layer3 (30-100μm) maps of OCTA in normal C57BL/6 mice and db/db mice. (b) Representative mask maps of OCTA of C57BL/6 and db/db mouse wall-healing models on days 0, 1, 3, 5, and 7. 4b. Relationship between blood vessel average area of wound area and time in different treatment groups. Supplementary Figure 5. Relationship between blood vessel average complexity, average diameter, and average skeleton density of wound area and time in different treatment groups (n=5). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, indicating statistically significant data between groups; ns indicates no statistical significance) Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 Apr, 2026 Reviews received at journal 13 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviews received at journal 26 Feb, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviews received at journal 24 Feb, 2026 Reviewers agreed at journal 20 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers invited by journal 17 Feb, 2026 Editor invited by journal 27 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 20 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8616724","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594039675,"identity":"329517b0-eac7-437f-a93b-17b453904a2c","order_by":0,"name":"Yaling Li","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yaling","middleName":"","lastName":"Li","suffix":""},{"id":594039676,"identity":"7561725b-d978-4a70-8ad9-79c1e607d59b","order_by":1,"name":"Hongjiu Zhang","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongjiu","middleName":"","lastName":"Zhang","suffix":""},{"id":594039677,"identity":"784ea579-3a70-4ea9-a279-9f1053896d25","order_by":2,"name":"Yongliang Ren","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongliang","middleName":"","lastName":"Ren","suffix":""},{"id":594039678,"identity":"5fbbeda7-ac28-4076-8a7f-30ae02e6bc13","order_by":3,"name":"Heng Wang","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Heng","middleName":"","lastName":"Wang","suffix":""},{"id":594039679,"identity":"e6381f02-f02f-4e33-8f3a-e3fb26aba279","order_by":4,"name":"Genmao Cao","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Genmao","middleName":"","lastName":"Cao","suffix":""},{"id":594039680,"identity":"762df470-6fc9-49cf-8b29-50a829bcd1b8","order_by":5,"name":"Keyi Fan","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Keyi","middleName":"","lastName":"Fan","suffix":""},{"id":594039681,"identity":"fd402fae-939d-440b-b8be-eb7f8ad82cb7","order_by":6,"name":"Chuanlong Lu","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chuanlong","middleName":"","lastName":"Lu","suffix":""},{"id":594039682,"identity":"7258ca36-91a7-4151-96ea-d13ea71d435a","order_by":7,"name":"Yuhang Zhang","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuhang","middleName":"","lastName":"Zhang","suffix":""},{"id":594039683,"identity":"3b2ccb12-e924-49ca-894a-9a4a9b4ccd7b","order_by":8,"name":"Jiang Han","email":"","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Han","suffix":""},{"id":594039684,"identity":"9239af02-79fb-4a08-b3fa-696e74855ff5","order_by":9,"name":"Honglin Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBACAxDxwICBsYGBgfEBsiB+LQkQLcwGyIIEtDCAtbBJEKXFnL338IuEgjuy/dLt1yp/tm1LbGBv3ibBUHMHpxbLnnNpFgkGz4xnzjlTdpvnzO3EBp5jZRIMx57hdtiNHDODBIPDiRtu5KTdZqgAapHIMZNgbDhMnJbCHwZALfJvCGoxfgDRkn6MgQdsCw8BLWfOmAED+bDxzBk5zNJAvxi38aQVWyQcw6PleI/xhw9/Dsv2S6Q//Piz7bZsP/vhjTc+1ODWwoCIDh5IdLCBiAR8GoCR/gFCsz/Ar24UjIJRMApGLAAAmj9eGVNDDMAAAAAASUVORK5CYII=","orcid":"","institution":"Second Hospital of Shanxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Honglin","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2026-01-16 08:42:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8616724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8616724/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103168041,"identity":"ab115db1-c166-4f4d-bd9a-9175521408d7","added_by":"auto","created_at":"2026-02-22 12:57:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":971525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdministration of Zn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e accelerates the repair process of wound healing.\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eRepresentative bright-field microscopy images of C57BL/6 and db/db mouse wound-healing models on days 0, 1, 3, 5, and 7. (Scale bar: 5mm) \u003cstrong\u003e(b)\u003c/strong\u003e Superposition images of wounds.\u0026nbsp; (Scale bar: 5mm) \u003cstrong\u003e(c)\u003c/strong\u003e Relationship between relative wound area and time in different treatment groups (n = 5). (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, indicating statistically significant data between groups; ns indicates no statistical significance)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/c4501fb36a4353b09dc5c54a.png"},{"id":103504885,"identity":"eb701a7b-3962-490b-a0ad-8786eeeea524","added_by":"auto","created_at":"2026-02-26 13:21:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1432698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLSCI reveals blood flow perfusion during wound healing\u003c/strong\u003e. \u003cstrong\u003e(a)\u003c/strong\u003eRepresentative pseudo-color images of LSCI of C57BL/6 and db/db mouse wall-healing models on days 0, 1, 3, 5, and 7. \u003cstrong\u003e(b)\u003c/strong\u003e Relationship between blood perfusion of wound area and time in different treatment groups (n = 5). (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, indicating statistically significant data between groups; ns indicates no statistical significance)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/7c3b6f89fdc5bae3e54541ee.png"},{"id":103168044,"identity":"c03cd797-b798-4d5a-8fe7-e5ed4a312363","added_by":"auto","created_at":"2026-02-22 12:57:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":862019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNIR-II imaging reveals changes in vascular structure during wound healing.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003eRepresentative pseudo-color images of NIR-II imaging of C57BL/6 and db/db mouse wall-healing models on days 0, 1, 3, 5, and 7. \u003cstrong\u003e(b)\u003c/strong\u003e Relationship between blood flow of wound area and time in different treatment groups (n = 5). (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, indicating statistically significant data between groups; ns indicates no statistical significance)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/abb28ae4f8b21e128614cb84.png"},{"id":103168046,"identity":"6b7822bc-cc51-4c5f-b0e7-9d1e932cb19e","added_by":"auto","created_at":"2026-02-22 12:57:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2285673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOCTA shows structural changes in microcirculation during the wound healing process.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003eRepresentative average area maps of OCTA of C57BL/6 and db/db mouse wall-healing models on days 0, 1, 3, 5, and 7. \u003cstrong\u003e(b)\u003c/strong\u003e Relationship between blood vessel average area and wound area over time in different treatment groups (n = 5). (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, indicating statistically significant data between groups; ns indicates no statistical significance)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/9684fc15a7eaf9be72ff79ac.png"},{"id":103168043,"identity":"09f51139-09ec-4040-ac09-822aa15160ca","added_by":"auto","created_at":"2026-02-22 12:57:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":990962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZn\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e promotes angiogenesis and tissue repair. (a) \u003c/strong\u003eRepresentative images of H\u0026amp;E staining of the wounds on day 7 posttreatment.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/7da3aac230aea676c4f6e697.png"},{"id":103509142,"identity":"e53f0f78-e17c-4f9c-a41c-24e6925691b2","added_by":"auto","created_at":"2026-02-26 13:56:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7717017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/f4942c0f-d887-4434-bb59-7e8989cc3ea0.pdf"},{"id":103168045,"identity":"97336732-fef3-4f09-b31b-db62d3893ec9","added_by":"auto","created_at":"2026-02-22 12:57:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25307221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary description\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1.\u003c/strong\u003e The transmission electron microscopy (TEM) image of Zn\u003csup\u003e2+\u003c/sup\u003e reagent. (Scale bar: 50nm)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2.\u003c/strong\u003e Gray-scale images and pseudo-color images of LSCI in normal C57BL/6 mice and db/db mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3.\u003c/strong\u003e Pseudo-color images of NIR-II imaging in normal C57BL/6 mice and db/db mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Representative all layers, layer1 (0-20μm), layer2 (20-30μm), and layer3 (30-100μm) maps of OCTA in normal C57BL/6 mice and db/db mice. \u003cstrong\u003e(b)\u003c/strong\u003e Representative mask maps of OCTA of C57BL/6 and db/db mouse wall-healing models on days 0, 1, 3, 5, and 7. 4b. Relationship between blood vessel average area of wound area and time in different treatment groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5.\u003c/strong\u003e Relationship between blood vessel average complexity, average diameter, and average skeleton density of wound area and time in different treatment groups (n=5). (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, indicating statistically significant data between groups; ns indicates no statistical significance)\u003c/p\u003e","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8616724/v1/9fe00b8ad88f79219e7dfbef.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Noninvasive assessment of the repair process in diabetic wound healing using multimodal imaging techniques","fulltext":[{"header":"1. Background","content":"\u003cp\u003eSkin wounds from burns, surgery, and chronic ulcers can be life-threatening. Diabetes mellitus (DM), characterized by hyperglycemia, leads to skin wounds in 19%-34% of patients[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Hyperglycemia-induced pathological changes, such as peripheral vascular disease and neuropathy, significantly impair wound healing[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], resulting in slow repair and increased infection risk[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, timely and accurate assessment of skin wounds is crucial for formulating effective and targeted treatment strategies.\u003c/p\u003e \u003cp\u003eWound healing involves hemostasis, inflammation, proliferation, and remodeling[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Notably, the cutaneous vasculature plays a crucial regulatory role in these phases, influencing both the process and outcome of wound healing[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, identifying and quantifying changes in cutaneous blood vessels is fundamental for assessing the status of wound healing and optimizing wound care management.\u003c/p\u003e \u003cp\u003eCurrently, the assessment of wound healing mainly relies on clinical techniques, including visual inspection and surface measurement, such as monitoring wound size, color, odor, exudate characteristics, and scab formation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, this method is limited to the skin surface and is largely influenced by the subjective experience of medical staff[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although histological analysis of biopsy tissue is the gold standard for assessing wound healing, it is an invasive procedure that causes secondary damage and increases the risk of infection[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, researchers have developed a variety of non-invasive detection techniques to assist in assessing wound healing, including magnetic resonance imaging (MRI), ultrasound imaging, polarization-sensitive optical coherence tomography (PS-OCT), and laser Doppler flowmetry (LDF)[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The above non-invasive techniques have limitations in terms of cost, operator dependence, and imaging depth. There is an urgent need to develop better non-invasive assessment solutions.\u003c/p\u003e \u003cp\u003eIn vivo tissue imaging is crucial for directly observing the dynamic changes in the complex wound repair process (such as skin angiogenesis). Laser speckle contrast imaging (LSCI) is an innovative optical imaging technique that can quantitatively and non-invasively evaluate the movement of red blood cells in the dermal microvasculature[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. LSCI has the advantages of short imaging time, high resolution, wide imaging range, non-invasiveness, and non-contact operation. It can be used to evaluate blood flow and perfusion dynamics, which helps predict the healing rate[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNear-infrared region (NIR) fluorescence imaging has the advantages of being non-radioactive and non-invasive, and has become a promising technique for monitoring blood perfusion. In the second near-infrared region (1000-1700nm, NIR-II), due to the significantly improved contrast between the pathological area and the background, spatial resolution, and penetration depth, non-invasive imaging of angiogenesis can be achieved[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Continuous monitoring and evaluation of wound healing through NIR-II fluorescence imaging may provide valuable information for patients' prognosis and care.\u003c/p\u003e \u003cp\u003eOptical coherence tomography angiography (OCTA) is a functional extension of the OCT imaging protocol[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It utilizes the hemodynamic changes caused by the movement of red blood cells to generate contrast, with the advantages of high resolution, no need for labeling, and non-invasiveness. Currently, it is widely used in clinical settings to examine vascular changes in retinopathy and the wound-healing process[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This method can perform three-dimensional imaging of the epidermal tissue structure and the vascular system, thus contributing to the monitoring of pathophysiological changes in skin wounds[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExisting studies have clearly demonstrated that zinc ions (Zn\u003csup\u003e2+\u003c/sup\u003e) play a crucial role in skin wound healing. Zn\u0026sup2;⁺ stimulates angiogenesis through vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), degrades the matrix through matrix metalloproteinases (MMPs), promotes collagen expression, and exerts anti-inflammatory effects[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study constructed a multimodal imaging platform integrating LSCI, NIR-II, and OCTA. Longitudinal imaging of full-thickness skin wounds in C57BL/6 and db/db mice enabled simultaneous dynamic evaluation of vascular structure and perfusion during diabetic wound repair. It quantified vascular remodeling and perfusion evolution, providing a novel quantitative tool for efficacy evaluation and prognosis prediction with clinical translation value.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal Surgery\u003c/h2\u003e \u003cp\u003e All animal procedures were approved by the Animal Care and Use Committee of the Second Hospital of Shanxi Medical University (DW2023053). Ten 8-week-old male C57BL/6 mice and ten 8-week-old male db/db mice (Nanjing Junke Bioengineering Co., Ltd.) were anesthetized via inhalation of isoflurane (3.8%). Following confirmation of sedation levels using the toe pinch test, the dorsal hair was shaved off. All surgical instruments were sterilized, and the surgical area was scrubbed with povidone-iodine. A 6mm biopsy punch was used to create a circular wound with a diameter of 6mm in the disinfected skin of the mouse's back[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Post-surgery, the mice were placed on a heating pad to recover to their normal state. Upon the conclusion of the experiments, all animals were anesthetized with isoflurane (2\u0026ndash;5%) and then euthanized by cervical dislocation, ensuring that the animals were in a painless state throughout the process. Moreover, animal care and all experimental procedures adhered to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 In Vivo Imaging\u003c/h2\u003e \u003cp\u003eStarting from the day of surgery (Day 0), in vivo imaging of the wound area was performed using LSCI, NIR-II, and OCTA every other day for a duration of 7 days. During the imaging process, each mouse was anesthetized with 3.8% isoflurane and subsequently immobilized on a heating pad maintained at 37℃, with continuous anesthesia administered to minimize bodily movements during imaging. The imaging was conducted under controlled room temperature (23\u0026ndash;24℃) to maintain stable blood flow in the wound area of the mice.\u003c/p\u003e \u003cp\u003eWe utilized a laser speckle contrast imager (RFLSI ZW, RWD Life Science CO, China) to quantify cutaneous blood flow perfusion. This system employs a laser wavelength of 785nm, with the laser head positioned 15cm above the skin, resulting in an image size of 2.8\u0026times;2.1cm and a resolution of 3.9 \u0026micro;m/pixel. The image acquisition time was set at 10 seconds. After anesthesia, the mice were placed on the imaging platform and allowed to acclimatize for 5 minutes before initiating the recording of skin blood flow. In vivo wound blood flow imaging of mice was performed using the RWD laser speckle imaging system coupled with LSCI v4.0 software (RWD Life Science).\u003c/p\u003e \u003cp\u003eWe employed an NIR-II imager (NIR-II, Beijing Digital Precision Medical Technology Co., China) to assess cutaneous vascular architecture and blood flow perfusion. NIR-II fluorescence was captured by an InGaAs short-wave infrared camera (Xenics Cheetah-640CL TE3), which was equipped with a filter wheel (1000nm, 1100nm, 1200nm, 1300nm, 1400nm, 1500nm, Thorlabs, USA) positioned in front of the camera, allowing only the passage of NIR-II fluorescence. The excitation laser wavelength was set at 808nm with a power output of 5000mW, and the image acquisition time was 2 minutes. After anesthesia, the mice were placed in a darkroom and injected with 200 \u0026micro;g of ICG (0.1mg/ml, Dandong Pharmaceutical Factory, Liaoning, China) via the tail vein, simultaneously initiating the fluorescence imaging process. In vivo wound vascular structure and perfusion of mice were acquired using NIR-II coupled DPM-IVFM-NIR-OF software for 2 minutes.\u003c/p\u003e \u003cp\u003eWe used an optical coherence tomography angiographer (Micro-VCC, Optoprobe, UK) to measure the microcirculation structure and blood flow within the skin. This system utilizes a swept-source laser with a central wavelength of 1060 nm, featuring an A-scan acquisition rate of 200 kHz and a scanning area of 8mm\u0026times;8mm. After anesthesia, the mice were placed on the imaging platform, and physiological saline was applied to the skin defect area to ensure close contact between the probe and the skin, thereby preventing the formation of air bubbles. In vivo imaging of wound microcirculation structure and blood flow was performed in mice using OCTA coupled OPTO-III scanning software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Image processing\u003c/h2\u003e \u003cp\u003eThe blood flow perfusion data obtained from LSCI were analyzed by calculating the mean perfusion units (PU) within the region of interest (ROI). We used the RFLSI-Analysis software to calculate the mean perfusion in the skin defect area. Specifically, the mean blood flow perfusion within the designated ROI (region over the skin defect) was extracted and recorded at three distinct time points, and the mean of these measurements was calculated to avoid potential temporal variability introduced by the equipment.\u003c/p\u003e \u003cp\u003eThe blood flow signals in the NIR-II window were analyzed by calculating the mean signal-to-background ratio (SBR) within the region of interest (ROI). We used the DPM-IVFM-NIR-OF software to calculate the mean SBR in the skin defect area. The mean blood flow signals within the designated ROI (region over the skin defect) were extracted and recorded at three distinct time points, and the mean of these measurements was calculated to mitigate potential temporal variability introduced by the equipment.\u003c/p\u003e \u003cp\u003eThe vascular-related parameters obtained from OCTA were analyzed by calculating the pixels within the region of interest (ROI), including: A\u0026thinsp;=\u0026thinsp;pixels occupied by the vascular area, S\u0026thinsp;=\u0026thinsp;pixels deemed as the length of the vessels, P\u0026thinsp;=\u0026thinsp;pixels within the perimeter of the vessel, and X\u0026thinsp;=\u0026thinsp;total pixels within the vessel map. Vascular parameters, including density, diameter, branching complexity, and skeleton density, were quantified from OCTA data using PyOct 8.0 software.\u003c/p\u003e \u003cp\u003eAverag_Area\u0026thinsp;=\u0026thinsp;A/X -- the ratio of the total image area occupied by the vascular system with the vessel area map to the total image area, indicating the vascular density within the analyzed region.\u003c/p\u003e \u003cp\u003eAverage_Skeleton_Density\u0026thinsp;=\u0026thinsp;S/X -- the ratio of vessel length as represented in the skeletonized vessel map to the total image area, providing insights into the distribution and compactness of the vascular network.\u003c/p\u003e \u003cp\u003eAverage_Diameter\u0026thinsp;=\u0026thinsp;A/S -- the ratio of the vessel area map to the skeletonized vessel map, providing the average vessel caliber\u003c/p\u003e \u003cp\u003eAverage_Complexity\u0026thinsp;=\u0026thinsp;P\u003csup\u003e2\u003c/sup\u003e/(4π*A) -- calculated using the circumference and vessel area maps, providing the degree of branching and intricacy within the vascular structure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Hematoxylin and eosin staining\u003c/h2\u003e \u003cp\u003eTo examine alterations in the skin tissue architecture, paraffin-embedded skin tissue sections (5-\u0026micro;m-thick) were stained with hematoxylin and eosin (H\u0026amp;E). Pathologic images were obtained using an optical microscope (Pannoramic MIDI, 3DHISTECH Ltd., Hungary).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eTwenty mice were subjected to wound healing experiments. They were all euthanized at the study endpoint (i.e., Day 7), with in vivo data utilized for statistical analysis of vascular structural and functional parameters. Quantitative data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons between groups were conducted using either one-way ANOVA or a t-test. GraphPad Prism 10.1.0 software was utilized for statistical analysis and graph generation. A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied to determine statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eWe used adult wild-type (C57BL/6) mice and type 2 diabetes (db/db) model mice to compare the healing process of their full-thickness skin wounds. A total of 20 mice were included in the experiment (10 in the C57BL/6 group and 10 in the db/db group). During the experiment, one db/db mouse that did not receive drug intervention unexpectedly died on Day 7. Therefore, we set the imaging endpoint on Day 7 after surgery.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Administration of Zn\u003csup\u003e2+\u003c/sup\u003e accelerates the repair process of wound healing\u003c/h2\u003e \u003cp\u003eFirstly, we established models of full-thickness skin defects (diameter: 6mm) in both C57BL/6 and db/db mice. Since the wound microenvironment is acidic and it has been demonstrated that Zn\u003csup\u003e2+\u003c/sup\u003e has an important role in promoting vascular angiogenesis and accelerating wound healing[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The treatment group received a reagent capable of releasing Zn\u003csup\u003e2+\u003c/sup\u003e under acidic conditions, administered every other day at a dose of 50\u0026micro;l (1mg/ml) post-induction of skin wounds. The structure of the Zn\u003csup\u003e2+\u003c/sup\u003e reagent is shown in the transmission electron microscopy (TEM) image \u003cb\u003e(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea depicts the healing process of skin wounds from Day 1 to Day 7 in C57BL/6 and db/db mice, respectively. Visually, skin wound healing in db/db mice was significantly impaired compared to C57BL/6 mice. At day 7, the wound healing area of db/db mice was significantly smaller than that of C57BL/6 mice. After Zn\u003csup\u003e2+\u003c/sup\u003e administration, both C57BL/6 and db/db mice exhibited accelerated wound healing rates \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther assessment of wound healing rates based on wound size revealed that the Zn\u003csup\u003e2+\u003c/sup\u003e-treated groups had significantly higher healing rates compared to their respective controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Specifically, the wound closure rates on Day 7 were 86% and 54% in the Zn\u003csup\u003e2+\u003c/sup\u003e-treated C57BL/6 and db/db mice, respectively, compared to 72% and 39% in their untreated counterparts (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), suggesting that Zn\u003csup\u003e2+\u003c/sup\u003e formulations can promote skin wound healing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 LSCI reveals blood flow perfusion during wound healing\u003c/h2\u003e \u003cp\u003eLSCI provides two-dimensional dynamic maps of blood flow perfusion based on fluctuations in speckle patterns induced by red blood cell motion, enabling the monitoring of hemodynamic changes throughout the wound healing process. Firstly, in vivo imaging was performed on normal C57BL/6 mice and db/db mice to obtain baseline information \u003cb\u003e(Figure S2)\u003c/b\u003e. Interestingly, in C57BL/6 mice, LSCI not only allowed for the visualization of overall skin microcirculatory blood flow perfusion but also revealed the structure of larger vessels. In contrast, due to alterations in skin structure in db/db diabetic mice, partial reflection of laser light occurred, leading to deviations in imaging results. Therefore, for db/db mice, LSCI imaging was focused on localized wound areas.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea shows a sequential series of LSCI images captured at days 0, 1, 3, 5, and 7 during the cutaneous wound healing process in both C57BL/6 mice and db/db mice, respectively. The type, severity, and stage of wound healing significantly impact the level of blood flow perfusion. A lower blood flow perfusion in the wound area compared to the surrounding tissues indicates the presence of eschar formation or an open epidermal injury. Conversely, a higher blood flow perfusion in the wound area compared to the surrounding tissues indicates the occurrence of wound healing. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, C57BL/6 mice exhibited a notable difference in blood flow perfusion between the wound and adjacent tissues, and Zn\u003csup\u003e2+\u003c/sup\u003e treatment increased perfusion within the wound area. Similarly, Zn\u003csup\u003e2+\u003c/sup\u003e administration augments blood flow perfusion in the wound region of db/db mice. Upon further quantification of the LSCI data, it was found that the wound region of C57BL/6 mice maintained a high level of blood flow perfusion from day 0 to 3, gradually declining from day 5. However, Zn\u003csup\u003e2+\u003c/sup\u003e treatment sustained this elevated perfusion level until day 7 in C57BL/6 mice. The same results were observed in the wound model of db/db mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 NIR-II imaging reveals changes in vascular structure during wound healing\u003c/h2\u003e \u003cp\u003eICG was injected into C57BL/6 mice and db/db mice via the tail vein for NIR-II fluorescence imaging. Firstly, NIR-II imaging was performed on normal C57BL/6 mice and db/db mice to obtain baseline information \u003cb\u003e(Figure S3)\u003c/b\u003e. NIR-II clearly showed the vascular morphology of the dorsal skin in C57BL/6 mice; db/db mice were less well imaged due to interference from adipose tissue and other factors, but the dorsal skin vascular structures could still be visualized. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows a series of NIR-II pseudo-color images of the wound healing process in C57BL/6 mice and db/db mice on days 0, 1, 3, 5, and 7, respectively. During the initial 0\u0026ndash;3 days, the vascular signal intensity in the wound area was elevated in C57BL/6 mice, with a downward trend observed in db/db mice. Compared with the control group, following Zn\u003csup\u003e2+\u003c/sup\u003e treatment, the vascular signal intensity in the wound area of both C57BL/6 mice and db/db mice continued to rise until day 5 and began to show a decreasing trend on day 7 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Interestingly, the NIR-II imaging revealed that wounds with higher signal intensities healed at a faster rate than those with lower signals, which is consistent with the pathologic features of wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 OCTA shows structural changes in microcirculation during the wound healing process\u003c/h2\u003e \u003cp\u003eOCTA is an emerging ophthalmic imaging technology that allows rapid and non-invasive reconstruction of the three-dimensional structure of retinal and choroidal vessels. By utilizing OCTA technology, we monitored model mice with wounds to obtain the perfusion of microcirculatory structures in the wound area. Firstly, OCTA imaging was performed on the dorsal skin of normal C57BL/6 and db/db mice to obtain baseline information. Based on the skin tissue structure, the OCTA volume was segmented into three layers using the semi-automatic segmentation software integrated with the OCTA instrument, with depths divided into layer 1 (0\u0026ndash;20\u0026micro;m), layer 2 (20\u0026ndash;30\u0026micro;m), and layer 3 (30\u0026ndash;100\u0026micro;m), respectively \u003cb\u003e(Figure S4a)\u003c/b\u003e. Among them, layer2 had the highest vascular density and best reflected the microcirculatory structure and blood flow changes during wound healing. Therefore, layer 2 was chosen for quantitative analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure S4b\u003c/b\u003e shows sequential mask maps on days 0, 1, 3, 5, and 7 during skin wound healing in C57BL/6 mice and db/db mice, which provides basic information on various vascular parameters. Based on this basic information, the OCTA analysis software can analyze vascular parameters, including vascular complexity, vascular diameter, vascular skeleton density, and vascular blood flow velocity. These vascular parameters are able to quantify the vascular responses, providing a comprehensive assessment of the wound healing process.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea is a pseudo-color map of the vascular area based on mask maps. In contrast to the uniform vascular distribution presented in the skin of normal mice, vasodilatation is evident around the wound site. As healing progresses, angiogenesis gradually migrates into the wound, forming neovascular sprouts. Compared with C57BL/6 mice, db/db mice had a significantly delayed establishment of microcirculation than C57BL/6 mice due to their hyperglycemic factors (P\u0026thinsp;=\u0026thinsp;0.0012). However, the rate of angiogenesis was accelerated after Zn\u003csup\u003e2+\u003c/sup\u003e administration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. During the healing process, vascular complexity, vessel diameter, and vascular skeleton density all increased over time, indicating enhanced microcirculation augmented blood perfusion within the wound area \u003cb\u003e(Figure S5)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Zn\u003csup\u003e2+\u003c/sup\u003e promotes angiogenesis and tissue repair\u003c/h2\u003e \u003cp\u003eAs mentioned above, previous studies have clearly demonstrated that Zn\u003csup\u003e2+\u003c/sup\u003e plays a crucial role in skin wound healing. We selected the wound-area tissues of C57BL/6 mice and db/db mice from the control group and the Zn\u003csup\u003e2+\u003c/sup\u003e-treated group on Day 7 for hematoxylin-eosin (H\u0026amp;E) staining to evaluate relevant histological changes, including angiogenesis, collagen deposition, and epithelialization \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Compared with the control group, the Zn\u003csup\u003e2+\u003c/sup\u003e-treated group showed increased angiogenesis in the wound area, higher levels of collagen deposition, and epithelialization. The collagen fiber bundles in the Zn\u003csup\u003e2+\u003c/sup\u003e-treated group were arranged more tightly and compactly, with abundant angiogenesis and continuous epithelialization. The same results were observed in both C57BL/6 mice and db/db mice. Compared with C57BL/6 mice, db/db mice had reduced angiogenesis, collagen deposition, and epithelialization in the wound area, leading to a delayed wound healing process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe wound healing process represents the body's inherent ability to restore tissue integrity after injury[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Wound healing in diabetic patients is usually complex, often accompanied by infection and ischemia[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Long-term elevated blood glucose can lead to vascular damage, insufficient blood perfusion, and prolonged wound healing time[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, it is particularly important to monitor vascular structure and blood perfusion around the wound site, along with wound interventions. In this study, we used LSCI, NIR-II, and OCTA multimodal imaging techniques to monitor skin wound healing in C57BL/6 mice and db/db mice, and combined with histopathologic examinations to evaluate the efficacy of these three imaging techniques for assessing the wound healing process. Through continuous monitoring of skin wounds in both C57BL/6 and db/db mice over 7 days, we observed that the wound healing rate of db/db mice significantly lagged behind that of C57BL/6 mice. Furthermore, Zn\u003csup\u003e2+\u003c/sup\u003e-treatment promoted angiogenesis and accelerated the wound healing process.\u003c/p\u003e \u003cp\u003eLaser speckle contrast imaging (LSCI) is a high-resolution and high-contrast optical imaging technique commonly employed to characterize hemodynamic variations in short-term physiological experiments[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. LSCI is commonly used in neurophysiological studies to dynamically image alterations in cerebral blood flow[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In recent years, there have also been related studies using LSCI to study skin and wound perfusion[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. LSCI can provide noninvasive real-time feedback on changes in perfusion and can monitor the microcirculation in the outer layer of the skin[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, we used an RFLSI ZW laser speckle blood flow imaging system designed based on LSCI technology with higher resolution to monitor and present the microcirculation blood flow perfusion distributions within living tissues in real time. Our findings showed that LSCI was able to monitor hemodynamic changes during wound healing, with quantification revealing the perfusion information of the wound area\u0026rsquo;s microcirculation. Notably, blood flow perfusion in the wound area increased from day 0 to day 3, and Zn\u003csup\u003e2+\u003c/sup\u003e treatment was observed to prolong this period of elevated perfusion, thereby facilitating wound healing.\u003c/p\u003e \u003cp\u003eThe second near-infrared region (NIR-II, 900\u0026ndash;1800 nm) imaging has emerged as a novel optical imaging modality with promising clinical application prospects in recent years[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It enables the visualization of clinically occult lesions and surrounding important structures with higher sensitivity and resolution, and has already achieved applications in some clinical fields, such as hepatectomy and pancreatic tumor resection, etc[\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Indocyanine green (ICG), the only cyanine dye approved by the Food and Drug Administration (FDA) in the United States, has demonstrated robust NIR-II imaging outcomes in both patients and small animals[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. By injecting ICG via the tail vein in mice and performing NIR-II imaging, we were able to comprehensively visualize the vascular structural changes within the wound area, thus reflecting the ability of wound healing. Surprisingly, we found that NIR-II signal values correlated with healing outcomes in our mouse model, with high signals suggesting good healing outcomes. This discovery has the potential to provide valuable insights for selecting optimal intervention timings in clinical wound management.\u003c/p\u003e \u003cp\u003eOptical coherence tomography angiography (OCTA) is a relatively new, non-invasive technique that provides high-resolution images of retinal and choroidal vascularization[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It has been reported in healthy subjects, in patients with different ocular and systemic diseases, as well as in diverse animal models[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. OCTA, which operates without the need for contrast agents, can show perfusion of microcirculatory tissues, which is useful for studying diseases in which microvascular morphology and perfusion change over time[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. By monitoring changes in microcirculatory structure and perfusion during wound healing using OCTA, we found that neovascular sprouts gradually migrated inward over time. Furthermore, OCTA provides many vascular parameters during wound healing, including vascular area density, vascular diameter, and vascular complexity, suggesting its potential as a future indicator for wound assessment.\u003c/p\u003e \u003cp\u003eIn this experiment, it was originally planned to conduct longer-term observations and multimodal imaging. However, a db/db mouse that did not receive drug intervention unexpectedly died on Day 7, resulting in the premature termination of the observation of this individual. This unexpected event limited the data integrity of this study. In subsequent studies, a longer observation period and more comprehensive imaging strategies will be employed to address this deficiency.\u003c/p\u003e \u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e plays a central role in enzymatic reactions, collagen synthesis, and antibacterial activities, and its toxicity is reduced, and the healing effect is remarkable after being compounded with polymers[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It should be noted that, as it was not the research objective of this study, the molecular mechanism by which Zn\u003csup\u003e2+\u003c/sup\u003e promotes wound healing was not explored. Future research can focus on the regulatory role of Zn\u003csup\u003e2+\u003c/sup\u003e in the diabetic wound microenvironment. In addition, the operating procedures of the multimodal imaging techniques involved in this study need to be further standardized, which is crucial for the translation of research findings into clinical applications.\u003c/p\u003e \u003cp\u003eThe multimodal imaging system integrating LSCI, NIR-II, and OCTA enables non-destructive visualization of tissue microstructure and microvasculature in murine skin, facilitating longitudinal monitoring of anatomical and vascular dynamics during wound healing. By synergistically complementing information across modalities, this approach provides a comprehensive perspective on healing progression and deepens mechanistic understanding of the process. We anticipate that this integrated platform will significantly advance wound intervention strategies and accelerate the development of novel therapeutics.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe developed imaging platform represents a significant advancement for predicting wound healing outcomes and evaluating treatment strategies for diabetic wounds. It provides a powerful tool for assessing the efficacy of novel drugs and shows potential for guiding optimal intervention timing in clinical settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eDM:\u003c/strong\u003e Diabetes Mellitus\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLSCI:\u003c/strong\u003e Laser Speckle Contrast Imaging\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNIR-II:\u003c/strong\u003e Second Near-infrared Region Imaging\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOCTA:\u003c/strong\u003e Optical Coherence Tomography Angiography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRI:\u003c/strong\u003e Magnetic Resonance Imaging\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePS-OCT:\u003c/strong\u003e Polarization-Sensitive Optical Coherence Tomography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLDF:\u003c/strong\u003e Laser Doppler Flowmetry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICG:\u0026nbsp;\u003c/strong\u003eIndocyanine Green\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH) and approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (Approval No. DW2023053). All measures were aimed at minimizing animal suffering and discomfort, including the use of appropriate anesthesia and analgesia during surgery, as well as providing a standard-compliant feeding environment and postoperative care.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work received the support from the Regional Cooperation Program of Shanxi Province, China (Grant No. 202204041101038); The Leading Talent Team Building Program of Shanxi Province, China (Grant No. 202204051002010); Construction and Demonstration of Molecular Diagnosis and Treatment Platform for Vascular Diseases in Shanxi Province, China (Grant No. SCP-2023-17); Translational Medicine Engineering Research Center for Vascular Diseases of Shanxi Province, China (Grant No. 2022017); Central government-guided local project, China (Grant No. YDZJSX2021C026); General Project of National Natural Science Foundation of China (Grant No. 81770695 and 81870354); Graduate Practical Innovation Project of the Education Department of Shanxi Province, China (Grant No.2023SJ145); Graduate Student Academic Innovation Project of the Education Department of Shanxi Province, China (Grant No. 2025XS309).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L., H.Z., and Y.R. contributed equally to this study. Y.L., H.Z., and Y.R. were responsible for the conception and design of the study, the acquisition, analysis, and interpretation of data, and the drafting of the manuscript. H.W. and G.C. contributed significantly to the acquisition and analysis of data. K.F. and C.L. provided overall guidance on the project, including the design and execution of the experiments. Y.Z. and J.H. were responsible for the statistical analysis of the data and for ensuring the accuracy and consistency of the results presented in the manuscript. H.D. oversaw the entire project, including the design, execution, and analysis of the study. He was also responsible for the final approval of the version. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLei, H. and D. 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healing over time.\u003c/em\u003e Quant Imaging Med Surg, 2018. \u003cstrong\u003e8\u003c/strong\u003e(2): p. 135-150.\u003c/li\u003e\n\u003cli\u003eYousefi, S., et al., \u003cem\u003eAssessment of microcirculation dynamics during cutaneous wound healing phases in vivo using optical microangiography.\u003c/em\u003e J Biomed Opt, 2014. \u003cstrong\u003e19\u003c/strong\u003e(7): p. 76015.\u003c/li\u003e\n\u003cli\u003eSun, X., et al., \u003cem\u003eConstruction of pH-Sensitive Multifunctional Hydrogel with Synergistic Anti-Inflammatory Effect for Treatment of Diabetic Wounds.\u003c/em\u003e Pharmaceutics, 2025. \u003cstrong\u003e17\u003c/strong\u003e(5).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmim","sideBox":"Learn more about [BMC Medical Imaging](http://bmcmedimaging.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmim/default.aspx","title":"BMC Medical Imaging","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"wound healing, diabetes mellitus, laser speckle contrast imaging (LSCI), second near-infrared region (NIR-II) imaging, optical coherence tomography angiography (OCTA)","lastPublishedDoi":"10.21203/rs.3.rs-8616724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8616724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe wound healing process in diabetic patients is complex and often accompanied by risks of infection and ischemia. Currently, clinical assessment mainly relies on visual examination and surface measurement, which have limitations such as strong subjectivity and the inability to conduct in-depth evaluations. In this study, we established an integrated multimodal non-invasive imaging platform to accurately evaluate the healing process of diabetic wounds and quantitatively assess the therapeutic potential of Zn\u0026sup2;⁺-based treatment methods in promoting microcirculation reconstruction.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe implemented a novel technical platform combining laser speckle contrast imaging (LSCI), second near-infrared region (NIR-II) imaging, and optical coherence tomography angiography (OCTA) to conduct longitudinal, high-resolution imaging of full-thickness skin wound healing in both normal and db/db diabetic mouse models. The platform enabled dynamic monitoring of vascular and structural changes throughout the healing process.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe multimodal imaging approach successfully provided comprehensive quantitative data: LSCI revealed real-time dynamic changes in cutaneous blood perfusion, NIR-II imaging delineated the spatial-temporal evolution of vascular network structures, while OCTA offered detailed characterization of internal wound microarchitecture and microvascular patterns. This integrated methodology permitted both qualitative and quantitative assessment of wound repair capacity with unprecedented resolution.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe developed imaging platform represents a significant advancement for predicting wound healing outcomes and evaluating treatment strategies for diabetic wounds. It provides a powerful tool for assessing the efficacy of novel drugs and shows potential for guiding optimal intervention timing in clinical settings.\u003c/p\u003e","manuscriptTitle":"Noninvasive assessment of the repair process in diabetic wound healing using multimodal imaging techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-22 12:57:06","doi":"10.21203/rs.3.rs-8616724/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-14T07:38:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-13T09:32:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326947157491006767930776894053142827854","date":"2026-03-02T13:36:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238398233422226576490762716013536842448","date":"2026-02-27T16:29:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T12:21:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216292387535543790840325109508804666993","date":"2026-02-25T20:07:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54433835793263045173689890384052401398","date":"2026-02-24T09:00:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-24T05:28:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43900252902756459915831382479565707461","date":"2026-02-20T20:31:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30612550489914411191434190625731330065","date":"2026-02-19T07:21:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-17T15:34:31+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-27T05:01:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T09:58:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T09:50:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Medical Imaging","date":"2026-01-20T09:32:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmim","sideBox":"Learn more about [BMC Medical Imaging](http://bmcmedimaging.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmim/default.aspx","title":"BMC Medical Imaging","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea54b1af-a584-4c21-be62-2165a86624eb","owner":[],"postedDate":"February 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T10:53:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-22 12:57:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8616724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8616724","identity":"rs-8616724","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}