Limb salvage surgery in diabetic foot infection: encouraging early results with a local antibiotic carrier

NL Vasukutty, S Mordecai, A Tarik, M Subramaniam and B Srinivasan

Citation: Vasukutty NL, Mordecai S, Tarik A et al (2022) Limb salvage surgery in diabetic foot infection: encouraging early results with a local antibiotic carrier. The Diabetic Foot Journal 25(2): 1–5

Key words - CERAMENT® injection - Diabetic foot ulcers - Osteomyelitis

Article points

1. The use of a local antibiotic is an attractive option for diabetic foot infection.

2. The authors present early results of a cohort of patients where a local antibiotic carrier was used in conjunction with other aspects of multidisciplinary care.

3. The uthors report a limb salvage rate of 94% and infection control of 88%.

Authors

      NL Vasukutty is Consultant Orthopaedic Surgeon, United Lincolnshire Hospitals NHS Trust, Boston, UK; S Mordecai is Consultant Orthopaedic Surgeon, East and North Hertfordshire Hospitals NHS Trust, Stevenage, UK; A Tarik is Consultant Diabetes Physician, United Lincolnshire Hospitals NHS Trust, Lincoln, UK; M Subramaniam is Consultant Vascular Surgeon, United Lincolnshire Hospitals NHS Trust, Boston, UK; B Srinivasan is Consultant Diabetes Physician, United Lincolnshire Hospitals NHS Trust, Lincoln, UK.

      Diabetic foot disease is associated with high morbidity and is one of the leading causes of lower limb amputation. The use of a local antibiotic carrier to augment debridement and reconstructive procedures is presented. Methods: The authors present early results of 48 feet in 47 patients from two centres in the UK. Their multidisciplinary protocol involved pre-operative assessment, debridement, culture-specific antibiotics and local antimicrobial management with an antibiotic-loaded biocomposite (CERAMENT G®, BONESUPPORT, Lund, Sweden). Of the 48 feet, 22 (46%) had various foot reconstructive procedures. Six patients had pre-operative revascularisation procedures. All patients were graded as either University of Texas 3B or 3D ulcers. Results: At a mean follow-up of 33 months (range 13–49 months), 42 feet (88%) were free of infection and 39 patients (83%) were mobilising. There were 28 wounds healed by secondary intention, 17 with primary closure and three required skin grafting. Three patients had non-healing and persisting ulcers at the most recent follow-up. Three patients had undergone below–knee amputation. The average time to wound healing was 16 weeks (range 3–24 weeks). A limb salvage rate of 94% was achieved. Conclusion: These are encouraging early results in a cohort of diabetic foot patients where CERAMENT G was used as a local antibiotic vehicle in conjunction with other aspects of multidisciplinary care. This is effective in preventing reinfection and avoiding multiple theatre visits.

生长因子调控创面修复的进展与思考

本文亮点：

(1) 总结了关于生长因子通过调控皮肤创面的微环境，从而促进创面愈合的国内研究进展。

(2) 总结了生长因子与免疫系统、神经系统以及脂肪组织的相互作用对创面愈合进程的影响。

肖健 张凡 温州医科大学附属第一医院创面修复科 温州医科大学创伤修复与再生医学中心 温州 医科大学药学院，温州 325035

通信作者：肖健，Email：该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【摘要】 生长因子在创面愈合中起重要调控作用，其主要通过激活相关信号通路加速创面愈合。中国科学家对生长因子的基础、临床研究至今已有 30 年历史，研发的生长因子系列药物被广泛应用于烧创伤及慢性难愈合溃疡的治疗。该文从免疫、神经、脂肪等多个角度阐述了生长因子对创面修复的前沿进展，并提出了该研究团队对生长因子调控创面修复的进一步思考。

【关键词】 伤口愈合； 皮肤； 生长因子； 调控

基金项目：国家自然科学基金优秀青年科学基金项目

（81722028）；温州市重大科技创新攻关项目（ZY2020023）

Progress and thoughts on the regulation of wound repair by growth factors

Xiao Jian, Zhang Fan. Department of Wound Repair, the First Affiliated Hospital of Wenzhou Medical University, Wound Repair and Regenerative Medicine Center, Wenzhou Medical University, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325035, China

Corresponding author: Xiao Jian, Email: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【Abstract】 Growth factors play an important role in wound healing, and they mainly accelerate wound healing by activating the related signal pathways. Chinese scientists have been conducting basic and clinical researches on growth factors for 30 years, with a series of growth factor drugs being developed and widely used in the treatment of burns and trauma and chronic refractory ulcers. This paper expounds the frontier progress of growth factors on wound healing from the perspectives of immunity, nerve, fat, and so on, and puts forward the further thoughts of the research team on the regulation of wound healing by growth factors.

【Key words】 Wound healing； Skin； Growth factor ； Regulation

Fund program: Natural Science Foundation of China Excellent Young Program of National (81722028); Wenzhou Science and Technology Innovation Project (ZY2020023)

Barriers and facilitators to reporting medical device-related pressure ulcers: A qualitative exploration of international practice

Ewa A. Crunden a,⁎, Peter R. Worsley a, Susanne B. Coleman c, Lisette Schoonhoven a,b a School of Health Sciences, University of Southampton, Highfield, Southampton SO17 1BJ, UK b University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands c University of Leeds, Leeds Institute of Clinical Trials Research, Leeds LS2 9JT, UK

ARTICLE INFO

Article history: Received 25 February 2022 Received in revised form 13 June 2022 Accepted 11 July 2022 Available online xxxx

Keywords: Medical device-related pressure ulcers. Reporting. Barriers and facilitators. Determinants of practice

abstract

Background: Pressure ulcers are a complex healthcare issue. Hospital-acquired pressure ulcers are used as proxy measurements for the quality and safety of nursing care. Medical device-related pressure ulcers are mostly facility acquired, but their reporting has only recently been widely adopted. Consequently, we do not yet know what factors impact their reporting by registered nurses.

Objectives: To identify and systematically report determinants of the practice of medical device-related pressure ulcers reporting using the Tailored Implementation for Chronic Diseases checklist.

Design: Descriptive, explorative design using semi-structured interviews to explore barriers and facilitators to reporting medical device-related pressure ulcers.

Setting: We undertook online, telephone, and face-to-face interviews with participants from 11 different countries.

Participants:We interviewed 17 participants who represented acute care (Adult, Paediatrics), academia, and industry. Eleven participants were healthcare professionals with more than 10 years' experience in wound care.

Methods: The interview recordings were transcribed and coded by the lead researcher. Data were analysed thematically using the codebook approach, and themes were developed inductively and deductively.

Results: Participants identified determinants of practice which clustered around four domains of the Tailored Implementation for Chronic Diseases checklist i) individual health professional factors, ii) professional interactions, iii) incentives and resources, and iv) capacity for organisational change. Knowledge, attitudes, workload, time, staffing, and perception of consequences, including financial, were identified as the main barriers to reporting. Factors supporting the practice were education, openness, and teamwork. Device procurement could take on characteristics of a barrier or facilitator depending on the organisation.

Conclusions: Reporting medical device-related pressure ulcers has been adopted in healthcare institutions worldwide. Understanding what drives the reporting practice enables improvements in incident reporting, which consequently can lead to improvements in the quality of nursing care and patient safety.

© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

基于单细胞 RNA 测序探讨小鼠全层皮肤 缺损创面中真皮成纤维细胞的 生长因子调控网络

本文亮点：

(1) 通过建立小鼠全层皮肤缺损创面模型并进行伤后 7 d 创面组织的单细胞 RNA 测序，定义了多个Fb 亚群，明确了创面愈合过程中的真皮 Fb 存在高异质性，了解到真皮 Fb 是创面愈合过程中细胞因子网络调控的主要靶点。

(2) 通过生物信息学及组织多重染色技术，了解到 FGF7 主要由间质祖细胞表达，并作用于多个真皮Fb 亚群，揭示了 FGF 信号通路在创面修复中新的调控途径。

孙礼祥 吴帅 张小薇 刘文杰 张凌娟

厦门大学药学院，细胞应激生物学国家重点实验室，厦门 361102   通信作者：张凌娟，Email：该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【摘要】

目的 基于单细胞 RNA 测序探讨小鼠全层皮肤缺损创面中真皮成纤维细胞（dFb）的异质性与生长因子调控网络。

方法 采用实验研究方法。取 5 只 8 周龄雄性健康 C57BL/6 小鼠（鼠龄、性别、品系下同）正常皮肤组织，另取 5 只背部全层皮肤缺损小鼠伤后 7 d 创面组织，用胶原酶 D 和 DNA 酶Ⅰ消化组织获得细胞悬液，用 10x Genomics 平台构建测序文库，用 Illumina Novaseq6000 测序仪进行单细胞 RNA 测序。采用 R4.1.1 软件的 Seurat 3.0 程序分析获得 2 种组织细胞的基因表达矩阵，采用按细胞群、细胞来源、标记皮肤中主要细胞基因分类的二维 tSNE 云图进行可视化展示。根据已有文献报道和 CellMarker 数据库检索情况，分析 2 种组织细胞的基因表达矩阵中标志基因表达情况，对各细胞群进行编号和定义。将基因表达矩阵和细胞分群信息导入 R4.1.1 软件的 CellChat 1.1.3 程序，分析 2 种组织中细胞间通信以及创面组织血管内皮生长因子（VEGF）、血小板衍生生长因子（PDGF）、表皮生长因子（EGF）、成纤维细胞生长因子（FGF）信号通路中的细胞间通信，2 种组织中FGF 的各亚型与 FGF 受体（FGFR）各亚型的两两配对（以下简称 FGF 配受体对）对 FGF 信号网络的相对贡献，2 种组织中相对贡献排名前 2 的 FGF 配受体对信号通路中的细胞间通信。取 1 只健康小鼠正常皮肤组织和 1 只全层皮肤缺损小鼠伤后 7 d 创面组织，行多重免疫荧光染色，检测 FGF7 蛋白的表达与分布及其与二肽基肽酶 4（DPP4）、干细胞抗原 1（SCA1）、平滑肌肌动蛋白（SMA）和 PDGF 受体 α（PDGFRα）的蛋白共定位表达。

结果 健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后 7 d 创面组织中均包含 25 个细胞群，但 2 种组织中各细胞群中细胞数不一致。PDGFRα、血小板内皮细胞黏附分子 1、淋巴管内皮透明质酸受体 1、受体型蛋白酪氨酸磷酸酶 C、角蛋白 10 和角蛋白 79 基因在二维tSNE 云图上均有各自明确的分布，分别指示特定的细胞群。将 25 个细胞群按 C0~C24 编号，分为 9 个dFb 亚群和 16 个非 dFb 群，dFb 亚群包括 C0 间质祖细胞、C5 脂肪前体细胞、C13 具有收缩能力的肌肉细胞相关成纤维细胞等，非 dFb 群包括 C3 中性粒细胞、C8 T 细胞、C18 红细胞等。与健康小鼠正常皮肤组织比较，全层皮肤缺损小鼠伤后 7 d 创面组织中细胞间通信更多更密集，其中细胞间通信强度排前 3 位的细胞群为 dFb 亚群 C0、C1、C2，这 3 个 dFb 亚群与创面组织中其他细胞群之间均有通信。在全层皮肤缺损小鼠伤后 7 d 创面组织中，VEGF 信号主要由 dFb 亚群 C0 发送、脉管相关细胞群 C19 和C21 接收，PDGF 信号主要由周细胞 C14 发送、多个 dFb 亚群接收，EGF 信号主要由角质形成细胞亚群C9 和 C11 发送、dFb 亚群 C0 接收，FGF 信号的主要发送者和接收者均为 dFb 亚群 C6。健康小鼠正常皮肤组织和全层皮肤缺损小鼠伤后 7 d 创面组织 FGF 信号网络中的 FGF 配受体对相对贡献，均是FGF7-FGFR1 排在首位，排名第 2 的分别是 FGF7-FGFR2、FGF10-FGFR1；与正常皮肤组织比较，创面组织 FGF7-FGFR1 信号通路中的细胞间通信更多，而 FGF7-FGFR2 和 FGF10-FGFR1 信号通路中的细胞间通信稍有减少或无明显变化 ；创面组织 FGF7-FGFR1 信号通路中的细胞间通信强于FGF7-FGFR2、FGF10-FGFR1 信号通路；在 2 种组织中，FGF7 信号均主要由 dFb 亚群 C0 与 C1 和 C2 发送、dFb 亚群 C6 和 C7 接收。与健康小鼠正常皮肤组织比较，全层皮肤缺损小鼠伤后 7 d 创面组织中FGF7 蛋白表达更多；在正常皮肤组织中，FGF7 蛋白主要表达于皮肤间质层且在靠近真皮白色脂肪组织 附 近 也 有 表 达 ；在 2 种 组 织 中 ，FGF7 蛋 白 与 DPP4、SCA1 蛋 白 共 定 位 表 达 于 皮 肤 间 质 层 中 ，与PDGFRα 蛋白共定位表达于 dFb 中，与 SMA 蛋白无共定位表达，其中创面组织中的共定位表达多于正常皮肤组织。

结论 小鼠全层皮肤缺损创面愈合过程中的 dFb 存在高异质性，为多种生长因子潜在的主要分泌或接收细胞群落，与生长因子信号通路之间存在紧密、复杂的联系；FGF7-FGFR1 信号通路是创面愈合过程中的主要 FGF 信号通路，靶向调控多个 dFb 亚群。

【关键词】 伤口愈合； 皮肤； 真皮； 成纤维细胞； 成纤维细胞生长因子； 单细胞 RNA 测序； 生长因子； 细胞间通信

基金项目：国家重点研发计划（2020YFA0112901）；国家自然科学基金面上项目（81971551）；国家自然科学基金青年科学基金项目（82103702）；中国博士后科学基金（2020M682095）

Investigation on the growth factor regulatory network of dermal fibroblasts in mouse full-thickness skin defect wounds based on single-cell RNA sequencing

Sun Lixiang, Wu Shuai, Zhang Xiaowei, Liu Wenjie, Zhang Lingjuan State Key Laboratory of Cell Stress Biology, School of Pharmacy, Xiamen University, Xiamen 361102, China Corresponding author: Zhang Lingjuan , Email: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【Abstract】

Objective To explore the heterogeneity and growth factor regulatory network of dermal fibroblasts (dFbs) in mouse full-thickness skin defect wounds based on single -cell RNA

Methods The experimental research methods were adopted . The normal skin tissue from 5 healthy 8-week-old male C57BL/6 mice (the same mouse age, sex, and strain below) was harvested, and the wound tissue of another 5 mice with full-thickness skin defect on the back was harvested on post injury day (PID) 7. The cell suspension was obtained by digesting the tissue with collagenase D and DNase Ⅰ , sequencing library was constructed using 10x Genomics platform, and single -cell RNA sequencing was performed by Illumina Novaseq6000 sequencer. The gene expression matrices of cells in the two kinds of tissue were obtained by analysis of Seurat 3.0 program of software R4.1.1, and two-dimensional tSNE plots classified by cell group, cell source, and gene labeling of major cells in skin were used for visual display. According to the existing literature and the CellMarker database searching, the expression of marker genes in the gene expression matrices of cells in the two kinds of tissue was analyzed, and each cell group was numbered and defined. The gene expression matrices and cell clustering information were introduced into CellChat 1.1.3 program of software R4.1.1 to analyze the intercellular communication in the two kinds of tissue and the intercellular communication involving vascular endothelial growth factor (VEGF), platelet -derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) signal pathways in the wound tissue, the relative contribution of each pair of FGF subtypes and FGF receptor (FGFR) subtypes (hereinafter referred to as FGF ligand receptor pairs) to FGF signal network in the two kinds of tissue, and the intercellular communication in the signal pathway of FGF ligand receptor pairs with the top 2 relative contributions in the two kinds of tissue.The normal skin tissue from one healthy mouse was harvested, and the wound tissue of one mouse with full-thickness skin defect on the back was harvested on PID 7. The multiple immunofluorescence staining was performed to detect the expression and distribution of FGF7 protein and its co -localized expression with dipeptidyl peptidase 4 (DPP4), stem cell antigen 1 (SCA1), smooth muscle actin (SMA), and PDGF receptor α (PDGFRα) protein.

Results Both the normal skin tissue of healthy mice and the wound tissue of full -thickness skin defected mice on PID 7 contained 25 cell groups , but the numbers of cells in each cell group between the two kinds of tissue were different . Genes PDGFRα, platelet endothelial cell adhesion molecule 1, lymphatic endothelial hyaluronic acid receptor 1, receptor protein tyrosine phosphatase C, keratin 10, and keratin 79 all had distinct distributions on two-dimensional tSNE plots, indicating specific cell groups respectively . The 25 cell groups were numbered by C0 − C24 and divided into 9 dFb subgroups and 16 non -dFb groups . dFb subgroups included C 0 as interstitial progenitor cells, C5 as adipose precursor cells, and C13 as contractile muscle cells related fibroblasts, etc. Non-dFb group included C3 as neutrophils, C8 as T cells, and C18 as erythrocytes, etc. Compared with that of the normal skin tissue of healthy mice, the intercellular communication in the wound tissue of full-thickness skin defected mice on PID 7 was more and denser, and the top 3 cell groups in intercellular communication intensity were dFb subgroups C0, C1, and C2, of which all communicated with other cell groups in the wound tissue. In the wound tissue of full-thickness skin defected mice on PID 7, VEGF signals were mainly sent by the dFb subgroup C0 and received by vascular related cell groups C19 and C21, PDGF signals were mainly sent by peripheral cells C14 and received by multiple dFb subgroups, EGF signals were mainly sent by keratinocyte subgroups C9 and C11 and received by the dFb subgroup C0, and the main sender and receiver of FGF signals were the dFb subgroup C6. In the relative contribution rank of FGF ligand receptor pairs to FGF signal network in the normal skin tissue of healthy mice and the wound tissue of full -thickness skin defected mice on PID 7, FGF7-FGFR1 was the top 1, and FGF7-FGFR 2 or FGF10 -FGFR 1 was in the second place, respectively ; compared with those in the normal skin tissue , there was more intercellular communication in FGF 7 - FGFR1 signal pathway , while the intercellular communication in FGF7 -FGFR2 and FGF10-FGFR 1 signal pathways decreased slightly or did not change significantly in the wound tissue; the intercellular communication in FGF7 -FGFR 1 signal pathway in the wound tissue was stronger than that in FGF7-FGFR2 or FGF10-FGFR1 signal pathway; in the two kinds of tissue, FGF7 signal was mainly sent by dFb subgroups C0, C1, and C2, and received by dFb subgroups C6 and C7. Compared with that in the normal skin tissue of healthy mouse, the expression of FGF7 protein was higher in the wound tissue of full-thickness skin defected mouse on PID 7; in the normal skin tissue, FGF7 protein was mainly expressed in the skin interstitium and also expressed in the white adipose tissue near the dermis layer; in the two kinds of tissue , FGF 7 protein was co -localized with DPP4 and SCA1 proteins and expressed in the skin interstitium , co -localized with PDGFR α protein and expressed in dFbs , but was not co -localized with SMA protein, with more co - localized expression of FGF 7 in the wound tissue than that in the normal skin tissue.

      Conclusions In the process of wound healing of mouse full-thickness skin defect wound, dFbs are highly heterogeneous, act as potential major secretory or receiving cell populations of a variety of growth factors , and have a close and complex relationship with the growth factor signal pathways. FGF7-FGFR1 signal pathway is the main FGF signal pathway in the process of wound healing, which targets and regulates multiple dFb subgroups.

【Key words】 Wound healing ; Skin; Dermis; Fibroblasts; Fibroblast growth factors; Single -cell RNA sequencing ; Growth factor; Intercellular communication Fund program : National Key Research and Development Program of China (2020YFA0112901); General Program of National Natural Science Foundation of China (81971551); Youth Science Fund Project of National Natural Science Foundation of China (82103702); China Postdoctoral Science Foundation (2020M682095)

Application of polyurethane foam dressing at split-thickness skin graft recipient site in patients with diabetic wounds: a case series

Luinio S Tongson

Aim: To investigate use of an advanced polyurethane foam dressing for recipient site wound healing after a split-thickness skin graft (STSG) for diabetic wound closure.

Methods: Graft viability, ease of use of the dressing, exudate management and wound infection status were assessed in 18 patients who underwent a STSG for diabetic wound closure and postoperative recipient site management with a polyurethane foam dressing (BETAplast Silver, Mundipharma). Results: Graft uptake was excellent in all 18 patients (100% viability at postoperative day 30). Clinicians assessed the dressing as easy to use at various anatomical sites, with good exudate management and a low infection rate. Conclusion: BETAplast Silver dressing supports recipient site wound healing after a STSG for diabetic wound closure.

Citation: Tongson LS (2022) Application of polyurethane foam dressing at split-thickness skin graft recipient site in patients with diabetic wounds: a case series. The Diabetic Foot Journal 25(2): 32–6

Key words - Diabetic foot ulcer - Polyurethane foam dressing - Split-thickness skin graft

Article points

1. BETAplast Silver, an advanced polyurethane foam dressing, was used for recipient site wound healing after split-thickness skin grafts for diabetic wound closure.

2. Graft viability, ease of use of the dressing, exudate management and wound infection status were assessed in 18 patients.

3. Clinicians assessed the dressing as easy to use over various anatomical sites, with good exudate management and low infection rate.

Authors

Luinio S Tongson is Head, Diabetic Foot and Wound Center, Capitol Medical Center, Quezon City, the Philippines; and Head, Dr James G Dy Wound Healing and Diabetic Foot Center, Chinese General Hospital and Medical Center, Manila, the Philippines

负载银和重组人碱性成纤维细胞生长因子的 甲基丙烯酸酐化明胶水凝胶对兔 深Ⅱ度烧伤创面的影响

本文亮点：

(1) 首 次 将 具 有 ECM 特 性 的 甲 基 丙 烯 酸 酐 化 明 胶 (GelMA)同 时 负 载 银 离 子 和 重 组 人 碱 性 FGF（rh-bFGF）制得含银+rh-bFGF-GelMA 水凝胶（以下简称复合水凝胶）。

(2) 该复合水凝胶不仅具有良好的抗菌性能，而且还可以促进创面组织上皮化，从而明显缩短兔深Ⅱ度烧伤创面愈合时间。

陈向军 吴兴 林欢欢 刘肇兴 刘沙

解放军联勤保障部队第 969 医院烧伤整形科，呼和浩特 010051

通信作者：陈向军，Email：该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【摘要】

目的 探讨负载银和重组人碱性成纤维细胞生长因子（rh-bFGF）的甲基丙烯酸酐化明胶（GelMA）水凝胶对兔深Ⅱ度烧伤创面的影响。 方法 采用实验研究方法。制备含不同浓度甲基丙烯酸酐（MA）的低浓度 MA 明胶（GelMA）材料、中浓度 GelMA 材料和高浓度 GelMA 材料，加入光引发剂后分别制得低浓度 GelMA 水凝胶、中浓度 GelMA 水凝胶和高浓度 GelMA 水凝胶。采用核磁共振波谱仪检测前述 3 种浓度 GelMA 材料的氢核磁共振谱并根据波谱图计算其取代度，采用场发射扫描电子显微镜（FESEM）检测前述 3 种浓度 GelMA 水凝胶的三维微观结构及孔径，样本数均为 9。根据前述筛选出的 MA 浓度合成含 10 种浓度银的 GelMA（含银 GelMA）溶液，将每种浓度的含银 GelMA 溶液均分为 3 份，加入光引发剂后分别暴露于紫外光下持续 20、25、35 s，制得相应的含银 GelMA 水凝胶。采用胶原酶降解法测定不同光交联时间含银 GelMA 水凝胶降解 12、24、36、48 h 的降解剩余率及彻底降解所需时长，样本数为 5。测定前述筛选出光交联时间下含 10 种浓度银 GelMA 水凝胶对金黄色葡萄球菌的抑菌圈直径反映其抑菌能力，样本数均为 5。以与含最低浓度银（即不含银）GelMA 水凝胶抑菌圈直径相比有统计学意义的含银 GelMA 水凝胶为有抑菌活性。选取具有抑菌活性的且载药浓度最低的含银 GelMA 水凝胶，采用 FESEM 检测其三维微观结构及孔径，采用能谱仪检测其内部银元素的存在情况，样本数均为 9。将冻干单纯 GelMA 水凝胶和冻干含银 GelMA 水凝胶分别浸没于磷酸盐缓冲液中 24 h，通过称重法计算并比较 2 种水凝胶的溶胀率，样本数为 5。根据预实验及前述实验结果，制备含银和 rh-bFGF 的 GelMA 水凝胶（简称复合水凝胶）。大体观察复合水凝胶的外观，并采用FESEM 检测其三维微观结构与孔径。取 30 只 4~6 个月龄、雌雄各半日本大耳兔，在其背部制作深Ⅱ度烧伤创面。以兔头侧为基准，将脊柱左侧创面作为复合水凝胶治疗组，右侧作为纱布对照组，2 组创面分别作相应处理。观察伤后 3、7、14、21、28 d 创面愈合情况；记录伤后 7、14、21、28 d 创面愈合面积并计算其愈合率，样本数为 30。对数据行重复测量方差分析、单因素方差分析、独立样本 t 检验。结果低浓度 GelMA 材料、中浓度 GelMA 材料及高浓度 GelMA 材料的取代度，差异明显（F=1 628.00，P<0.01）。低浓度 GelMA 水凝胶存在疏松、不规则三维空间网状结构，孔径为（60±17）μm；中浓度 GelMA 水凝胶的三维空间网络、孔径大小均较均匀规则，孔径为（45±13）μm；高浓度 GelMA 水凝胶的三维空间网状结构致密、层次混乱，孔径为（25±15）μm。3 种 GelMA 水凝胶孔径大小差异有统计学意义（F=12.20，P<0.01），选取（MA）中浓度为后续材料制作浓度。相同光交联时间下的含不同浓度银 GelMA 水凝胶的降解性基本一致；20、25、35 s 光交联时间下含银 GelMA 水凝胶降解 12 h 的降解剩余率分别为（74.2±1.7）%、（85.3±0.9）%、（93.2±1.2）%，降解 24 h 的降解剩余率分别为（58.3±2.1）%、（65.2±1.8）%、（81.4±2.6）%，降解 36 h 的降解剩余率分别为（22.4±1.9）%、（45.2±1.7）%、（68.1±1.4）%，降解 48 h 的降解剩余率分别为（8.2±1.7）%、（32.4±1.3）%、（54.3±2.2）%；20、25、30 s 光交联时间下含银GelMA 水凝胶彻底降解所需时间分别为（50.2±2.4）、（62.4±1.4）、（72.2±3.2）h，差异有统计学意义（F=182.40，P<0.01），选取 25 s 作为后续光交联时间。低浓度至高浓度的 10 种含银 GelMA 水凝胶对金黄色葡萄球菌的抑菌圈直径依次为（2.6±0.4）、（2.5±0.4）、（3.2±0.4）、（12.1±0.7）、（14.8±0.7）、（15.1±0.5）、（16.2±0.6）、（16.7±0.5）、（16.7±0.4）、（16.7±0.6）mm，基本呈浓度依赖性升高趋势，总体比较差异有统计学意义（F=428.70，P<0.01），与含最低浓度银 GelMA 水凝胶相比，其他有抑菌活性的含低浓度至高浓 度 银 GelMA 水 凝 胶 的 抑 菌 圈 直 径 均 明 显 增 大（t 值 分 别 为 26. 35、33. 84、43. 65、42. 17、49. 24、55. 74、43. 72，P<0. 01）。对金黄色葡萄球菌抑菌圈直径为（12.1±0.7）mm 的含银 GelMA 水凝胶具有抑菌活性且载药浓度最低，选取该含银浓度为后续材料制作浓度。含银 GelMA 水凝胶的微观形貌为规律的趋于平行线性的条索状结构，孔径为（45±13）μm，且含有银元素。浸没 24 h，含银 GelMA 水凝胶的溶胀率与单纯 GelMA 水凝胶相近（P>0.05）。复合水凝胶呈无色清亮透明状；其三维结构为规则、均匀的网格状，内部存在细丝网状结构，孔径为（40±21）μm。伤后 3 d，复合水凝胶组兔创面可见大量坏死组织及渗出物；纱布对照组兔创面可见散在结痂，亦可见少量坏死组织及渗出物。伤后 7 d，复合水凝胶组兔创面已明显缩小，纱布对照组兔出现创面存在与纱布粘连情况。伤后 14 d，复合水凝胶组兔创面红润、可见肉芽组织生长；纱布对照组兔创面基底呈苍白色、血运差。伤后 21 d，复合水凝胶组兔创面完全愈合，纱布对照组兔创面出现愈合趋势。伤后 28 d，复合水凝胶组兔创面部位可见新生毛发，纱布对照组兔仍残存椭圆形创面。伤后 7、14、21、28 d，复合水凝胶组兔创面愈合率均明显大于纱布对照组（t 值分别为 2.24、4.43、7.67、7.69，P<0.05 或 P<0.01）。 结论 中浓度 GelMA 水凝胶在溶胀性、可降解性方面具有良好的理化特性，筛选出的含银 GelMA 水凝胶具有抑菌活性且载药浓度最低，制得的复合水凝胶可明显缩短兔深Ⅱ度烧伤创面愈合时间。

【关键词】 敷料，水胶体； 成纤维细胞生长因子； 伤口愈合； 皮肤； 甲基丙烯酸酐化明胶； 纳米银

基金项目：内蒙古自治区自然科学基金（2018MS08002）

Effects of methacrylic anhydride gelatin hydrogel loaded with silver and recombinant human basic fibroblast growth factor on deep partial-thickness burn wounds in rabbits

Chen Xiangjun, Wu Xing, Lin Huanhuan, Liu Zhaoxing, Liu Sha Department of Burn Orthopaedics, the 969th Hospital of PLA Joint Logistic Support Force, Hohhot 010051, China

Corresponding author: Chen Xiangjun, Email: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。

【Abstract】

      Objective To investigate the effects of methacrylic anhydride gelatin (GelMA) hydrogel loaded with silver and recombinant human basic fibroblast growth factor (rh-bFGF) on deep partial -thickness burn wounds in rabbits . Methods The experimental research method was adopted. Low -concentration GelMA materials , medium -concentration GelMA materials and high -concentration GelMA materials containing different concentrations of methacrylic anhydride (MA) were prepared, after adding photoinitiator, low-concentration GelMA hydrogels, medium-concentration GelMA hydrogels, and high-concentration GelMA hydrogels were obtained, respectively. The nuclear magnetic resonance spectroscopy was performed to detect the hydrogen nuclear magnetic resonance spectra of the above-mentioned three concentrations of GelMA materials , and to calculate the degree of substitution according to the spectrum diagram . The three -dimensional microstructure and pore size of 3 types of above -mentioned GelMA hydrogels were detected by field emission scanning electron microscopy (FESEM), with 9 samples measured. According to the selected concentration of MA , ten kinds of solutions of GelMA with different concentration of silver (silver -containing GelMA) were synthesized , and the silver-containing GelMA solution of each concentration was divided into three parts, and then exposed to ultraviolet light lasting for 20, 25, and 35 s, respectively. After adding photoinitiator, the corresponding silver-containing GelMA hydrogels were obtained. The residual degradation rate of silver -containing GelMA hydrogel with different photocrosslinking times was detected by collagenase degradation method at degradation of 12, 24, 36, and 48 h; and the time required for complete degradation was detected, and the sample number was 5. The inhibition zone diameter of GelMA hydrogel under above screened photocrosslinking times containing 10 concentrations of silver against Staphylococcus aureus was measured to reflect its antibacterial ability, and the sample numbers were all 5. The silver-containing GelMA hydrogel with statistical significance compared with the antibacterial circle diameter of the silver-containing GelMA hydrogel containing the lowest concentration (no silver) was considered as having antibacterial activity. The three -dimensional microstructure and pore size of the silver-containing GelMA hydrogels with antibacterial activity and the lowest drug concentration selected were detected by FESEM, and the sample numbers were all 9. The freeze-dried alone GelMA hydrogel and the freeze-dried silver-containing GelMA hydrogel were soaked in phosphate buffer solution for 24 h, respectively, then the swelling rate of the two GelMA hydrogel were calculated and compared by weighing method , and the sample number was 5. GelMA hydrogel containing silver and rh -bFGF, namely compound hydrogel for short , was prepared according to the preliminary experiment and the above experimental results. The appearance of the composite hydrogel was observed in general, and its three-dimensional microstructure and pore size were detected by FESEM. The deep partial-thickness burn wound was made on the back of 30 rabbits (aged 4-6 months, female half and half). Meanwhile, with the rabbit head as the benchmark, the wounds on the left side of the spine were treated as composite hydrogel treatment group, and the wounds on the right side were treated as gauze control group, and which were treated accordingly. On post injury day (PID) 3, 7, 14, 21, and 28, the healing of wounds in the two groups was observed. On PID 7, 14, 21, and 28, the wound healing area was recorded and the healing rate was calculated, with a sample number of 30. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance , and independent sample t test.

Results

      The substitution degree among low-concentration GelMA materials , medium-concentration GelMA materials, and high-concentration GelMA materials was significantly different (F=1 628.00, P<0.01). The low-concentration GelMA hydrogel had a loose and irregular three-dimensional spatial network structure with a pore size of (60±17) μm; the medium-concentration GelMA hydrogel had a relatively uniform three -dimensional spatial network and pore size with a pore size of (45±13) μm; the high -concentration GelMA hydrogel had the dense and disordered three -dimensional spatial network with a pore size of (25±15) μm, the pore sizes of 3 types of GelMA hydrogels were significantly differences (F=12.20, P<0.01), and medium concentration of MA was selected for the concentration of subsequent materials . The degradability of silver -containing GelMA hydrogels with different concentrations of the same photocrosslinking time was basically same. The degradation residual rates of silver-containing GelMA hydrogels with 20, 25, and 35 s crosslinking time at 12 h were (74.2±1.7)%, (85.3±0.9)%, and (93.2±1.2)%, respectively; the residual rates of degradation at 24 h were (58.3±2.1)% , (65.2±1.8)% , and (81.4±2.6)% , respectively; the residual rates of degradation at 36 h were (22.4±1.9)% , (45.2±1.7)% , and (68.1±1.4)% , respectively; the residual rates of degradation at 48 h were (8.2±1.7)% , (32.4±1.3)% , and (54.3±2.2)% , respectively, and 20, 25, and 30 s photocrosslinking time required for complete degradation of silver-containing GelMA hydrogels were ( 50.2± 2.4), (62.4±1.4), and (72.2±3.2) h, and the difference was statistically significant ( F=182.40, P<0.01), 25 s were selected as the subsequent photocrosslinking time . The antibacterial diameters of 10 types of silver -containing GelMA hydrogels against Staphylococcus aureus from low to high concentrations were (2.6± 0.4), (2.5±0.4), (3.2±0.4), (12.1±0.7), (14.8±0.7), (15.1±0.5), (16.2±0.6 ), (16.7±0.5), (16.7±0.4), and (16.7± 0.6) mm, respectively, and which basically showed a concentration -dependent increasing trend, and the overall difference was statistically significant ( F=428.70, P<0.01). Compared with the silver - containing GelMA hydrogel with the lowest concentration, the antibacterial circle diameters of other silver -containing GelMA hydrogels with antibacterial ability from low to high concentration were significantly increased (with t values of 26.35, 33.84, 43.65, 42.17, 49.24, 55.74, and 43.72, respectively, P<0.01). The silver-containing GelMA hydrogel with the antibacterial diameter of (12.1±0.7) mm had the lowest antibacterial activity against Staphylococcus aureus and the lowest drug loading concentration, and the concentration of silver was selected for the concentration of subsequent materials. The microscopic morphology of the silver -containing GelMA hydrogel containing silver element with a pore size of (45±13) μm had a regular and linear strip -like structure. After soaking for 24 h, the swelling ratio of silver-containing GelMA hydrogel was similar to that of alone GelMA hydrogel. The composite hydrogel was colorless, clear and transparent, and its three-dimensional microstructure was a regular and uniform grid, with a filament network structure inside, and the pore size of (40±21) μm. On PID 3, a large amount of necrotic tissue and exudate of rabbit wound in  composite hydrogel group were observed, and scattered scabs, a small amount of necrotic tissue and exudate of rabbit wound in gauze control group were observed. On PID 7, the area of rabbit wound in composite hydrogel group was significantly reduced, and adhesion of rabbit wound and gauze in gauze control group was observed. On PID 14, In composite hydrogel group, the rabbit wound surface was ruddy, and the growth of granulation tissue was observed, and in gauze control group, the rabbit wound base was pale, and the blood supply was poor. On PID 21, the rabbit wounds in composite hydrogel group healed completely, and rabbit wound in gauze control group had healing trend. On PID 28, new hair could be seen on rabbit wound surface in composite hydrogel group; oval wound of rabbit in gauze control group still remained. On PID 7, 14, 21, and 28, the wound healing areas of rabbit in composite hydrogel group were significantly larger than those in gauze control group (with t values of 2.24, 4.43, 7.67, and 7.69, respectively, P<0.05 or P<0.01).

Conclusions

      The medium-concentration GelMA hydrogel has good physical and chemical properties in terms of swelling and degradability. The screened silver-containing GelMA hydrogels had the lowest antibacterial activity and the lowest drug loading concentration. Composite hydrogel can significantly shorten the healing time of deep partial -thickness burn wounds in rabbits.

【Key words】

Bandages , hydrocolloid; Fibroblast growth factors ; Wound healing; Skin; Methacrylic anhydride gelatin ; Nanosilver

Fund program: Natural Science Foundation of Inner Mongolia Autonomous Region of China (2018MS08002)