Effects of three-dimensional bioprinting antibacterial hydrogel on full-thickness skin defect wounds in rats / 中华烧伤杂志

Objective: To explore the effects of three-dimensional (3D) bioprinting gelatin methacrylamide (GelMA) hydrogel loaded with nano silver on full-thickness skin defect wounds in rats. Methods: The experimental research method was adopted. The morphology, particle diameter, and distribution of silver nanoparticles in nano silver solution with different mass concentrations and the pore structure of silver-containing GelMA hydrogel with different final mass fractions of GelMA were observed by scanning electron microscope and the pore size was calculated. On treatment day 1, 3, 7, and 14, the concentration of nano silver released from the hydrogel containing GelMA with final mass fraction of 15% and nano silver with final mass concentration of 10 mg/L was detected by mass spectrometer. At 24 h of culture, the diameters of inhibition zone of GelMA hydrogel containing final mass concentration of 0 (no nano silver), 25, 50, and 100 mg/L nano silver against Staphylococcus aureus and Escherichia coli were detected. Fibroblasts (Fbs) and adipose stem cells (ASCs) were isolated respectively by enzymatic digestion using the discarded prepuce after circumcision from a 5-year-old healthy boy who was treated in the Department of Urology of the Second Affiliated Hospital of Zhejiang University School of Medicine in July 2020, and the discarded fat tissue after liposuction from a 23-year-old healthy woman who was treated in the Department of Plastic Surgery of the Hospital in July 2020. The Fbs were divided into blank control group (culture medium only), 2 mg/L nano sliver group, 5 mg/L nano sliver group, 10 mg/L nano sliver group, 25 mg/L nano sliver group, and 50 mg/L nano sliver group, which were added with the corresponding final mass concentrations of nano sliver solution, respectively. At 48 h of culture, the Fb proliferation viability was detected by cell counting kit 8 method. The Fbs were divided into 0 mg/L silver-containing GelMA hydrogel group, 10 mg/L silver-containing GelMA hydrogel group, 50 mg/L silver-containing GelMA hydrogel group, and 100 mg/L silver-containing GelMA hydrogel group and then were correspondingly treated. On culture day 1, 3, and 7, the Fb proliferation viability was detected as before. The ASCs were mixed into GelMA hydrogel and divided into 3D bioprinting group and non-printing group. On culture day 1, 3, and 7, the ASC proliferation viability was detected as before and cell growth was observed by live/dead cell fluorescence staining. The sample numbers in the above experiments were all 3. Four full-thickness skin defect wounds were produced on the back of 18 male Sprague-Dawley rats aged 4 to 6 weeks. The wounds were divided into hydrogel alone group, hydrogel/nano sliver group, hydrogel scaffold/nano sliver group, and hydrogel scaffold/nano sliver/ASC group, and transplanted with the corresponding scaffolds, respectively. On post injury day (PID) 4, 7, 14, and 21, the wound healing was observed and the wound healing rate was calculated (n=6). On PID 7 and 14, histopathological changes of wounds were observed by hematoxylin eosin staining (n=6). On PID 21, collagen deposition of wounds was observed by Masson staining (n=3). Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Bonferroni correction, and independent sample t test. Results: The sliver nano particles in nano silver solution with different mass concentrations were all round, in scattered distribution and uniform in size. The silver-containing GelMA hydrogels with different final mass fractions of GelMA all showed pore structures of different sizes and interconnections. The pore size of silver-containing GelMA hydrogel with 10% final mass fraction was significantly larger than that of silver-containing GelMA hydrogels with 15% and 20% final mass fractions (with P values both below 0.05). On treatment day 1, 3, and 7, the concentration of nano silver released from silver-containing GelMA hydrogel in vitro showed a relatively flat trend. On treatment day 14, the concentration of released nano silver in vitro increased rapidly. At 24 h of culture, the diameters of inhibition zone of GelMA hydrogel containing 0, 25, 50, and 100 mg/L nano silver against Staphylococcus aureus and Escherichia coli were 0, 0, 0.7, and 2.1 mm and 0, 1.4, 3.2, and 3.3 mm, respectively. At 48 h of culture, the proliferation activity of Fbs in 2 mg/L nano silver group and 5 mg/L nano silver group was both significantly higher than that in blank control group (P<0.05), and the proliferation activity of Fbs in 10 mg/L nano silver group, 25 mg/L nano silver group, and 50 mg/L nano silver group was all significantly lower than that in blank control group (P<0.05). Compared with the that of Fbs in 0 mg/L silver-containing GelMA hydrogel group, the proliferation activity of Fbs in 50 mg/L silver-containing GelMA hydrogel group and 100 mg/L silver-containing GelMA hydrogel group was all significantly decreased on culture day 1 (P<0.05); the proliferation activity of Fbs in 50 mg/L silver-containing GelMA hydrogel group was significantly increased (P<0.05), while the proliferation activity of Fbs in 100 mg/L silver-containing GelMA hydrogel group was significantly decreased on culture day 3 (P<0.05); the proliferation activity of Fbs in 100 mg/L silver-containing GelMA hydrogel group was significantly decreased on culture day 7 (P<0.05). The proliferation activity of ASCs in 3D bioprinting group show no statistically significant differences to that in non-printing group on culture day 1 (P>0.05). The proliferation activity of ASCs in 3D bioprinting group was significantly higher than that in non-printing group on culture day 3 and 7 (with t values of 21.50 and 12.95, respectively, P<0.05). On culture day 1, the number of dead ASCs in 3D bioprinting group was slightly more than that in non-printing group. On culture day 3 and 5, the majority of ASCs in 3D bioprinting group and non-printing group were living cells. On PID 4, the wounds of rats in hydrogel alone group and hydrogel/nano sliver group had more exudation, and the wounds of rats in hydrogel scaffold/nano sliver group and hydrogel scaffold/nano sliver/ASC group were dry without obvious signs of infection. On PID 7, there was still a small amount of exudation on the wounds of rats in hydrogel alone group and hydrogel/nano sliver group, while the wounds of rats in hydrogel scaffold/nano sliver group and hydrogel scaffold/nano sliver/ASC group were dry and scabbed. On PID 14, the hydrogels on the wound surface of rats in the four groups all fell off. On PID 21, a small area of wounds remained unhealed in hydrogel alone group. On PID 4 and 7, the wound healing rates of rats in hydrogel scaffold/nano sliver/ASC group were significantly higher than those of the other three groups (P<0.05). On PID 14, the wound healing rate of rats in hydrogel scaffold/nano sliver/ASC group was significantly higher than the wound healing rates in hydrogel alone group and hydrogel/nano sliver group (all P<0.05). On PID 21, the wound healing rate of rats in hydrogel alone group was significantly lower than that in hydrogel scaffold/nano sliver/ASC group (P<0.05). On PID 7, the hydrogels on the wound surface of rats in the four groups remained in place; on PID 14, the hydrogel in hydrogel alone group was separated from the wounds of rats, while some hydrogels still existed in the new tissue of the wounds of rats in the other three groups. On PID 21, the collagen arrangement in the wounds of rats in hydrogel alone group was out of order, while the collagen arrangement in the wounds of rats in hydrogel/nano sliver group, and hydrogel scaffold/nano sliver/ASC group was relatively orderly. Conclusions: Silver-containing GelMA hydrogel has good biocompatibility and antibacterial properties. Its three-dimensional bioprinted double-layer structure can better integrate with new formed tissue in the full-thickness skin defect wounds in rats and promote wound healing.

A case of chronic refractory wound in the abdomen caused by residual foreign body / 中华烧伤杂志

A 59-year-old male patient with local sinus tract formation due to residual foreign body was admitted to the Second Affiliated Hospital of Zhejiang University College of Medicine on December 17, 2018. The examination showed that the residual foreign body was the component of a sticky cloth implanted when the patient underwent appendectomy 27 years ago. Hypertrophic scar developed at the right-lower abdominal incision for appendectomy 23 years ago and the secondary infection after cicatrectomy resulted in non-healing of the wound. The chronic refractory wound healed completely after surgical treatment in our hospital after this admission. The postoperative pathological examination revealed local inflammatory granuloma. This case suggests that chronic refractory wound is likely to form when secondary infection occurs following the surgical procedure near the implant, and aggressive surgery is an effective way to solve this problem.

Development and progress of sixty years and stepping firmly to the future / 中华烧伤杂志

Since its establishment for 60 years, Department of Burns of the Second Affiliated Hospital of Zhejiang University School of Medicine has grown into a famous regional burn center in China under the leading of the pioneers and through the efforts of several generations. The department has distinctive disciplinary features in burn care, nutritional support, scar prevention and treatments, standard management of chronic wound, and skin tissue engineering research, making positive contribution to the development of burn medicine in China.

Advances in the research of negative-pressure wound therapy inducing the vascularization of dermal substitute / 中华烧伤杂志

In clinical practice, skin defects resulted from various acute and chronic diseases occur frequently. Dermal substitute (DS), known as dermal regenerative template, is used more and more widely, but the slow process of vascularization limits its clinical application. At present, there are many strategies developed to enhance the process of vascularization, such as modifying the structure of dermal scaffolds, prevascularization by seeding stem cells and/or endothelial cells. Recently, negative-pressure wound therapy (NPWT) emerged and rapidly became popular in promoting wound healing due to its intrinsic advantages. Furthermore, some researchers introduced this technique to accelerate the vascularization process of DS. This paper represents a comprehensive overview on the efficiency of NPWT in different combination models, and the related mechanism.

Influence of collagen/fibroin scaffolds containing silver nanoparticles on dermal regeneration of full-thickness skin defect wound in rat / 中华烧伤杂志

Objective@#To explore the influence of collagen/fibroin scaffolds containing silver nanoparticles on dermal regeneration of full-thickness skin defect wound in rat.@*Methods@#Eighty-one collagen/fibroin scaffolds containing silver nanoparticles (with the mass concentration of silver nanoparticles as 10 mg/L) and 81 collagen/fibroin scaffolds without silver nanoparticles were produced respectively with freeze-drying method and enrolled as silver nanoparticles scaffold group (SNS) and control scaffold group (CS). Nine scaffolds in each group were cultured with human fibroblasts. At post culture hour (PCH) 2, 12, and 24, the human fibroblasts adherent to the scaffolds (n=3) in two groups were counted. Four full-thickness skin defect wounds were reproduced on the back of each one of the 36 SD rats. The rats were divided into groups SNS (wounds were transplanted with collagen/fibroin scaffolds containing silver nanoparticles) and CS (wounds were transplanted with collagen/fibroin scaffolds without silver nanoparticles) according to the random number table, with 18 rats in each group. In post surgery week (PSW) 1, 2, and 4, 6 rats in each group were sacrificed respectively for general observation, observation of histological structure, inflammatory cell infiltration, and collagen deposition with HE staining, count of CD68 positive cells with immunohistochemical staining, and mRNA expressions of interleukin-6 (IL-6) and IL-10 with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with analysis of variance of factorial design, t test, and Bonferroni correction.@*Results@#(1) At PCH 2, 12, and 24, the numbers of human fibroblasts adherent to the scaffolds in the two groups were close (with t values from 1.77 to 2.60, P values above 0.05). (2) In PSW 1, no obvious symptom of infection was observed in wound or wound edge of rats in group SNS with obvious vascularization of scaffolds, while obvious symptoms of infection were observed in wounds of rats in group CS with some scaffolds exfoliated. In PSW 2, the scaffolds were firmly attached to the wounds of rats in group SNS, while obvious contracture was observed in the wounds of rats in group CS with a lot of scaffolds exfoliated. In PSW 4, the scaffolds covered the wounds of rats in group SNS with obvious epithelization on the surface of the scaffolds, while all the scaffolds exfoliated, leaving obvious contracture of residual wounds of rats in group CS. (3) In PSW 1 and 2, compared with those in group CS, more collagen secretion and tissue regeneration and less inflammatory cell infiltration in the scaffolds were observed in the wounds of rats in group SNS. In PSW 4, obvious epithelization was observed in the wounds of rats in group SNS, while inflammatory cell infiltration was observed without obvious epithelization in the wounds of rats in group CS. (4) In PSW 1, the number of CD68 positive cells in the wounds of rats in group SNS [(54±10) /mm2] was similar to that in group CS [(78±7) /mm2, t=1.52, P>0.05]. In PSW 2 and 4, the numbers of CD68 positive cells in the wounds of rats in group SNS [(154±10) and (77±7) /mm2] were significantly less than those in group CS [(268±16) and (136±13) /mm2, with t values respectively 7.31 and 3.83, P values below 0.01] respectively. (5) Except for the expression in PSW 4 (t=1.23, P>0.05), the mRNA expressions of IL-6 in the wounds of rats in group SNS in PSW 1 and 2 were significantly lower than those in group CS (with t values respectively 13.12 and 4.65, P values below 0.01). Except for the expression in PSW 1 (t=3.08, P<0.05), the mRNA expressions of IL-10 in PSW 2 and 4 in the wounds of rats in the two groups were similar (with t values respectively 2.14 and 0.49, P values above 0.05).@*Conclusions@#Besides good biocompatibility, collagen/fibroin scaffolds containing silver nanoparticles have obvious effect in modulating inflammation, thus they can accelerate dermal regeneration induced by collagen/fibroin scaffolds for wound repair.

Design and biological evaluation of poly-lactic-co-glycolic acid (PLGA) mesh/collagen-chitosan hybrid scaffold (CCS) as a dermal substitute / 中华烧伤杂志

<p><b>OBJECTIVE</b>To design and construct a kind of dermal regeneration template with mesh, and to preliminarily evaluate its biological characteristics.</p><p><b>METHODS</b>PLGA mesh was integrated into CCS with freeze-drying method for constructing PLGA mesh/CCS composite (PCCS). The micromorphologies and mechanical properties among PLGA mesh, CCS, and PCCS were compared. PCCS and CCS was respectively implanted into subcutaneous tissue of SD rats (PCCS and CCS groups, 9 rats in each group). The tissue samples were collected at post operation week (POW) 1, 2, and 4 for histopathological and immunohistochemical observation. Protein levels of CD68, MPO, IL-1beta, IL-10 were examined by Western blot, with expression of gray value. Data were processed with one-way analysis of variance and t test.</p><p><b>RESULTS</b>Three-dimensional porous structure of PCCS was similar to that of CCS. Mechanical property of PLGA mesh and PCCS was respectively (3.07 +/- 0.10), (3.26 +/- 0.15) MPa, and they were higher than that of CCS [(0.42 +/- 0.21) MPa, F = 592.3, P < 0.0001)]. The scaffolds were filled with newly formed tissue in PCCS group at POW 2, while those in CCS group were observed at POW 4. A large accumulation of macrophages was observed in both groups, especially at POW 2, and more macrophage infiltration was observed in CCS group. The protein level of IL-10 in PCCS group at POW 2 was obviously higher than that in CCS group, while the protein levels of CD68, MPO, IL-1beta were significantly decreased as compared with those in CCS group (with t value from -4.06 to 2.89, P < 0.05 or P < 0.01).</p><p><b>CONCLUSIONS</b>PCCS has excellent mechanical property with appropriate three-dimensional porous structure. Meanwhile, it can rapidly induce formation of new tissue and vascularization, and it has a prospect of serving as a dermal substitute.</p>

Vascularization of poly-lactic-co-glycolic acid knitted mesh/collagen-chitosan hybrid scaffold / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of poly-lactic-co-glycolic acid (PLGA) knitted mesh/collagen-chitosan hybrid scaffold (PCCS) on angiogenesis, and to explore the relative mechanisms.</p><p><b>METHODS</b>PLGA knitted mesh was integrated into collagen-chitosan scaffold (CCS) to construct PCCS with freeze-lyophilizing method, and CCS was made with the same method. The characteristics of morphology and water absorbing capacity among PCCS, PLGA knitted mesh, and CCS were compared in vitro. PCCS and CCS was respectively implanted into subcutaneous tissue of back on both sides in 24 SD rats, and the tissue specimens were harvested at post operation week (POW) 1, 2, and 4 according to the random number table to evaluate the level of angiogenesis by histopathological and immunohistochemical examinations. The expression levels of alpha smooth muscle actin (alpha-SMA) and vascular endothelial growth factor (VEGF) mRNA were examined by real-time quantitative RT-PCR. Data were processed with t test.</p><p><b>RESULTS</b>(1) PLGA knitted mesh was closely integrated with sponge of collagen-chitosan in PCCS, and the porous structure of PCCS was similar to that of CCS. (2) Compared with that of PCCS [(506 +/- 15)%], the water absorbing rate of CCS and PLGA knitted mesh was respectively increased and decreased [(627 +/- 21)%, (195 +/- 15)%, with t value respectively 3.8, 11.9, P < 0.05 or P < 0.001]. (3) The scaffolds were filled with newly formed tissue in CCS at POW 4, while those in PCCS were observed at POW 2 with more homogeneous and abundant collagen. (4) Blood vessels could be induced, and they grew into scaffolds along with prolongation of implantation time in PCCS and CCS. The number of mature blood vessels in PCCS at POW 1, 2, 4 [(10.7 +/- 3.2), (18.6 +/- 2.1), and (30.3 +/- 4.5) branches per square centimeter] was respectively higher than that in CCS [(5.4 +/- 0.9), (10.8 +/- 4.2), and (23.6 +/- 1.7) branches per square centimeter, with t value respectively 4.6, 4.4, 4.5, P values all below 0.01]. (5) The expression levels of alpha-SMA and VEGF mRNA in PCCS at POW 1, 2, 4 were significantly higher than those in the CCS (with t(alpha-SMA) value respectively 1.26, 1.63, 2.17, with t(VEGF) value respectively 5.52, 2.07, 1.78, P values all below 0.01).</p><p><b>CONCLUSIONS</b>PCCS is able to induce the ingrowth of blood vessels rapidly and promote their maturity. The mechanical properties and microstructures of scaffolds play synergistic role in the process of angiogenesis.</p>