Acellular porcine dermal matrix produced with different methods and an experimental study on its transplantation to skin wound.]

OBJECTIVE: To observed the effect of healing quality of composite skin grafting consisting of acellular porcine dermal matrix combined with autologous split-thickness skin graft. METHODS: Porcine skin was treated with dispase II/triton X-100 or hyperosmotic saline/sodium-dodecyl-sulfate (SDS) respectively, and acellular porcine dermal matrix I (APDMI) and APDM II were obtained. Sixty-three Sprague-Dawley rats with full-thickness skin defects on the back were separately covered with APDMT + split-thickness autologous skin, or APDM II + split-thickness autologous skin. The quality of wound healing was observed, the rates of survival and contraction of the grafts were calculated, the tissue samples were harvested for histological examination, and compared with that of autologous split-thickness skin graft. RESULTS: The wound healing quality of composite skin I, and II was good. There was no significant difference in the rate of survival and contraction of the grafts between the two composite skin grafting groups. It was indicated by histological examination intact basal membrane. There was no significant difference in the survival rate between composite skin grafting groups and autologous split-thickness skin at the 6 th week after operation, but the contraction rates of the grafts in the composite skin groups were lower. CONCLUSION: Full-thickness skin defect can be healed by covering with acellular porcine dermal matrix produced by two methods combined with split-thickness autolograft, and it can help improve the quality of wound healing.     

异体表皮干细胞与猪脱细胞真皮构建复合皮移植6例

2005-01/2006-10在南昌大学第一附属医院烧伤科就诊的深度烧伤患者6例,均为四肢Ⅲ度烧伤创面.异种(猪)脱细胞真皮与异体表皮干细胞构建的复合皮由南昌大学第一附属医院烧伤中心提供.清创消毒后,6例患者均进行复合皮移植、自体刃厚皮移植,共移植复合皮12块,自体刃厚皮96块.复合皮移植是将复合皮剪成1.5 cm×1.5 cm,粗糙面向下、光滑面向上植于创面上;自体刃厚皮移植是将大片自体刃厚皮铺于矾土林纱布上,剪成1.5cm×1.5 cm,植于复合皮的四周.移植后3 d揭去创面外层纱布,见复合皮及自体刃厚皮与创面粘连好、湿润,呈淡红色:7 d复合皮及自体刃厚皮大部分成活,随后成活的皮片向四周扩展,至14 d大部分皮片接近融合,21 d时创面已基本愈合.移植的复合皮、自体刃厚皮成活数量比较,差异无显著性意义(P＞0.05).因复合皮扩展情况不如自体刃厚皮,所以复合皮移植后的创面愈合面积小于自体刃厚皮移植(P＜0.01).在临床观察中6例患者暂末发生不良事件和副反应.提示表皮干细胞和(猪)脱细胞真皮构建的人工复合皮移植到受区,可建立血液循环,并保持一定的活力,达到覆盖创面、防止感染、防止体液流失的目的.     

Reduced wound contraction after grafting of full-thickness burns with a collagen and chondroitin-6-sulfate (GAG) dermal skin substitute and coverage with biobrane

Full-thickness burns destroy both the epidermal and dermal tissues of the skin. This study evaluates a collagen and chondroitin-6-sulfate dermal skin substitute (graft) that was applied to excised full-thickness burns and covered with Biobrane. Experimental conditions included: (a) no burn, subcutaneous implantation of the graft; (b) burn, excision, graft, coverage with Biobrane and bandages; (c) burn, excision, no graft, coverage with Biobrane and bandages; (d) burn only. forty-one days post-surgery, subcutaneous implantation (N = 3) of the graft caused no detectable contraction or necrosis of the overlying skin, whereas all burn wounds contracted. Measurements of wounds (percentage of original wound size) showed statistically significant differences between the following treatments; (a) graft plus Biobrane (N = 10), 34%; (b) no graft plus Biobrane (N = 9), 25%; (c) untreated burns (N = 6), 16%. Semi-quantitative evaluation of time to healing indicated by spontaneous detachment of Biobrane from wounds showed that grafted, excised wounds healed in an average of 2.7 weeks, while ungrafted, excised wounds required an average of 4.3 weeks to heal. Histological appearance of healed wounds after grafting and coverage with Biobrane resembles undamaged skin without epidermal adnexal structures. Excision of full-thickness burn eschar, followed by grafting with a collagen and chondroitin-6-sulfate dermal skin substitute and coverage with Biobrane provides reduced wound contraction within a six-week period of observation compared to non-excised wounds. Both more rapid and more complete wound healing took place compared to excised wounds that were not grafted.     

The application of acellular dermal matrix allograft in functional position of patients with post-barns plastic operation

AIM: To investigate the effect of allogeneic acellular dermal matrix(ADM) on cograft in joint functional positions of patients with post-burn plastic operation. METHODS: 9 patients vrith hypertrophic scar and joint dysfunction after severe burns were used After pre-treating with trypsin and TritonX-100, 13 reticulated ADM were overlapped with autogenous ultrathin split-thickness skin grafts (USTS), and were transplanted to the scar excision wounds in the joints of four limbs at the same time. The neighbouring autogenous thin split-thickness skin grafts(TSTS) were used as control RESULTS: The composite skin grafts as well as the controls were all survived The rejection and hypertrophic scars were not found during (1-5) years follow-up studies. The appearance, fiber and function of composite skin grafts were near to normal skins. CONCLUSION: The ADM could be used to joint functional positions of patients with post-burn hypertrophic scan and could produce satisfactory plastic results as dermal substitute.     

微孔异种无细胞真皮复合移植用于烧伤创面烧伤创面F功能评估

目的 观察微孔异种无细胞真皮 (acellular xeno- drmal matrix,ADM)与自体皮片复合移植的临床效果. 方法 将打孔的无细胞真皮与自体的大张刃厚皮、网状皮、邮票皮微粒皮等复合移植 ,用于深度烧伤创面的修复 ,观察自体皮片存活率及创面愈合的色素沉着、弹性与柔韧性等 ,并与单纯自体皮片移植部位进行比较. 结果 微孔无细胞真皮与自体刃厚皮、网状皮及邮票皮移植 ,存活率达 100% ,而与微粒皮移植存活率较低.与单纯自体皮片移植相比 ,复合移植后 3个月 ,色素沉着浅 ,富有弹性与柔韧性 ,明显改善创面愈合质量. 结论 微孔无细胞真皮具有完整的基底膜结构 ,胶原排列规则有序 ,可重建高质量真皮层 ,提高柔韧性、耐磨与耐压性.     

全层皮肤缺损创面移植异种脱细胞真皮基质与薄自体皮重塑基底膜的实验观察

背景异种脱细胞真皮基质与薄自体皮复合移植修复全层皮肤缺损创面取得了良好的效果,但对移植后基底膜的重塑尚不十分清楚.目的观察异种(猪)脱细胞真皮基质与薄自体皮复合移植模型中基底膜重塑的变化规律.设计随机对照实验研究.地点和材料动物实验在上海市烧伤研究所完成,指标检测在上海第二医科大学细胞生物学教研室完成.清洁级雄性SD大鼠(由中国医学科学院上海实验动物中心提供),体质量200～250 g.干预将SD大鼠随机分为两组,每组42只.所有动物均饲养在层流房间内,恒定的湿度与温度,单笼饲养.A组单纯移植薄自体皮组;B组脱细胞猪真皮基质(江苏启东医疗用品研究所提供)+薄自体皮复合移植.将异种(猪)脱细胞真皮基质与薄自体皮复合移植于大鼠创面,分别于移植后1,2,3,4,8,12,16周留取标本,采用免疫组化、透射电镜等方法.主要观察指标观察基底膜中层粘连蛋白的变化规律及12周时基底膜的超微结构,同时以单纯薄自体皮移植进行对照.结果B组移植后1,2,3,4,8,12,16周,层粘连蛋白的表达分别为88.32±2.72,89.22±2.16,88.84±3.43,93.49 ±4.59,87.57±3.33,95.87±1.84,86.57±2.45,A组分别为78.96±1.68,85.37±5.41,82.49±3.73,84.27±2.69,72.88±3.57,88.19±3.36,82.82±2.86,12周时复合皮移植组基底膜结构清晰连续,而单纯移植薄自体皮组基底膜模糊、不连续.结论异种脱细胞真皮基质与薄自体皮复合移植后,基底膜中层粘连蛋白表达增高,可能有利于复合皮移植后基底膜的重塑,增强表皮和真皮之间的连接,进而改善创面愈合质量.     

Healing characteristics of expanded autografts on wounds covered with homograft and Biobrane temporary wound dressing

This study compared the healing characteristics of expanded autografts on wounds with interstices that were closed with cryopreserved cadaver homograft split-thickness skin and wounds with interstices that were closed with the synthetic skin substitute, Biobrane temporary wound dressing. Nine paired wounds in four patients with large burns were used in this study. When Biobrane temporary wound dressing adhered to a wound, epithelial migration did not proceed until it was removed. Although wounds covered with homograft immediately had the appearance of healed wounds, biopsy specimens showed evidence of a delay in epithelial migration. Although these results indicate impaired epithelial migration with the use of both materials, we concluded that homograft offered an advantage because the wounds that were covered with it remained closed during the entire reepithelialization process.     

Promotion of dermal regeneration using pullulan/gelatin porous skin substitute

Tissue‐engineered dermal substitutes represent a promising approach to improve wound healing and provide more sufficient regeneration, compared with current clinical standards on care of large wounds, early excision, and grafting of autografts. However, inadequate regenerative capacity, impaired regeneration/degradation profile, and high cost of current commercial tissue‐engineered dermal regeneration templates hinder their utilization, and the development of an efficient and cost‐effective tissue‐engineered dermal substitute remains a challenge. Inspired from our previously reported data on a pullulan/gelatin scaffold, here we present a new generation of a porous pullulan/gelatin scaffold (PG2) served as a dermal substitute with enhanced chemical and structural characteristics. PG2 shows excellent biocompatibility (viability, migration, and proliferation), assessed by in vitro incorporation of human dermal fibroblasts in comparison with the Integra® dermal regeneration template (Control). When applied on a mouse full‐thickness excisional wound, PG2 shows rapid scaffold degradation, more granulation tissue, more collagen deposition, and more cellularity in comparison with Control at 20 days post surgery. The faster degradation is likely due to the enhanced recruitment of inflammatory macrophages to the scaffold from the wound bed, and that leads to earlier maturation of granulation tissue with less myofibroblastic cells. Collectively, our data reveal PG2's characteristics as an applicable dermal substitute with excellent dermal regeneration, which may attenuate scar formation.     

Application of split-thickness dermal grafts in deep partial- and full-thickness burns: a new source of auto-skin grafting

Early tangential excision of nonviable burn tissue, followed by immediate skin grafting with autograft or allograft, has resulted in the improvement of burn patient survival. The aim of this study was to add split-thickness dermal grafts (STDGs) as a new source of auto-skin grafting tool to our reconstructive armamentarium in deep partial- and full-thickness burns and soft tissue defects. The authors successfully applied STDGs along with split-thickness skin grafts as a new source of auto-skin grafting in 11 deep partial- and full-thickness burns over a period of 1 year without any significant donor site morbidity. Dermal graft take was complete in all but one patient. There was no donor site healing problem, and donor site epithelization was completed generally 1 week later than split-thickness skin graft by semi-open technique. Autologous split-thickness skin grafting still remains the standard therapy for burn wound closure but may be in limited availability in severe burns. The authors conclude that STDGs may be a new source of auto-skin grafting tool in extensive deep partial- and full-thickness burns.     

Weak direct current accelerates split-thickness graft healing on tangentially excised second-degree burns

We have examined the effects of direct current (DC) conducted through silver-nylon dressings on the healing time and morphologic maturation of split-thickness grafts placed on tangentially excised deep partial-thickness burn wounds. Male guinea pigs (n = 120) were used as the experimental hosts. The DC-treated animals required 2 days for complete revascularization of their grafts; control animals required 7 days (p less than 0.01). The DC-treated animals had increased epithelial proliferation at the graft-wound interface as compared with controls (p less than 0.01). Grafts from DC-treated animals were firmly adherent within 4 days, whereas graft adherence in controls was weak before 7 days after grafting. At 3 months after grafting, control animal grafts had mild contraction with moderate hair loss and thick subepidermal fibrosis; the grafts in DC-treated animals expanded with the growth of the animals and had abundant hair growth and significantly reduced dermal fibrosis (p less than 0.01).     

Clinical trials of a living dermal tissue replacement placed beneath meshed, split-thickness skin grafts on excised burn wounds.

We evaluated the ability of Dermagraft (Advanced Tissue Sciences, La Jolla, Calif.), a living tissue analog that is composed of human neonatal fibroblasts, which are grown on a polyglactin acid Vicryl mesh (Ethicon Inc., Somerville, N.J.), to function as a dermal replacement when placed beneath meshed, expanded split-thickness skin grafts (MESTSGs). Full-thickness burn wounds in 17 patients with burns (mean age, 31 years; range, 6 to 69 years; mean burn size, 23.8% total body surface area) were excised to subcutaneous fat (nine patients), to fascia (three patients), or to a combination of deep dermis and fat (five patients). Dermagraft was placed over the experimental sites, which were then covered with MESTSGs. Paired control sites on each patient received MESTSGs only. The results showed that "take" of MESTSGs on control sites was slightly better than take on experimental sites that contained the Dermagraft; however, the differences were not statistically significant. Mesh interstices epithelialized over the surface of the full-thickness wound (control sites) or over the surface of Dermagraft (experimental sites). Wound biopsy specimens demonstrated no evidence of rejection of the cultured allogeneic fibroblasts and minimal inflammatory reaction to the Vicryl fibers. Evidence of continuous basement membrane formation at the epithelial-Dermagraft junction, which was identified by immunohistochemical staining for laminin and type IV collagen, was seen by day 14 beneath the healed epithelium in the skin graft interstices. The Vicryl fibers were hydrolyzed in the wound over a 2-to-4-week period, although some expulsion of fibers occurred as the healing epithelium advanced to close the MESTSG interstices. Elastic fibers were not seen in neodermal tissue in either control or experimental wounds at periods of up to 1 year after grafting. Further trials with this living tissue replacement are in progress.     

Overexpression of vascular endothelial growth factor accelerates early vascularization and improves healing of genetically modified cultured skin substitutes.

Cultured skin substitutes (CSS) lack a vascular plexus, leading to slower vascularization after grafting than split-thickness skin autograft. CSS containing keratinocytes genetically modified to overexpress vascular endothelial growth factor (VEGF) were previously shown to exhibit enhanced vascularization up to 2 weeks after grafting to athymic mice. The present study examines whether enhanced vascularization compared with controls persists after stable engraftment is achieved and analyzes VEGF expression, wound contraction, and engraftment. Control and VEGF-modified (VEGF+) CSS were grafted onto full-thickness wounds in athymic mice. VEGF expression was detected in VEGF+ CSS 14 weeks after grafting. Graft contraction was significantly lower in VEGF+ CSS compared with controls, suggesting more stable engraftment and better tissue development. Positive HLA-ABC staining, indicating persistence of human cells, was seen in 86.7% (13/15) of grafted VEGF+ CSS, compared with 58.3% (7/12) of controls. Differences in dermal vascularization between control and VEGF+ grafts were significant 1 week after surgery, but not at later times. However, the distribution of vessels was different, with more vessels in the upper dermis of VEGF+ grafts. These results suggest that VEGF overexpression in genetically modified CSS acts to accelerate early graft vascularization and can contribute to improved healing of full-thickness skin wounds.     

无细胞异体真皮基质在烧伤后整形患者功能部位的应用

AIM:To investigate the effect of allogeneic acellular dermal matrix(ADM) on cograft in joint functional positions of patients with post burn plastic operation. METHODS:9 patients with hypertrophic scar and joint dysfunction after severe burns were used. After pre treating with trypsin and TritonX 100, 13 reticulated ADM were overlapped with autogenous ultrathin split thickness skin grafts(USTS), and were transplanted to the scar excision wounds in the joints of four limbs at the same time. The neighbouring autogenous thin split thickness skin grafts(TSTS) were used as control.RESULTS:The composite skin grafts as well as the controls were all survived. The rejection and hypertrophic scars were not found during (1- 5) years follow up studies. The appearance, fiber and function of composite skin grafts were near to normal skins. CONCLUSION:The ADM could be used to joint functional positions of patients with post burn hypertrophic scars and could produce satisfactory plastic results as dermal substitute.     

异种(猪)无细胞真皮基质与自体微粒皮复合移植修复深度烧伤创面的实验研究

目的　观察异种 (猪 )无细胞真皮支架与自体微粒皮加同种异体皮复合移植修复全层皮肤缺损的效果。方法　 4 8只 SD大鼠背部全层皮肤缺损创面 ,分别行异种 (猪 )无细胞真皮支架与自体微粒皮移植(复合皮组 )和单纯微粒皮移植 (对照组 ) ,术后定期观察创面愈合情况并行创面愈合率和收缩率的计算 ,同时取创面组织进行组织学观察。结果　复合皮组的创面愈合情况良好 ,未见明显挛缩 ,皮肤弹性较好 ,两组异体皮成活情况及创面愈合情况相近 ,但是复合皮组创面收缩率显著低于对照组。组织学观察复合皮组上皮化良好 ,胶原纤维排列有序 ,基底膜结构完整。结论　异种 (猪 )无细胞真皮支架与自体微粒皮复合移植能修复全层皮肤缺损 ,改善创面愈合质量。     

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin‐induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha‐smooth muscle actin and transforming growth factor‐beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing.     

复合皮移植修复创面的临床观察

目的 观察移植以小肠黏膜下层（SIS）为真皮替代物体外构建的复合皮修复创面的效果。方法制取家猪空肠黏膜下层为真皮替代物,致密层表面种植自体表皮细胞,构建人工复合皮,移植复合皮修复37例皮肤肉芽组织创面;观察复合皮早期成活率,并分别于移植后第1周、第2周取复合皮标本作组织学观察。结果表皮细胞在SIS表面定位、生长,24例移植的复合皮早期成活良好,SIS内新生血管形成,炎性细胞浸润,无移植排斥反应。结论以SIS为真皮替代物构建的复合皮,有可能成为一种新型的创面修复材料。     

脱细胞异体敷料皮在烧伤治疗中的应用

目的:探讨脱细胞异体敷料皮在烧伤创面应用的效果。方法:2003—2004年在15例烧伤患者中应用脱细胞异体敷料皮覆盖烧伤创面及切削痂创面,分期自体植皮。结果:所有创面顺利愈合,取得良好效果。结论:脱细胞异体敷料皮使用简便,能促进创面愈合及提高植皮成功率,是一种良好的创面覆盖材料。     

异体脱细胞真皮基质作为组织工程皮肤真皮支架的可行性

背景:组织工程皮肤是目前研究皮肤损伤修复重建的重要手段之一,异体脱细胞真皮基质不存在免疫原性,在异体移植时不会发生排斥反应,是比较理想的真皮替代物。目的:观察异体脱细胞真皮基质的组织相容性。方法:以正常人体真皮组织作为对照,通过体外、体内细胞毒性实验检测异体脱细胞真皮基质的组织相容性,以膨胀度、饱和含水量及生物力学分析检测异体脱细胞真皮基质的亲水性及机械性能。结果与结论:真皮基质中未见任何细胞成分,其网孔直径介于100～180μm之间。脱细胞真皮基质组饱和含水量为(69.6±3.97)%,膨胀度2.30±0.42,最大断裂力为(3.082±0.046)N,与对照组相比,差异无显著性意义(P>0.05)。体内外细胞毒性检测,未见明显细胞生长抑制及免疫排斥反应。提示异体脱细胞真皮基质机械性能接近正常皮肤,组织相容性好,免疫排斥反应小,是构建组织工程皮肤理想的真皮材料。     

The 1999 clinical research award. Cultured skin substitutes combined with Integra Artificial Skin to replace native skin autograft and allograft for the closure of excised full-thickness burns

Prompt and permanent closure of excised full-thickness burns remains a critical factor in a patient's recovery from massive burn injuries. Hypothetically, Integra Artificial Skin (Integra) may replace the need for allografts for immediate wound coverage, and cultured skin substitutes (CSS) that contain stratified epithelium may replace the need for autografts for definitive wound closure. To test this hypothesis, 3 patients with full-thickness burns of greater than 60% of their total body surface areas had their eschar excised within 14 days of admission. Integra was applied, and a skin biopsy was collected from each patient for the preparation of CSS. At 3 weeks or more after the application of the Integra and the collection of skin biopsies, the outer silastic cover of the Integra was removed and CSS were grafted. The CSS were irrigated with nutrients and antimicrobials for 6 days and then dressed with antimicrobial ointment and cotton gauze. Treated wounds were traced on days 14 and 28 after the grafting of CSS for determination of engraftment and wound closure, respectively. Cost analysis was not performed. Engraftment on postoperative day (POD) 14 was 98%+/-1% (mean +/- standard error of the mean), the ratio of closed:donor areas on POD 28 was 52.3+/-5.2, and no treated sites required regrafting. The histology of the closed wounds showed stable epithelium that covered a layer of newly formed fibrovascular tissue above the reticulated structure of the degrading Integra. The clinical outcomes of the closed wounds after POD 28 demonstrated smooth, pliable, and hypopigmented skin. Two patients who had received CSS grafts over Integra on their backs were positioned supine on air beds from POD 8 or POD 9 with minimal graft loss because of mechanical loading. One patient with a full-thickness burn of 88% of the total body surface area was covered definitively at 55 days postburn. These results demonstrate that the combination of CSS and Integra can accomplish functionally stable and cosmetically acceptable wound closure in patients with extensive full-thickness burns. This combination of alternatives to the conventional grafting of split-thickness skin permits the substitution of cadaveric allograft with Integra and the substitution of donor autograft with CSS. This approach to the closure of excised full-thickness burns is expected to reduce greatly the time to definitive closure of burn wounds and to reduce the morbidity associated with the harvesting of donor sites for split-thickness skin autografts.     

应用真皮模板改善创面愈合质量的研究

目的:动态观察真皮模板应用后对创面微血管形成的影响,为真皮模板应用改善创面愈合质量提供依据。方法:选取2001/2002Ш度小面积烧伤患者,采用真皮模板(异体无细胞真皮基质)加自体薄皮复合移植的方法,修复Ш度烧伤患者切痂后创面,并以单纯自体薄皮移植作为对照,分别于术后1,2,3,4,6周取材,实验组与对照组各15例次,采用苏木精-伊红染色观察、免疫组化技术检测标本中CD34的表达。结果:在术后1～6周,随着创面的修复,真皮模板组CD34表达逐渐降低,而薄皮组CD34持续高表达,且有逐渐升高趋势,其结果与HE染色所见一致。两组比较具有显著性差异。结论:真皮模板与自体薄皮复合移植后,可以抑制创面毛细血管超常增生,从而减少瘢痕形成,改善创面愈合质量。