Tissue-Engineered Products for Skin Regenerative Medicine

In a general wound healing process, foreign bodies and tissue detritus have to be broken down and then a new tissue is produced. However, the new tissue formation sometimes fails to proceed under the impaired conditions such as burn injury and intractable skin ulcer. A major obstruction to wound healing is infection. Another obstruction to wound healing is deficiency of growth factors. The endogenous levels of growth factors are reduced in some chronic wounds. To improve these wound conditions, researchers have been trying to create several types of artificial skins. The tissue-engineered products include three prime constituents, i.e., cells, growth factors, and materials. In this review, the practical design of tissue-engineered products for skin regenerative medicine is introduced. The first design makes it possible to release silver sulfadiazine (AgSD) from a wound dressing. The second design makes it possible to release Epidermal Growth Factor (EGF) from a wound dressing or a skin care product composed of hyaluronic acid spongy sheet containing bioactive ingredients. The third design makes it possible to release several types of growth factors from allogeneic fibroblasts within cultured dermal substitute. This tissue-engineered product is prepared by seeding allogeneic fibroblasts into a collagen and hyaluronic acid spongy sheet. Although allogeneic cells are rejected gradually in immune system, they are able to release some types of growth factors, thereby regenerating a damaged tissue. The clinical study demonstrates that these tissue-engineered products are promising for the treatment of burn injury and intractable skin ulcer.

Angiogenesis in tissue-engineered nerves evaluated objectively using MICROFIL perfusion and micro-CT scanning

Angiogenesis is a key process in regenerative medicine generally, as well as in the specific field of nerve regeneration. However, no convenient and objective method for evaluating the angiogenesis of tissue-engineered nerves has been reported. In this study, tissue-engineered nerves were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds to bridge 10-mm sciatic nerve defects in rats. Four weeks after surgery, three-dimensional blood vessel reconstructions were made through MICROFIL perfusion and micro-CT scanning, and parameter analysis of the tissue-engineered nerves was performed. New blood vessels grew into the tissue-engineered nerves from three main directions： the proximal end, the distal end, and the middle. The parameter analysis of the three-dimensional blood vessel images yielded several parameters, including the number, diameter, connection, and spatial distribution of blood vessels. The new blood vessels were mainly capillaries and microvessels, with diameters ranging from 9 to 301 μm. The blood vessels with diameters from 27 to 155 μm accounted for 82.84% of the new vessels. The microvessels in the tissue-engineered nerves implanted in vivo were relatively well-identified using the MICROFIL perfusion and micro-CT scanning method, which allows the evaluation and comparison of differences and changes of angiogenesis in tissue-engineered nerves implanted in vivo.

Bioengineered skin organoids:from development to applications

Signifcant advancements have been made in recent years in the development of highly sophisticated skin organoids.Serving as three-dimensional(3D)models that mimic human skin,these organoids have evolved into complex structures and are increasingly recognized as efective alternatives to traditional culture models and human skin due to their ability to overcome the limitations of two-dimensional(2D)systems and ethical concerns.The inherent plasticity of skin organoids allows for their construction into physiological and pathological models,enabling the study of skin development and dynamic changes.This review provides an overview of the pivotal work in the progression from 3D layered epidermis to cyst-like skin organoids with appendages.Furthermore,it highlights the latest advancements in organoid construction facilitated by state-of-the-art engineering techniques,such as 3D printing and microfuidic devices.The review also summarizes and discusses the diverse applications of skin organoids in developmental biology,disease modelling,regenerative medicine,and personalized medicine,while considering their prospects and limitations.

Vital roles of stem cells and biomaterials in skin tissue engineering

Tissue engineering essentially refers to technology for growing new human tissue and is distinct from regenerative medicine. Currently, pieces of skin are already being fabricated for clinical use and many other tissue types may be fabricated in the future.Tissue engineering was first defined in 1987 by the United States National Science Foundation which critically discussed the future targets of bioengineering research and its consequences. The principles of tissue engineering are to initiate cell cultures in vitro, grow them on scaffolds in situ and transplant the composite into a recipient in vivo. From the beginning, scaffolds have been necessary in tissue engineering applications. Regardless, the latest technology has redirected established approaches by omitting scaffolds. Currently, scientists from diverse research institutes are engineering skin without scaffolds. Due to their advantageous properties, stem cells have robustly transformed the tissue engineering field as part of an engineered bilayered skin substitute that will later be discussed in detail. Additionally, utilizing biomaterials or skin replacement products in skin tissue engineering as strategy to successfully direct cell proliferation and differentiation as well as to optimize the safety of handling during grafting is beneficial. This approach has also led to the cells' application in developing the novel skin substitute that will be briefly explained in this review.

Realization Feature of Mesenchymal Dermal Cells Tissue Engineering Construction Response in Granulating Wound Transplantation in Relation with Time-Frame

Derma is progenitor cells sours, that are able to differentiate further in several mesodermal lineage and neural and endodermal lineage. Culture conditions, skin taking site and culture medium composition considerably contribute to it. Spheroid cultured mesenchymal dermal cells contribution to skin regeneration in granulating wound in rat model was estimated.

Noncoding RNAs and Their Potential Therapeutic Applications in Tissue Engineering

Tissue engineering is a relatively new but rapidly developing field in the medical sciences. Noncoding RNAs(ncRNAs) are functional RNA molecules without a protein-coding function; they can regulate cellular behavior and change the biological milieu of the tissue. The application of ncRNAs in tissue engineering is starting to attract increasing attention as a means of resolving a large number of unmet healthcare needs, although ncRNA-based approaches have not yet entered clinical practice. In-depth research on the regulation and delivery of ncRNAs may improve their application in tissue engineering.The aim of this review is: to outline essential ncRNAs that are related to tissue engineering for the repair and regeneration of nerve, skin, liver, vascular system, and muscle tissue; to discuss their regulation and delivery; and to anticipate their potential therapeutic applications.

Use of autologous tissue engineered skin to treat porcine full-thickness skin defects

Objective. To explore a feasible method to repair full-thickness skin defects utilizing tissue engineered techniques. Methods： The Changfeng hybrid swines were used and the skin specimens were cut from the posterior limb girdle region, from which the keratinocytes and fibroblasts were isolated and harvested by trypsin, EDTA, and type II collagenase. The cells were seeded in Petri dishes for primary culture. When the cells were in logarithmic growth phase, they were treated with trypsin to separate them from the floor of the tissue culture dishes. A biodegradable material, Pluronic F-127, was prefabricated and mixed with these cells, and then the cell-Pluronic compounds were seeded evenly into a polyglycolic acid （PGA）. Then the constructs were replanted to the autologous animals to repair the full-thickness skin defects. Histology and immunohistochemistry of the neotissue were observed in 1, 2, 4, and 8 postoperative weeks. Results. The cell-Pluronic F-127-PGA compounds repaired autologous full-thickness skin defects 1 week after implantation. Histologically, the tissue engineered skin was similar to the normal skin with stratified epidermis overlying a moderately thick collageneous dermis. Three of the structural proteins in the epidermal basement membrane zone, type IV collagen, laminin, and type VII collagen were detected using immunohistochemicai methods. Conclusions ： By studying the histology and immunohistochemistry of the neotissue, the bioengineered skin graft holds great promise for improving healing of the skin defects.

In vitro comparison of human plasma-based and self-assembled tissue-engineered skin substitutes:two different manufacturing processes for the treatment of deep and difficult to heal injuries

Background:The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes(hbTESSs)designed for the treatment of severely burned patients.These are the scaffold-free self-assembled skin substitute(SASS)and the human plasma-based skin substitute(HPSS).Methods:Fibroblasts and keratinocytes from three humans were extracted from skin biopsies(N=3)and cells from the same donor were used to produce both hbTESS models.For SASS manu-facture,keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced(n=12),while for HPSS production,keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial(Fibrin),plasma combined with hyaluronic acid(Fibrin-HA)or plasma combined with collagen(Fibrin-Col)(n/biomaterial=9).The production time was 46-55 days for SASSs and 32-39 days for HPSSs.Substitutes were characterized by histology,mechanical testing,PrestoBlue™-assay,immunofluorescence(Ki67,Keratin(K)10,K15,K19,Loricrin,type IV collagen)and Western blot(type I and IV collagens).Results:The SASSs were more resistant to tensile forces(p-value<0.01)but less elastic(p-value<0.001)compared to HPSSs.A higher number of proliferative Ki67+cells were found in SASSs although their metabolic activity was lower.After epidermal differentiation,no significant difference was observed in the expression of K10,K15,K19 and Loricrin.Overall,the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs.Conclusions:This study demonstrates,for the first time,that both hbTESS models present similar in vitro biological characteristics.However,mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.

Reversibly immortalized keratinocytes(iKera)facilitate re-epithelization and skin wound healing:Potential applications in cell-based skin tissue engineering

Skin injury is repaired through a multi-phase wound healing process of tissue granulation and re-epithelialization.Any failure in the healing process may lead to chronic non-healing wounds or abnormal scar formation.Although significant progress has been made in developing novel scaffolds and/or cell-based therapeutic strategies to promote wound healing,effective management of large chronic skin wounds remains a clinical challenge.Keratinocytes are critical to re-epithelialization and wound healing.Here,we investigated whether exogenous keratinocytes,in combination with a citrate-based scaffold,enhanced skin wound healing.We first established reversibly immortalized mouse keratinocytes(iKera),and confirmed that the iKera cells expressed keratinocyte markers,and were responsive to UVB treatment,and were non-tumorigenic.In a proof-of-principle experiment,we demonstrated that iKera cells embedded in citrate-based scaffold PPCN provided more effective re-epithelialization and cutaneous wound healing than that of either PPCN or iKera cells alone,in a mouse skin wound model.Thus,these results demonstrate that iKera cells may serve as a valuable skin epithelial source when,combining with appropriate biocompatible scaffolds,to investigate cutaneous wound healing and skin regeneration.

Recent advancements in applications of chitosan-based biomaterials for skin tissue engineering

The use of polymer based composites in the treatment of skin tissue damages,has got huge attention in clinical demand,which enforced the scientists to improve the methods of biopolymer designing in order to obtain highly efficient system for complete restoration of damaged tissue.In last few decades,chitosan-based biomaterials have major applications in skin tissue engineering due to its biocompatible,hemostatic,antimicrobial and biodegradable capabilities.This article overviewed the promising biological properties of chitosan and further discussed the various preparation methods involved in chitosan-based biomaterials.In addition,this review also gave a comprehensive discussion of different forms of chitosan-based biomaterials including membrane,sponge,nanofiber and hydrogel that were extensively employed in skin tissue engineering.This review will help to form a base for the advanced applications of chitosan-based biomaterials in treatment of skin tissue damages.

壳多糖-胶原-糖胺聚糖凝胶人工皮肤的初步研究

目的 研制一种新型的胶原凝胶类人工皮肤。方法 制备壳多糖 -胶原 -糖胺聚糖 (GAGs) -成纤维细胞真皮替代物 (DE) ,观察成纤维细胞 (FB)在凝胶中的生长情况和不同因素对凝胶收缩的影响 ,并绘制 FB生长曲线和凝胶收缩曲线。了解不同含量壳多糖对 FB和角质形成细胞 (KC)生长的影响 ,不同含量壳多糖 DE的抗感染能力。再于“成熟”的 DE表面接种 KC,先浸没培养 ,后行气液界面培养 ,构建完整的人工皮肤。对 DE和人工皮肤行组织学和电镜分析。结果 DE收缩度与 FB数量呈正比 ,终末收缩度与胶原蛋白浓度成反比 ;FB在凝胶中 2～ 9天呈指数增生。 DE基质配方对 FB的生长无明显抑制作用 ,但可促进 KC的生长 ,对金黄色葡萄球菌的抑制作用随壳多糖含量增大而增强。扫描电镜示 DE有丰富的微孔结构。人工皮肤组织学观察见类似于正常皮肤 ,有分化良好的表皮和致密的真皮。结论壳多糖 -胶原 - GAGs胶原凝胶人工皮肤是一种新型、有一定抗感染能力的活人工皮肤。

3D-SC人工皮肤材料体内降解实验研究

目的测试3D-SC人工皮肤材料在动物体内的降解性。方法将一定大小的材料真空干燥后植入动物背部皮下,分阶段取出植入材料,观察材料及周围组织情况,将取出的材料真空干燥至恒重。计算材料的质量损失百分率。结果未见材料周围组织异常,第3天材料质量增加3.39%,以后逐渐降低,第7、14、28、42、56、70、84、98天材料质量损失百分率分别为2.06%、7.58%、14.31%、26.83%、37.45%、38.08%、85.45%、87.26%。结论3D-SC人工皮肤材料是一种具有优良生物降解性能的材料,降解产物无毒性,在体内降解过程中表现出较好的生物相容性。70d内降解相对较慢,70d后降解速度加快,降解产物被动物吸收。

Protein-spatiotemporal partition releasing gradient porous scaffolds and anti-inflammatory and antioxidant regulation remodel tissue engineered anisotropic meniscus

Meniscus is a wedge-shaped fibrocartilaginous tissue,playing important roles in maintaining joint stability and function.Meniscus injuries are difficult to heal and frequently progress into structural breakdown,which then leads to osteoarthritis.Regeneration of heterogeneous tissue engineering meniscus(TEM)continues to be a scientific and translational challenge.The morphology,tissue architecture,mechanical strength,and functional applications of the cultivated TEMs have not been able to meet clinical needs,which may due to the negligent attention on the importance of microenvironment in vitro and in vivo.Herein,we combined the 3D(three-dimensional)-printed gradient porous scaffolds,spatiotemporal partition release of growth factors,and anti-inflammatory and anti-oxidant microenvironment regulation of Ac2-26 peptide to prepare a versatile meniscus composite scaffold with heterogeneous bionic structures,excellent biomechanical properties and anti-inflammatory and anti-oxidant effects.By observing the results of cell activity and differentiation,and biomechanics under anti-inflammatory and anti-oxidant microenvironments in vitro,we explored the effects of anti-inflammatory and anti-oxidant microenvironments on construction of regional and functional heterogeneous TEM via the growth process regulation,with a view to cultivating a high-quality of TEM from bench to bedside.

壳聚糖-明胶海绵与脐带间充质干细胞修复皮肤伤口的差异

背景:脐带间充质干细胞有多向分化和自我复制能力,在一定条件下可以向骨细胞、软骨细胞、平滑肌细胞、神经细胞以及血管内皮细胞分化,有助于表皮再植和血管生成。目的:比较脐带间充质干细胞与壳聚糖-明胶海绵在伤口愈合方面的作用。方法:将30只雄性SD大鼠等分为模型组、壳聚糖-明胶海绵组和脐带间充质干细胞组。3组大鼠均在背部建立3 cm×3 cm全层皮肤缺损创面。造模后1 d起,模型组连续15 d皮肤伤口边缘注射PBS;壳聚糖-明胶海绵组连续15 d在皮肤伤口边缘注射壳聚糖凝胶;脐带间充质干细胞组在皮肤伤口边缘注射脐带间充质干细胞。结果与结论:与模型组相比,壳聚糖-明胶海绵组和脐带间充质干细胞组大鼠创面愈合率较高,表皮厚度降低,胶原纤维面积升高;且脐带间充质干细胞组效果优于壳聚糖-明胶海绵组。说明壳聚糖-明胶海绵和脐带间充质干细胞均可有效修复皮肤损伤,且脐带间充质干细胞效果更佳。

聚乳酸-羟基乙酸共聚物和胶原膜对体外培养人表皮角质细胞分泌细胞因子的影响

目的探讨聚乳酸-羟基乙酸共聚物(PLGA)和胶原膜对表皮角质细胞分泌细胞因子的影响及其在创面修复中的可能作用机制。方法实验组分为PLGA组和胶原膜组,表皮角质细胞以3×107cm-2密度分别种植于PLGA和胶原膜上;对照组为单层表皮角质细胞培养。于第7天和第14天扫描电镜下观察细胞生长状况;种植后第1,3,5,7,14天收集细胞培养上清液,检测乳酸脱氢酶(LDH)水平观察细胞损伤程度,应用ELISA法检测培养上清液中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素8(IL-8)的水平。结果表皮角质细胞种植后第7天,扫描电镜下可见细胞粘附于PLGA和胶原膜呈原有三维结构稳定均一生长,第14天有纤维丝状细胞外基质生成。实验组和对照组各时相点LDH水平差别无统计学意义。PLGA组和胶原膜组表皮角质细胞分泌IL-1β、IL-6和IL-8水平与对照组比较均有不同程度上升,其差别具有统计学意义(P<0.05或P<0.01)。结论PLGA和胶原膜在构建组织工程皮肤中作为一种生物支架,增强表皮角质细胞分泌IL-1β、IL-6和IL-8能力,对加速创面愈合可能起着积极作用。

神经系统在调控皮肤创伤修复和瘢痕愈合中的重要作用

背景:皮肤创伤修复是一个极其复杂的过程,神经系统在调控创伤修复和瘢痕愈合方面发挥重要作用。目的:对皮肤创伤修复过程中神经系统的作用,包括相关因子、多种细胞对损伤皮肤修复的作用进行综述。方法:检索PubMed及中国知网数据库2003-2024年发表的文献,英文检索词为“skin wound healing,nervous system,nerve regeneration,growth factor”,中文检索词为“皮肤创伤修复,皮肤神经支配,神经肽,生长因子,细胞协同作用”。最终筛选出83篇文献进行分析。结果与结论:①皮肤受到自主神经和感觉神经的密集支配,越来越多的证据表明,皮肤神经系统不仅负责向中枢神经系统传递感觉信息,而且在包括伤口愈合在内的各种皮肤功能中发挥重要的作用。②神经系统作用于皮肤创伤愈合的不同阶段,能够调节血管再生,促进相关生长因子及神经递质释放,和不同类型细胞协同作用从而促进皮肤创面愈合;因此,研究神经系统作用的潜在机制成为促进皮肤创伤愈合的有力靶点。

皮肤组织工程种子细胞-表皮干细胞提供来源选择的研究

目的寻找表皮基底层细胞中含有较高干细胞比例的供皮区。方法选择不同供皮部位，利用免疫组织化学的方法，以K19和整合素β1作为表皮干细胞鉴定标记。结果皮肤基底层表皮干细胞以包皮及阴囊最多，其次是臀部，背部多于胸部。四肢近端外侧多于四肢近端内侧，头皮、手掌及足底皮肤基底层干细胞数量较少。结论从组织工程种子细胞的供应来源考虑，以男性包皮最佳，在客观上也容易获得。

壳聚糖-明胶支架材料的制备及性能研究

目的研究用于组织修复的壳聚糖-明胶支架材料的制备及其性能,为其进一步的细胞培养以及组织工程皮肤的临床应用积累数据。方法将壳聚糖与明胶在一定条件下共混,预冻,冷冻干燥并交联得到支架材料,考察其性能。结果制备过程中,预冻溶液的固含量、壳聚糖与明胶的配比以及预冻温度等参数对支架材料的微观结构、表观密度、含水量和力学性能有较大影响。结论采用冷冻干燥法可以成功地制备壳聚糖-明胶支架材料,通过控制其反应条件可以调整支架孔隙结构和性能,从而得到满足需要的支架材料。