人工毛乳头异种移植诱导大鼠足垫毛囊形成

目的观察人头皮毛乳头细胞微囊（人工毛乳头）异种移植诱导大鼠足垫毛囊形成的能力。方法以海藻酸钠-多聚赖氨酸-海藻酸钠（alginate- polylysine - alginate, APA）微囊包裹分离培养的毛乳头细胞；对体外培养1、4周的毛乳头细胞微囊及无APA的微囊对照组行组织学观察；取培养4周的毛乳头细胞微囊移植至大鼠足垫皮下，6周后取材行组织学检查。结果毛乳头细胞微囊体外培养1周后，毛乳头细胞周围出现细胞外基质；4周后，囊中形成“类毛乳头样结构”；人头皮毛乳头细胞微囊移植至大鼠足垫6周后，移植部位及其周围皮下有大量毛囊及皮脂腺结构形成。结论人工毛乳头诱导并参与了无毛区域新生毛囊及皮脂腺的组织构成。     

微囊化人头皮毛乳头细胞诱导小鼠耳毛囊再生的研究

目的 观察人头皮毛乳头细胞海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate，APA）微囊是否具备诱导小鼠耳部毛囊再生的功能；寻找理想的微囊直径。方法 以APA微囊包裹体外分离培养的人头皮毛乳头细胞；将毛乳头细胞微囊移植至小鼠耳部皮下，6周后局部取材行组织学检查；共聚焦显微镜下观察葡聚糖-荧光素在APA微囊中的扩散速度和扩散方式，并对比相同时间、不同直径的APA微囊中葡聚糖-荧光素的强度，综合分析确定最佳的微囊直径。结果 组织学检查显示：移植部位皮下有密集的同心圆状毛囊结构形成，其数量、大小、分化程度等与对照组明显不同。荧光素以同心圆状、逐层渗透的方式向APA微囊中扩散；相同时间内荧光强度比较：小囊组〉中囊组〉大囊组。结论 微囊化毛乳头细胞具备诱导毛囊再生的生理功能；微囊理想的直径是400μm。     

人工毛乳头异种移植诱导大鼠足垫毛囊形成

目的观察人头皮毛乳头细胞微囊(人工毛乳头)异种移植诱导大鼠足垫毛囊形成的能力。方法以海藻酸钠-多聚赖氨酸-海藻酸钠(alginate-polylysine-alginate,APA)微囊包裹分离培养的毛乳头细胞;对体外培养1、4周的毛乳头细胞微囊及无APA的微囊对照组行组织学观察;取培养4周的毛乳头细胞微囊移植至大鼠足垫皮下,6周后取材行组织学检查。结果毛乳头细胞微囊体外培养1周后,毛乳头细胞周围出现细胞外基质;4周后,囊中形成“类毛乳头样结构”;人头皮毛乳头细胞微囊移植至大鼠足垫6周后,移植部位及其周围皮下有大量毛囊及皮脂腺结构形成。结论人工毛乳头诱导并参与了无毛区域新生毛囊及皮脂腺的组织构成。     

毛乳头细胞诱导毛囊形成的研究

目的 探讨培养的毛乳头细胞在体内外条件下诱导毛囊形成的可能性。方法 采用酶消化法获得毛乳头细胞、真皮鞘细胞、毛囊上、下段及球部细胞，进行毛囊组织工程重建，或用游离细胞混合移植于棵鼠，组织学观察毛囊形成情况。结果 毛囊间表皮细胞、毛囊上段上皮细胞、下段上皮细胞和球部细胞在间质细胞凝胶上均可形成双层结构的组织工程皮肤，在真皮鞘细胞胶原凝胶上毛囊的上、下段上皮细胞形成了毛囊结构，移植于棵鼠后8周毛乳头细胞胶原凝胶诱导毛囊上、下段细胞形成了毛囊。低代毛乳头细胞与毛囊上皮细胞混合移植形成了数量较多、结构典型的毛囊，并有肉眼可见的毛发纤维产生。结论 毛囊的真皮成分细胞即毛乳头细胞、真皮鞘细胞在体内、外均具有诱导毛囊形成的能力，通过与毛囊上皮细胞之间的相互作用，可诱导毛囊形成。     

毛发诱导结合自体毛囊单位移植技术进行毛发定向重建的研究

目的 将异位诱导形成的毛发组织进行移植,实现毛发的定向重建.方法 制备新生C57鼠的皮肤细胞悬液注射于裸鼠体内,诱导形成皮下异位毛发组织,取出后在体视显微镜下分离成单个毛囊单位,移植至裸鼠体表,并进行大体观察和组织学检测.结果 按照预定密度移植后的毛发成活良好并呈周期性生长,组织学可见各时期相应的毛囊形态.结论 诱导形成的毛发组织可以作为移植物,通过毛囊单位移植的方式,实现毛发组织密度和方向的可控性重建.     

毛乳头细胞和表皮细胞构建带附属器的组织工程皮肤

目的根据毛囊形成原理,以人毛乳头细胞和表皮细胞为种子细胞,构建带附属器的组织工程皮肤替代物。方法分实验组和对照组,实验组皮肤替代物真皮层接种人毛乳头细胞和表皮细胞;对照组真皮层不接种任何细胞。在裸鼠背部作一圆形全层皮肤缺损,将两组皮肤替代物气-液界面培养5 d后移植到裸鼠背部。观察创面愈合情况,并在移植后第4、6、8周取材,进行组织学观察。结果皮肤替代物移植后2周,实验组创面已完全愈合,移植后4周对照组创面才完全愈合,并且对照组创面收缩比实验组明显。移植后6周,实验组真皮层内见到毛囊样结构和皮脂腺结构,对照组未见毛囊样结构和皮脂腺样结构。结论将人的毛乳头细胞和表皮细胞共同种植在皮肤替代物的真皮层,可以构建出带附属器的组织工程皮肤替代物,这种替代物对创面的修复能力更强。     

新生鼠皮肤细胞以小室法移植后构建毛囊发育模型

目的 构建一种简便、可靠、直观的毛囊发育模型以检测毛囊细胞的毛发诱导能力,探讨毛囊形态发生和周期循环的分子机制.方法 于裸鼠背部植入一开放小室,将新生C57BL/6鼠皮肤中的真皮和表皮细胞分离,并按一定的比例混合移植至小室内,1周后拆除小室,观察毛发形成及毛发拔除后的再生情况.结果 细胞移植后1周,创面湿润无明显收缩,中间有淡红色半透明组织形成.移植后2周,创面完全愈合.移植后3周有黑色毛发长出皮肤.移植后4周,浓密黑色毛发垂直于皮肤表面生长,石蜡切片HE染色见毛囊结构发育完整.毛发拔除后1周能够再生出新的毛发.细胞以不同的比例移植,真皮细胞数量为1 × 107、表皮细胞数量降至1 ×106时,毛囊重构的效率并无明显改变.表皮细胞数量为1×107、真皮细胞数量降至5×106或者更少时,再生毛发的数量明显减少,两者单独移植均无毛发生长.结论 新生鼠皮肤细胞以小室法移植后可以构建毛囊发育的完整模型,并可用于检测毛囊细胞的诱导能力,以研究毛囊形态的发生和周期循环的分子机制.     

小鼠皮肤表皮和真皮细胞共移植诱导毛囊再生实验研究

目的探讨绿色荧光蛋白(green fluorescent protein,GFP)标记的C57小鼠皮肤表皮和真皮细胞共移植诱导裸鼠毛囊重建的实验研究。方法取C57-GFP新生及成年小鼠分离表皮与真皮,分别制备成年小鼠原代真皮-表皮混合细胞(按1∶1比例混合)、成年小鼠原代真皮细胞、新生小鼠第3代真皮细胞,荧光显微镜观察细胞激发绿色荧光情况,免疫细胞化学检测培养的新生小鼠第3代真皮细胞毛囊干细胞标志。取Balb/c裸鼠,分别将成年小鼠原代真皮-表皮混合细胞(A组)、成年小鼠原代真皮细胞(B组)、新生小鼠第3代真皮细胞(C组)及生理盐水(D组)移植至裸鼠皮下,术后4、5、6周行大体观察,6周时取移植部位皮肤行GFP免疫组织化学染色观察各组毛囊形成情况。结果荧光显微镜观察示,分离、培养的C57-GFP新生及成年小鼠表皮及真皮细胞均能激发绿色荧光;免疫细胞化学检测培养的第3代真皮细胞中毛囊干细胞标志,显示波形蛋白、α-平滑肌肌动蛋白强阳性,表明细胞多为真皮鞘细胞;部分细胞表达毛乳头细胞标志CD133、多能聚糖及毛囊隆突部标志角蛋白15。裸鼠皮下移植实验:大体观察示A组可见接种部位皮下呈灰黑色,但未见毛发穿出皮肤表面;B、C、D组裸鼠皮肤均无着色。免疫组织化学染色示,A组形成新的、完整的毛囊结构或参与形成毛囊;B组GFP阳性细胞多表达于毛囊真皮鞘、外根鞘;C组GFP阳性细胞主要分布于毛囊真皮鞘、外根鞘、毛乳头,在汗腺中也有表达;D组GFP染色呈阴性。结论小鼠皮肤的表皮和真皮细胞在裸鼠体内相互作用可形成完整的毛囊结构,而仅移植新鲜分离或培养的真皮细胞则主要参与毛囊形成,无法形成完整的毛囊结构。     

微囊化新生猪甲状旁腺细胞异种移植的实验研究

目的 探讨微囊化新生猪甲状旁腺细胞异种移植治疗大鼠甲状旁腺功能低下症的效果。方法 应用微囊化技术，制备微囊化（海藻酸钠－聚赖氨酸－海藻酸钠生物微胶囊）新生猪甲状旁腺细胞，32只去甲状旁腺的Wistar大鼠随机分成微囊组、非微囊组、空囊组和对照组，分别移植微囊化新生猪甲状旁腺细胞、甲状旁腺细胞、空微囊及生理盐水。移植后监测血钙及甲状旁腺素水平40周，40周后回收移植物，透射电镜检查。结果 移植后，微囊组大鼠血钙及甲状旁腺素水平恢复到正常范围内，直至观察结束时（40周），透射电镜检查显示移植物存活良好；非微囊组、空囊组和对照组大鼠的血钙及甲状旁腺素水平无改善。结论 微囊化新生猪甲状旁腺细胞异种移植在不用免疫抑制剂情况下，可以在大鼠体内存活，且有功能；海藻酸钠－聚赖氨酸－海藻酸钠生物微胶囊对免疫活性细胞及抗体具有屏蔽作用。     

用毛囊隆突细胞构建复合皮的实验观察

目的 以人毛囊隆突细胞为种子细胞体外构建组织工程复合皮，在体观察其功能性修复全层皮肤缺损的可行性。方法 胶原酶消化法体外分离培养人毛囊隆突细胞和毛乳头细胞，实验分为A、B两组。A组将毛囊隆突细胞与毛乳头细胞按1：2混合，接种于胶原包被的聚羟基乙酸纤维支架中；B组单纯接种相同数量的毛乳头细胞。而后覆盖角质形成细胞膜片，构成组织工程复合皮，移植于裸鼠全层皮肤缺损创面。观察创面愈合情况，分别于术后2、4、6周在光学显微镜下观察移植物组织学变化。结果 组织工程复合皮能够有效修复A、B两组裸鼠全层皮肤缺损。术后2周，A、B组创面均可见完整的表皮及真皮结构。术后4-6周，A组复合皮表皮层明显增厚并形成基膜的钉突，可见毛囊样结构；B组仅表皮层有所增厚但基膜平整，未见钉突和毛囊结构形成。结论 以聚羟基乙酸真皮基质为支架，用角质形成细胞、毛囊隆突细胞和毛乳头细胞共同构建的组织工程复合皮，可以有效修复裸鼠全层皮肤缺损。其中毛囊隆突细胞参与了创面解剖修复，同时可能引导组织结构和功能的修复。     

Role of hair papilla cells on induction and regeneration processes of hair follicles

Hair dermal papilla cells are specialized mesenchymal cells that exist in the dermal papilla located at the bottom of hair follicles. These cells play pivotal roles in hair formation, growth, and cycling. Hair follicle formation is usually directed by an aggregation of dermal mesenchymal cells, the origin of dermal papilla cells, in the embryonic skin. We noticed that cultured dermal papilla cells also have hair-forming activity and do not lose the activity even after long-term cultivation, if they are cultured with conditioned medium from keratinocytes obtained from the sole or with a medium containing fibroblast growth factor. The secreted factors from keratinocytes and fibroblast growth factor are, therefore, important for maintaining the cellular properties of dermal papilla cells. Even if the hair bulb, including the hair matrix and the dermal papilla, has been removed from vibrissal follicles in vivo, the new hair matrix and papilla can regenerate from the rest of the follicle, and eventually a hair shaft regrows. It has been reported that hair bulb regeneration does not occur when the lower half of a hair follicle is removed. However, new hair bulbs were formed in the remaining upper halves of vibrissal follicles if the amputated follicles had been implanted under the kidney capsule. The formed bulbs were small and pelage-type, not large vibrissa-type. Histological studies showed that the new dermal papillae were derived from dermal sheath cells surrounding upper follicular epidermis, and the new hair matrices were produced from the follicular epidermis. Moreover, the upper halves of vibrissal follicles reformed large vibrissa-type bulbs when they were associated with dermal papillae or cultured papilla cells and implanted in the kidney. Thus, dermal papilla cells and probably dermal sheath cells have the ability to induce and form hair bulbs under preferred environmental conditions. Attempts to identify the genes and proteins associated with hair-forming activity of dermal papilla cells have been carried out. We and other groups successfully isolated the molecules that were specifically expressed in dermal papilla cells. The nature of the hair-producing factors could be understood through the studies of these molecules.     

Induction of hair follicle regeneration in rat ear by mi-croencapsulated human hair dermal papilla cells

Objective: To induce hair follicle regeneration in rat ear by microencapsulated dermal papillae (DP) cells.Methods: Intact dermal papillae were obtained from human scalp follicles which were digested with collagenase I. The human hair DP cells were encapsulated with alginate-polylysine-alginate (APA) by a high-voltage electric field droplet generator. The diameters of the DP cell microcapsules were optimized by regulating the voltage, the distance be-tween the needle head and the solution surface and the injection speed. Then DP cell microencapsulations were xenotransplanted into ears of 20 SD rats with a novel method. One rat was killed every week at the postoperative 2-12 weeks and the implantation sites were biopsied for histo-logical observation.Results: The DP cell microencapsulations were found in a group of round, smooth and transparent microcapsules under a phase-contrast microscope. The optimal combina-tion of parameters to obtain 0.4 mm DP cell microcapsules was voltage 7.0 kV, injection speed 55 mm/h, and distance 10mm. After 4-12 weeks, 18 of 20 DP cell microcapsule implan-tations had produced high-density hair. Histological obser-vation indicated that both large follicles and sebaceous gland structures were formed in the rat ear within 3-12 weeks.Conclusions: These findings show that the DP cell microencapsulation maintain the capacity for initiating the follicle regeneration and can be considered as a substitute for fresh isolated dermal papillae.     

GMP-compliant culture of human hair follicle cells for encapsulation and transplantation

Human hair follicle cells, both bulge and dermal papilla cells, were isolated and cultured in a GMP cell factory, in order to obtain an in vitro hair follicle source for encapsulation end transplantation in alopecia regenerative cell therapy. An in vitro model, constituted by organotypic cultures of human skin sample, was set up to simulate the dermal-epidermal interaction between bulge cells and dermal papilla cells, evaluating the possible new follicles formation and the regenerative potentiality of these hair follicle cells. Both the bulge and dermal papilla cells show an excellent cellular proliferation as well as an abundant extracellular matrix production. The immunofluorescence investigation revealed the positivity of both cell lines to CK15 and CD200, whereas both cell lines were negative to CD71 and Oct-4. The pool of cultured bulge and dermal papilla cells was injected into the deep dermis; at day 28 of culture, some organized areas with a higher cell density can be observed: the cells self-organize into papilla-like lengthened aggregates. In samples in which the follicular cells have been seeded on the dermis surface, an epidermis-like homogeneous monolayer on the dermis surface can be seen, therefore showing a potentiality of these cells for epidermis regeneration. These data show the efficacy of a cellular isolation and amplification approach to obtain an in vitro human hair follicle regenerative source on industrial scale in a GMP cell factory. The results also proved an intrinsic potentiality of follicular cells to in vitro recreate the epidermis for tissue engineering purposes. Thus, it is feasible to produce bioengineered hair follicles in a GMP cell factory, for encapsulation and transplantation in alopecic patients.     

Viability of Isolated Single Hair Follicles Preserved at 4°C

Background. Hair follicle preservation for the purpose of delayed application would help us to transplant hair follicles more efficiently.
Methods. Isolated single hair follicles were preserved at 4°C in four different solutions. Viability of preserved follicles was judged by organ culture and cell culture. In addition, a small number of hair follicles were transplanted into athymic mice.
Results. By cell culture, both dermal papilla and outer root sheath cells could be cultivated after 7 days of preservation. Hair follicles preserved for 48 hours showed a significant increase of hair shafts in organ culture. Those preserved for 7 days regrew well when transplanted into athymic mice.
Conclusion. Preservation of hair follicles at 4°C could be one option to prepare many follicular units at one time for transplantation.     

Hair transplantation in mice: Challenges and solutions

Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well‐established human hair‐restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip‐side microdissected, and implanted individually into needle hole incisions in the back skin of immune‐deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck‐induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP‐positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip‐side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.     

Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells

OBJECTIVE: To investigate the influence of hair follicle dermal papilla cells (DPCs) on biological features of composite skin. METHODS: In the test group, xenogeneic acellular dermal matrix was employed as the frame, DPCs were seeded on the subcutaneous side, and epithelial stem cells onto the dermal papilla side of the dermal frame so as to construct a composite skin. In the control group, there was no DPC in the frame. The two kinds of composite skin were employed to cover skin defects on the back of the nude mice. Wound healing was observed 4 weeks after grafting and area was analyzed and contraction rate was calculated. The tissue samples in the grafted area were harvested for HE staining and the state of the composite skin was observed. The stress-strain curve of the sampled skin was measured, so as to calculate the maximal breaking power of the sample. The data were collected and statistically analyzed. RESULTS: HE staining indicated that the epithelial depth was increased (more than 10 layers of cells) in test group, with only 6-7 layers in control group. The skin contraction rate in test group on the 4th week after skin grafting (3.94+/-0.013)% was much lower than that in control group (29.07+/-0.018)% (P<0.05). It was indicated by biomechanical test that the stress-strain curve of the composite skin in the test group was closer to that of normal nude mice skin in comparison to that in control group. The maximal breaking force of the composite skin in test group was (1.835+/-0.035)N (Newton), while that in control group was (1.075+/-0.065)N (P<0.01). CONCLUSION: Reconstruction of epidermis in composite skin was promoted by dermal DPCs seeded in the dermal matrix frame. As a result, there was less skin contraction in the composite skin with DPCs, so that the biological characteristics of the skin were improved.