研究背景

先天性子宫发育异常及创伤等继发子宫缺损严重影响患者的生活质量及生育能力。组织工程技术在重建子宫功能方面取得了一定进展，但主要是针对组织结构相对简单及组织缺损面积小于1 cm的子宫内膜的修复，有良好的修复效果和转化应用潜能。然而，子宫大面积全层缺损的重建和修复仍是领域内亟待解决的问题并面临严重挑战。

脱细胞基质材料（Decellularized matrix, dECM）因保留天然组织结构和细胞外基质在组织修复中展现较好的临床效果，但在子宫全层大面积缺损中尚未有研究。子宫因有多层组织结构，其再生修复的要求不同，根据子宫组织的特性，对脱细胞基质材料进一步改进有望满足不同组织结构再生的需求，进而实现子宫全层大面积缺损的有效修复。我们基于脱细胞技术得到双层胶原支架材料，胶原支架由光滑层（支持上皮细胞生长和抑制粘连）和粗糙层（支持间质细胞的生长）构成。将粗糙面对合塑形，得到两面光滑，中间粗糙的三维夹心结构的胶原支架，以适应内脏器官的全层结构特征。碱性成纤维细胞生长因子（Basic fibroblast growth factor, bFGF）可以有效的促进有丝分裂，也是分化和组织形态发生的关键诱导分子，可以促进包括成纤维细胞、血管内皮细胞和平滑肌细胞的分化和增殖，对组织再生和伤口愈合具有重要作用。我们将碱性成纤维细胞生长因子结合于胶原支架（bFGF-胶原支架），构建了组织工程移植物用于子宫大面积缺损的结构重建。

研究目的

构建三维胶原支架负载bFGF的组织工程移植物，用于修复兔子宫较大面积的全层缺损；评价bFGF-胶原支架对子宫各层结构的修复和再生效果，并初步探讨修复机制；评价对妊娠结局的影响。

研究方法

bFGF，将其交联至胶原支架，对材料进行质谱测序鉴定支架蛋白及肽段成分；

兔子宫缺损的长度为5cm、宽度为周长2/3，构建子宫大面积缺损模型，随机分配至正常对照组、子宫切除组、原位缝合对照组、胶原支架组及bFGF-胶原支架组，进行子宫重建手术；

重建1个月组织形态学评估：基于大体观评价子宫粘连；基于H&E染色、  Masson染色和免疫组化方法，评价子宫的组织结构、胶原含量以及Ki67阳性细胞增殖情况；转录组测序初步探索修复调控机制；

重建3个月组织形态学评估：包括组织结构、胶原含量、panCK阳性上皮细胞生长、E-cadherin阳性上皮细胞的连接、间质细胞波形蛋白表达情况。

重建6个月组织形态学评估：宫腔造影显示宫腔形态，基于H&E染色评价组织结构、管腔周长和腺体密度，基于Masson染色比较子宫内膜厚度和胶原含量，基于免疫组化染色评价CD31阳性血管密度、SMA阳性肌层厚度、孕激素受体表达模式、上皮细胞Ckpan表达和间质细胞波形蛋白表达情况。

重建子宫妊娠结局评价：子宫重建6个月后进行交配，观察是否妊娠，妊娠胎数以及分娩结局。

研究结果

重建1个月时：bFGF-胶原支架组的细胞增殖强于正常对照和子宫切除对照组；bFGF-胶原支架组的胶原含量显著低于子宫切除对照组（p=0. 01），但仍高于正常子宫对照组（p=0.01），转录测序提示bFGF-胶原支架组上调基因富集在免疫调控通路。

重建3个月时：bFGF-胶原支架组胶原含量显著低于子宫切除对照组（p=0.01），略高于正常对照组，差异无统计学意义（p=0.24）；bFGF-胶原支架组子宫CKpan、E-cadherin和Vimentin表达模式与正常对照组子宫相似。

重建6个月时：bFGF-胶原支架组胶原含量显著低于子宫切除对照组（p=0.01），与正常对照组相似（p=0.93）；bFGF-胶原支架组的腺体密度（p=0.05）、CD31血管密度（p=0.05）、SMA阳性肌层厚度（p=0.08）均高于子宫切除对照组，与正常对照组子宫相似（P值分别为0.78、0.99和0.98）；bFGF-胶原支架组子宫的CKpan、Vimentin和PR表达模式与正常对照组子宫相似。

妊娠结局：正常对照组、原位缝合对照组、子宫切除对照组、胶原支架组和bFGF-胶原支架组重建子宫的妊娠率分别为100%、33.3%、0%、30%和50%。

研究结论

脱细胞的三维胶原支架负载bFGF构建的组织工程移植物可以引导原位子宫种子细胞的生长，重建5cm长的全层子宫缺损，组织工程移植物重建的子宫壁有完整的结构，实现了上皮、血管、腺体以及肌层等各层结构重建；

转录组测序提示免疫浸润及免疫调控在bFGF-胶原支架组重建子宫早期发挥重要作用；

组织工程移植物重建后的子宫对孕激素有反应性，实现了功能重建

组织工程移植物重建后的子宫妊娠率达到50%；

Background

Uterine abnormalities, such as endometrial adhesions, damage of corpus uteri, or congenital uterine dysplasia, are common factor that causes female infertility. Tissue engineering technology has made some progress in reconstructing uterine function, but it mainly focuses on repairing endometrium with relatively simple tissue structure and tissue defect area less than 1 cm, which has good repair effects and potential for translational application. However, the reconstruction and repair of large-scale full-thickness defects of the uterus are still difficult in the field and face serious challenges.

Decellularized matrix (dECM) has shown good potential in tissue repair such as skin injury due to its preservation of natural tissue structure and extracellular matrix, but has not been studied in large-scale full-thickness defects of the uterus. The regeneration of different tissues requires different materials, and further improvement of decellularized matrix materials based on tissue characteristics is expected to meet the needs of different tissue regeneration, thereby achieving effective repair of large-area full-thickness defects in the uterus. We obtained a double-layer collagen scaffold material based on decellularization technology. The collagen scaffold consists of a smooth layer (supporting epithelial cell crawling) and a rough layer (supporting interstitial cell growth). On the basis, we fold the decellularized matrix in half to make the rough surfaces in contact, forming a three-dimensional collagen scaffold with smooth surfaces on both sides and rough in the middle, to adapt to the full layer structural characteristics of visceral organs. Basic fibroblast growth factor (bFGF) can effectively promote mitosis and is a key inducer of differentiation and tissue morphogenesis. It can promote the differentiation and proliferation of fibroblasts, vascular endothelial cells, and smooth muscle cells, playing an important role in tissue regeneration and wound healing. We crosslinked bFGF onto collagen scaffold and constructed an innovative tissue engineering graft.

Objective

We construct a three-dimensional collagen scaffold loaded with bFGF as a tissue engineering graft for repairing large full-thickness defects. This study is based on a rabbit model of full-thickness uterine defect to evaluate the effectiveness of tissue engineering graft for repairing large uterine defect, and preliminarily explore the mechanism of regeneration; Further evaluate the impact on pregnancy outcomes and the impact on pregnancy outcomes.

Methods

Decellularized matrix material was obtained from the pig bladder after decellularization treatment and bFGF with collagen binding domain was construct. bFGF was crosslinked onto collagen scaffold. Mass spectrometry sequencing was performed to identify scaffold protein and peptide components.

Rabbits were divided the into two parts, one for histological evaluation and the other for pregnancy function evaluation. Further, the rabbits were randomly assigned to a normal control group, a hysterectomy group, a uterine in-situ suturing group, collagen scaffold group, and a bFGF collagen scaffold group. A full-thickness defect with a length of 5 cm and a width of 2/3 of the circumference was removed from the rabbit uterus, to construct a model of large-scale uterine defect. And then uterine reconstruction surgeries were performed in these models. Postoperative histological and pregnancy function evaluations were performed, as follows.

Histological evaluation for one month after reconstruction: uterine adhesions based on macroscopic evaluation, evaluation of the tissue structure based on H&E staining, collagen content based on Masson staining, Ki67 positive cell proliferation of the uterus based on immunohistochemical staining.

Histological evaluation for three months after reconstruction: uterine adhesions based on macroscopic evaluation, evaluation of the tissue structure based on H&E staining, collagen content based on Masson staining and evaluation of growth of CKpan positive epithelial cells, junctions of E-cadherin positive epithelial cells, and expression of vimentin in stromal cells based on immunohistochemical staining.

Histological evaluation for six months after reconstruction: uterine adhesions based on macroscopic evaluation. Hysterography displayed the morphology of the uterine cavity. Evaluation of the tissue structure, lumen circumference, and glandular density based on H&E staining. Evaluation of endometrial thickness and collagen content based on Masson staining. Evaluation of CD31 positive vascular density, SMA positive muscle layer thickness and the expression pattern of progesterone receptor, CKpan and vimentin expression based on immunohistochemical staining.

Evaluation of pregnancy outcomes after uterine reconstruction: Six months after uterine reconstruction, mating was performed to collect data including pregnancy, number of fetal rabbits in each pregnancy and delivery outcomes.

Results

After one month of reconstruction, the cell proliferation in the bFGF collagen scaffold group was stronger than that in the normal control and hysterectomy control groups; The collagen content in the bFGF collagen scaffold group was significantly lower than that in the hysterectomy control group (p=0.01), but still higher than that in the normal uterine control group (p=0.01).

At 3 months of fine reconstruction, the collagen content in the bFGF collagen scaffold group was significantly lower than that in the hysterectomy control group (p=0.01) and slightly higher than that in the normal control group, with no statistically significant difference (p=0.24); The expression patterns of CKpan, E-cadherin, and Vimentin in the uterus of the bFGF collagen scaffold group were similar to those of the normal control group uterus.

At 6 months of fine reconstruction, the collagen content in the bFGF collagen scaffold group was significantly lower than that in the hysterectomy control group (p=0.01), similar to that in the normal control group (p=0.93); The glandular density (p=0.05), CD31 vascular density (p=0.05), and SMA positive muscle layer thickness (p=0.08) of the bFGF collagen scaffold group were higher than those of the hysterectomy control group and similar to those of the normal control group (P values of 0.78, 0.99, and 0.98, respectively); The expression patterns of CKpan, Vimentin, and PR in the uterus of the bFGF collagen scaffold group were similar to those of the normal control group uterus.

The pregnancy rates of reconstructed uterus in the normal control group, in situ suture control group, hysterectomy control group, collagen scaffold group, and bFGF collagen scaffold group were 100%, 33.3%, 0%, 30%, and 50%, respectively.

Conclusion

The tissue engineering graft constructed with decellularized collagen scaffold loaded with bFGF can guide the growth of in situ uterine seed cells and reconstruct a 5cm full-thickness uterine defect.

The uterine wall reconstructed by tissue engineering graft has a complete structure, achieving epithelial reconstruction, vascular reconstruction, glandular reconstruction, and muscular reconstruction.

The uterus reconstructed by tissue engineering graft is responsive to progesterone, achieving functional reconstruction.

The pregnancy rate of the uterus reconstructed by tissue engineering graft reaches 50%.

Based on the results, tissue engineering grafts have achieved breakthroughs in relatively large area reconstruction and full layer reconstruction in the field of tissue engineering uterine regeneration, which may help achieve uterine reconstruction and fertility function for patients with uterine dysplasia or defects.