Biomimetic scaffold‐based stem cell transplantation promotes lung regeneration

Abstract Therapeutic options are limited for severe lung injury and disease as the spontaneous regeneration of functional alveolar is terminated owing to the weakness of the inherent stem cells and the dyscrasia of the niche. Umbilical cord mesenchymal‐derived stem cells (UC‐MSCs) have been applied to clinical trials to promote lung repair through stem cell niche restruction. However, the application of UC‐MSCs is hampered by the effectiveness of cell transplantation with few cells homing to the injury sites and poor retention, survival, and proliferation in vivo. In this study, we constructed an artificial three‐dimensional (3D) biomimetic scaffold‐based MSCs implant to establish a beneficial regeneration niche for endogenous stem cells in situ lung regeneration. The therapeutic potential of 3D biomimetic scaffold‐based MSCs implants was evaluated by 3D culture in vitro. And RNA sequencing (RNA‐Seq) was mapped to explore the gene expression involved in the niche improvement. Next, a model of partial lung resection was established in rats, and the implants were implanted into the operative region. Effects of the implants on rat resected lung injury repair were detected. The results revealed that UC‐MSCs loaded on biomimetic scaffolds exerted strong paracrine effects and some UC‐MSCs migrated to the lung from scaffolds and had long‐term retention to suppress inflammation and fibrosis in residual lungs and promoted vascular endothelial cells and alveolar type II epithelial cells to enter the scaffolds. Then, under the guidance of the ECM‐mimicking structures of scaffolds and the stimulation of the remaining UC‐MSCs, vascular and alveolar‐like structures were formed in the scaffold region. Moreover, the general morphology of the operative lung was also restored. Taken together, the artificial 3D biomimetic scaffold‐based MSCs implants induce in situ lung regeneration and recovery after lung destruction, providing a promising direction for tissue engineering and stem cell strategies in lung regeneration.


| INTRODUCTION
End-stage lung disease remains the leading cause of death in the industrialized world owing to the complex nature and slow regeneration capacity of the lungs. 1 Unlike tissues with a rapid renewal capacity, which have well-defined hierarchies of stem cells and differentiated cells, the mature lung of mammals maintains homeostasis with a slow cellular turnover, and regeneration and renewal of the lung depend on multiple cell populations. 2,3 The stem/progenitor cell populations in the trachea, bronchioles, and alveolar contribute to lung maintenance and perform regeneration and repair upon injury, but the dyscrasia of niche in the injured area is not sufficient to continuously support the slow regeneration process, resulting in the failure of repair. 4 Thus, a beneficial regenerative microenvironment for endogenous stem cells is vitally necessary for situ lung regeneration.
Mesenchymal stem cells (MSCs) have emerged as an attractive measure for regenerative medicine. 5,6 Umbilical cord-derived MSCs (UC-MSCs) are a promising MSC source for clinical cell-based treatments because of their strong paracrine effects, immunomodulation, high proliferative potential, low immunogenicity, abundant supply, and painless collection. 7,8 There is growing evidence that UC-MSCs play a vital role in repairing multiple tissues such as peripheral nerves, skin, bones, kidneys, liver, brain, and endometrium. [9][10][11][12][13][14][15] In lungs, considering the complexity in terms of structural and cellular diversity, the unique ability of UC-MSCs to interact with multiple cell types to maintain and promote the survival of injured cells makes them a promising approach for lung regeneration. [16][17][18] It has been reported that the use of UC-MSCs and/or their secretome as a therapeutic strategy to improve lung transplantation. Although the mechanisms are not fully defined yet, it has been widely demonstrated in both preclinical and clinical studies that the therapeutic effects of MSCs are mediated to mitigate fibrosis, inflammation, ischemia-reperfusion injury, bronchiolitis obliterans syndrome, and promote the repair of residual endogenous lung stem/progenitor cells, and improve ex vivo lung perfusion during lung transplantation. 19 tions for lung repair following injury. 22 These paracrine effects are mediated by factors such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), which are closely related to lung immunomodulation, epithelial stem cell niches, and angiogenesis. [23][24][25][26] However, the application of UC-MSCs is hampered by the effectiveness of cell transplantation with poor cell retention, survival, and proliferation in vivo. 27 In fact, only 1%-20% of transplanted cells survive, which restricts the clinical therapy potential. 28 Decellularized lung scaffolds are one representative approach to artificial whole lung scaffolds. In a previous study, a decellularized lung scaffold seeded with epithelial and endothelial cells was perfused with air and blood, which generated gas exchange in a bioreactor. When implanted into mice, this artificial lung system provided gas exchange for a short period. 29 Various procedures to construct lung acellular scaffolds have been investigated, which extended the time of gas exchange. Among the decellularization processes, collagen was largely preserved, resulting in the loss of other extracellular matrix proteins such as glycosaminoglycans and elastin. 30 Therefore, collagen might play an essential role in lung regeneration. Collagen as the main component of the extracellular matrix is a kind of natural biomaterial that has been widely used for tissue engineering scaffolds in the field of tissue regeneration because of its biodegradability, low antigenicity, and good biocompatibility. 31,32 A collagen scaffold not only supports the framework of tissue but also regulates cell adhesion, migration, and differentiation. 33 In past studies, collagen scaffolds loaded with MSCs have reduced the loss and death of MSCs and were applied to the regeneration of other tissues including nerve, endometrium, heart, and bone. [34][35][36][37] In the present study, we prepared a porous three-dimensional (3D) collagen scaffold with pore diameters ranging from 50 to 100 μm, which had similar structure characteristics to the lung. Then, passage three UC-MSCs were uniformly loaded on the 3D collagen scaffold. Thus, an artificial stem cell niche was established. Next, the collagen/UC-MSCs scaffold was implanted orthotopically into a partial lung resection model. The results demonstrated that collagen/UC-MSCs scaffold exerted strong paracrine effects in vitro, promoted the behaviors of alveolar stem/progenitor cells and endothelial cells, suppressed inflammation and fibrosis, restored the general morphology, and formed alveolar-like structures in vivo, suggesting a clinical potential for lung repair (Scheme 1).

| Ethics and human UC-MSCs isolation-culture
Human umbilical cord tissue was obtained from full-term fetuses after normal vaginal delivery at the Obstetrics Department of the Southwest Hospital. The study was approved by the Animal Ethics Committee of the Institute of Genetics and Development, Chinese Academy of Sciences. And the approved protocol number is IGDB-2018-IRB-007. Particularly, the object of this study was rats, and did not harm the donor's health. And to protect the privacy of donors, informed consent was obtained from the parents and families, and it was a single-blind trial for us, where the sex of the umbilical cord was randomized. In this series of experiments presented below, we detected the karyotype of the cell line, suggesting that the sex of the cell line was male. The procedure for UC-MSCs isolation was carried out according to our previous study. 5 The isolated UC-MSCs were maintained at 37 C in a 5% CO 2 humidified atmosphere with LG-DMEM/F-12 medium (11330032, Gibco, USA) supplemented with 10% fetal bovine serum (10099-141, Gibco, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin. Human UC-MSCs at passages three to five were used for the following experiments.

| Human UC-MSCs differentiation assays
Adipogenic and osteogenic differentiation assays at the fourth passage were conducted to detect the multilineage differentiation potential of the isolated human UC-MSCs. UC-MSCs were induced to differentiate using an Adipogenesis Differentiation Kit (A1007001, Gibco) and Osteogenesis Differentiation Kit (A1007201, Gibco). Lipid accumulation was detected at 7-14 days after induction culture by Oil Red O staining. For osteogenic differentiation, the calcium deposition was detected at 21-28 days after induction culture by Alizarin Red S staining.

| Human UC-MSCs culture on the 3D collagen scaffolds
3D collagen scaffolds were prepared from bovine skin tissue as described previously. 24 Scaffolds were rinsed with culture medium and placed on 48-well culture plates. After soaking overnight at 37 C, excess fluid was removed from the scaffolds using a sterile cotton swab. Then, 50 μL of a UC-MSCs suspension (5 Â 10 5 cells/cm 2 scaffold) was dripped equally onto each scaffold to observe histological and cellular morphology. The cell-seeded scaffolds were incubated for 1 h at 37 C in a humidified atmosphere with 5% CO 2 and then maintained in a complete culture medium for the following experiments.
UC-MSCs-seeded scaffolds were collected after 3, 12, 24, and 48 h of culture. Some samples were fixed in 4% paraformaldehyde overnight, dehydrated in graded alcohol solutions, and embedded in paraffin.   All animal studies that meet ARRIVE guidelines were randomly divided into three groups with different treatments to observe the scaffold region at 7, 30, and 60 days post-operation. In the sham control group, the incision was made to expose the middle lobe of the right lung in the fifth intercostal space, but there was no injured site made in the lung. In the blank 3D collagen group, the excised lung was replaced with a blank collagen scaffold loaded with 100 μL phosphate buffer. In the 3D collagen scaffold/UC-MSCs group, the excised lung was replaced with a collagen scaffold seeded with 100 μL of a UC-MSCs suspension (1 Â 10 6 cells/cm 2 scaffold). At the indicated time points, the rats were sacrificed by acute blood loss. Random numbers were generated using the standard = RANDBETWEEN(1,80) function in Microsoft Excel.
The surgical procedures for the lung injury model are described in our previous study. 24 Following intraperitoneal anesthesia with chloral hydrate (40 mg/kg body weight), skin preparation, and noninvasive mechanical ventilation, a 2-cm incision was made in the anterior chest to expose the middle lobe of the right lung in the fifth intercostal space. Then, lung tissue with a volume of 6 Â 3 Â 2 mm was removed, and the same volume of a 3D collagen scaffold seeded with UC-MSCs was implanted into the lung tissue gap. Then, the surgery site was irrigated with normal saline, and the musculature and skin were closed in separate layers with sutures. Finally, the rats were returned to their cages and kept warm to promote recovery from anesthetization.
The details of the UC-MSCs seeded operating procedure are as follows: (1) The isolated fresh UC-MSCs were passaged about 2-5 generations and used for morphological observation, flow cytometry analysis, differentiation ability detection, and karyotype analysis for cell identification. The obtained high-quality UC-MSCs were partially frozen in liquid nitrogen for reserve at the concentration of 1 Â 10 6 cells/mL (frozen storage solution: 90% fetal bovine serum and 10% dimethylsulfoxide). 38 (2) Another part of the fresh, high-quality UC-MSCs were seeded in a complete medium for amplification culture.
(3) Sterilized 3D collagen scaffolds with a volume of 6 Â 3 Â 2 mm were rinsed with culture medium and soaked overnight at 37 C, excess fluid was removed and then transferred to a new 6-well plate.

| Histological analysis
Rats were anesthetized by an intraperitoneal injection of chloral hydrate (40 mg/kg body weight) at 7, 30, and 60 days post-surgery.
The operative region of lung tissue was collected and fixed in 4% paraformaldehyde overnight, dehydrated in graded alcohol solutions, and embedded in paraffin. Sections of 5 μm in thickness were prepared transversally and stained using standard hematoxylin-eosin and Masson's trichrome staining protocols.

| Micro-CT testing
Rats were anesthetized with 10% chloral hydrate and placed in the chamber of a computed tomography (CT) scanner for small animals (Quantum GX; PerkinElmer, Massachusetts, USA). CT scanning was performed and image acquisition was conducted under respiratory gating. The total lung volume was calculated using Analyze 11.0 software.

| Statistical analysis
Data were presented as mean ± standard deviation (SD). Normality tests were performed before comparison between groups. Differences between groups were compared using a two-tailed Student's t-test or ANOVA. Statistics were calculated with Graph Pad Prism 6 software. Differences were considered significant as *p < 0.05 and **p < 0.01.

| Characterization of collagen/UC-MSCs scaffolds
As shown in Figure 1a group were focused on the VEGF and MAPK signaling pathways associated with the tissue regeneration mechanisms. Furthermore, the KEGG analysis indicated that the key enrichment signaling pathways such as PPAR and IκB signaling pathways, were also centered on relieving inflammation and apoptosis. Notably, the fibrosis-associated signaling pathways, TGFβ signaling pathways, were significantly inhibited after UC-MSCs were loaded (Figure 2j).
In brief, we constructed an artificial 3D biomimetic scaffold-based MSCs implant which had good biocompatibility and paracrine capacity. It may have a great potential for lung regeneration with the potential to promote endothelial and epithelial cell survival, proliferation, and migration, as well as downregulate inflammation, apoptosis, and fibrosis.

| UC-MSCs migration and long-term survival and proliferation and injured lungs
CM-Dil, a long-term cellular labeling dye, was used to track UC-MSCs.
As shown in Figure 3b

| Vascularization on collagen/UC-MSCs scaffolds
Alveolar capillaries exist between adjacent alveolar walls and fill the alveolar septum, are an essential part of lung tissue for gas exchange.
The emergence of new capillaries is an important step in alveolar formation and functional recovery. 40 Next, we investigated angiogenesis by CD31 immunofluorescence staining in scaffolds after implantation

| General morphology recovery of the injured lung and alveolar structures on collagen/UC-MSCs scaffolds
Finally, functional recovery of the injured lung was evaluated by micro-CT scanning that was used to assess the lung's general morphology ( Figure 8a) and calculate the lung volume (Figure 8b). The results showed that, although the general morphology of the injured lung was restored at 60 days post-surgery, the surgical gap still existed in the collagen group, whereas the lung morphology was obviously restored in the collagen/UC-MSCs group. The lung volume of the collagen group was also significantly lower than that in the collagen/UC-MSCs group at 60 days post-surgery.
The functional unit for gas exchange of alveoli mainly consists of AT I cells and microvascular. Therefore, double immunofluorescence  previous study, MSCs supported epithelial integrity during homeostasis and promoted epithelial regeneration after lung injury, which were attributed to the cellular element of stem cell niche. 55 An acellular lung scaffold is the most common artificial lung, which provides an adhesion surface and growth environment for epithelial and endothelial cells, which are essential noncellular components for a stem cell niche.
Collagens, especially collagen type I, are the main component of most acellular lung scaffolds and widely used in tissue regeneration. 56 In our study, we developed a 3D culture system by preparing a 3D porous collagen scaffold loaded with UC-MSCs and demonstrated that the collagen scaffold was suitable for cell adhesion, nutrient and oxygen delivery, cell survival, proliferation, and even long-term cell retention, which was confirmed by tracking UC-MSCs using CM-Dil labeling in vivo and greatly improved the therapeutic efficiency of UC-MSCs.
Paracrine signaling, including immunomodulation, is believed to be one of the major mechanisms for UC-MSCs-mediated tissue repair.
Destruction of the lung alveolus-capillary membrane barrier is the primary characteristic of acute lung injury. 57 In previous studies, VEGF 44 and HGF 25 alleviated such destruction by antagonizing vascular inflammation and promoting the survival of endothelial cells and angiogenesis. Other studies have also indicated that HGF ameliorates lung injury and restores the integrity and permeability of endothelial cells in emphysema and fibrosis models. 58,59 In our 3D cultures, the expression levels of VEGF and HGF were significantly increased compared with two-dimensional (2D)-cultured cells. In addition, gene expression differences between 2D-and 3D-cultured cells were detected by RNA sequencing, which showed upregulation of lung recovery and regeneration-related genes and signaling pathways.