Perfusability and immunogenicity of implantable pre-vascularized tissues recapitulating native capillary network

doi:10.21203/rs.3.rs-2325499/v1

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Perfusability and immunogenicity of implantable pre-vascularized tissues recapitulating native capillary network

https://doi.org/10.21203/rs.3.rs-2325499/v1

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Vascularization is a key pre-requisite to engineered anatomical scale three dimensional (3-D) constructs to ensure their nutrient and oxygen supply upon implantation. Presently, engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels, which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix (ECM) environments. Anatomical scale perfusable constructs are critically needed for translational applications. To overcome this challenge, we previously developed prevascularized ECM sheets with long and oriented dense microvascular networks. The present study further evaluated the patency, perfusability and innate immune response toward these pre-vascularized constructs. Macrophage-co-cultured pre-vascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism. Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation. This completely biological pre-vascularized construct has a great potential to serve as a building block to engineer perfusable anatomical scale tissues.

Biological sciences/Stem cells/Mesenchymal stem cells

Biological sciences/Stem cells/Regeneration

Pre-vascularization

Micro-vasculature

Mesenchymal stem cells

Extracellular matrix

Fluorescence lifetime imaging

Recent progress in the field of regenerative medicine has advanced the fabrication of tissue analogues to replace damaged or diseased tissues, and for disease modelling. One of the long-standing challenges in current approaches is the limited size of engineered tissues, restricted to up to a few hundred microns in thickness due to the lack of anatomical scale built-in microvasculature [1]. As for millimeter-scale or larger three-dimensional (3-D) tissue constructs, the spontaneous growth of host micro-vessels toward the implants occurs at the rate of several tenths of microns per day [2]. At this rate, the complete vascularization of an implant sizing several millimeters will require several weeks or months depending on the implant site and biomaterials involved [2]. Hence, a built-in and dense microvasculature with anastomotic capability, which enables nutrient and oxygen supply, is crucial to engineered 3-D tissue analogues or constructs to ensure their survival and regenerative potential post transplantation. To address this challenge, advanced micro-scaffolding, surface-patterning, and 3-D printing techniques have designed and fabricated vascular network with diameters of several tens of microns and above [3–5]. Although these approaches are promising for disease modelling and drug screening, the micro-vessels generated by these strategies are oversized and/or not sufficiently dense compared with native vasculatures, and are therefore not able to ensure the survival of implanted organoids or tissue constructs [3]. Advanced microfluidics-based approaches have emerged to fabricate microvascular self-assemblies inside an ECM-mimicking hydrogel that is surrounded by two large vessels in a microdevice [6–8]. Although perfusion was successfully achieved in the micro-vessels, the microscale of the engineered tissues has limited their applications to in vitro platforms for disease modeling and drug screening [6–8]. Moreover, the patency, perfusion and immunogenicity of these self-assembled vasculature has not been validated in vivo. Presently, anatomical scale perfusable constructs are critically needed for translational applications. Rapid anastomosis of the engineered microvasculature with host vessels must also occur to ensure the perfusion and functional performance of the pre-vascularized constructs post implantation [9].

Advanced microscopy and high-resolution imaging techniques have revealed that most native tissues including skin [10], cardiac [11, 12] and skeletal muscle [13] have microvasculature and ECM components organized in a precise architecture to achieve maximal function. To attain such a microvasculature, we previously developed a physiological angiogenesis-mimicking approach in which human endothelial cells (ECs) self-assembled into a microvascular network, with the support of human mesenchymal stem cell (hMSCs), under precisely controlled oxygen concentrations [14, 15]. hMSCs were adopted as a bulk cell in the system because they have been recognized for their strong immunomodulatory and regenerative properties via paracrine mechanisms that includes secretion of cytokines, growth factors, exosomes and microvesicles [16]. Additionally, the excellent safety profile of allogenic hMSCs have enabled their infusion in human clinical trials for the treatment of autoimmune and severe viral diseases, with over 10,000 patients during the past two decades [17, 18]. These superior properties have identified hMSCs as an ideal cell type to be involved in diverse tissue engineering applications and interventions. To guide the organization of hMSCs, we have created a highly aligned nanofibrous extracellular matrix (ECM) scaffold [19], which is robust and large, with an area up to 25 cm², allowing us to engineer a transplantable hMSC/ECM sheet with tunable structures, independent of any external dynamic culture system. By further seeding ECs, highly dense and mature microvasculature with the same order of magnitude as native capillaries were formed on the hMSC/ECM sheet [14, 20], which can be utilized as building blocks to engineer 3D pre-vascularized tissue constructs by layering the pre-vascularized cell sheets or integrating the pre-vascularized cell sheets into the engineered 3D constructs. Besides directly promoting tissue regeneration after implantation, hMSCs also act as perivascular cells that support and mature the newly assembled micro-vessels on the pre-vascularized hDF-ECM sheet [17, 21, 22]. However, one of the major concerns related to static-culture self-assembled micro-vessels, which have a diameter < 20 µm, is their patency and perfusability in vitro and in vivo. In addition, the immunogenicity of pre-vascularized constructs must be evaluated to ensure their long-term survival and functionality post implantation.

Herein, the objective of the current work was to evaluate immune-compatibility, patency, and anastomotic capability of the structured microvascular networks self-assembled on hDF-ECM scaffolds. Immunogenicity of the engineered pre-vascularized construct was evaluated via in vitro characterization of macrophage response to the constructs by label-free fluorescence lifetime imaging (FLIM) along with cytokine release profile and regulatory metabolic protein expression [23]. Finally, anastomotic ability, perfusability and immunogenicity of the engineered pre-vascularized tissues were evaluated in vivo via subcutaneous implantation in athymic nude rats, which have a robust innate immune system and have been well-accepted to evaluate tissue-implants in vivo [24–28]. Upon implantation, the pre-vascularized constructs exhibited blood perfusion within 3 days of implantation. Moreover, the constructs revealed a hypoimmunogenic nature without active ongoing inflammation in vivo over 28 days. Overall, the patency, perfusability, and immunogenicity of pre-vascularized tissue constructs strongly confirmed its potential as a building block to fabricate anatomical scale 3-D pre-vascularized tissues for diverse tissue-engineering applications.

Integrity of engineered microvascular networks upon macrophage co-culture

Anisotropic and isotropic hDF-ECM sheets were used as a scaffold to support and guide micro-vessel formation in aligned or random orientations, respectively (Fig. S1). These hDF-ECM can be developed in a desired size and shape. hDF-ECM can be stored at -80ºC and can readily be used to engineer pre-vascularized constructs. HMSCs and HUVECs were co-cultured on hDF-ECM scaffolds under optimal oxygen concentrations, to develop self-assembled microvascular networks as illustrated in Fig. 1. To mimic innate immune responses towards the pre-vascularized constructs in vitro, PMA-stimulated THP-1 macrophage were seeded onto pre-vascularized constructs (hMSC/EC/MP) or hMSC-only constructs (hMSC/MP) (Fig. 1).

Prior to the co-culture with pre-vascularized constructs, THP-1 monocytes were differentiated into macrophages via PMA (50 ng/mL) stimulation. Upon PMA stimulation, THP-1 cells showed increased cell adhesion, cell spreading and expression of macrophage specific markers (CD68, CD11b, CD14, EGR2) as observed via immunofluorescence (IF) staining (Fig. S2A). In addition, the mRNA expression of macrophage-specific markers (CD14, CD68, CD80) were also significantly increased (p < 0.001) (Fig. S2B), indicating successful differentiation of monocytes into macrophages. The representative density and morphology of THP-1 derived macrophages co-cultured onto aligned and random hMSC-only constructs (hMSC/MP) can be observed in Fig S2C. Macrophages co-cultured onto the hMSC construct for 3 and 7 days can distinctively be observed via IF staining targeting CD68 marker (Fig S2C).

Microvascular networks co-cultured with macrophages were characterized via measurement of (1) vessel length, (2) vessel diameter, (3) intercapillary distance and (4) percentage area covered by micro-vessels at days 3 and 7 post macrophage seeding (Fig. 2). Aligned and randomly organized pre-vascularized constructs without macrophages (hMSC/EC) were used as a control to evaluate aforementioned characteristics of the microvascular network (Fig. 2A). At day 3, microvascular networks co-cultured without (hMSC/EC) or with macrophages (hMSC/EC/MP) showed overall similar characteristics in both aligned and random groups. The hMSC/EC (386 ± 296 µm) group showed increased vessel length compared to hMSC/EC/MP (305 ± 255 µm) on aligned constructs, but no significant difference was observed on random constructs (Fig. 2B). Aligned constructs showed significantly increased vessel length compared with random constructs in the presence (hMSC/EC/MP) (p < 0.01) or absence of macrophages (hMSC/EC) (p < 0.0001) due to the anisotropic orientation of micro-vessels (Fig. 2B). However, post 7 days of macrophage seeding, the vessel length in hMSC/EC/MP group (183 ± 116 µm in align group and 141 ± 60 µm in random group) was significantly reduced compared with the control (hMSC/EC (without macrophages) in both aligned (250 ± 206 µm) (p < 0.05) and random (244 ± 233 µm) (p < 0.0001) groups (Fig. 2B). No significant difference in the percentage area covered by micro-vessels was observed between aligned and random constructs co-cultured with macrophages (hMSC/EC/MP) at day 3, however, at day 7, the hMSC/EC/MP group showed a significant decrease in the percentage area in both aligned (4.23 ± 2.45 µm²) (p < 0.0001) and randomly organized (4.07 ± 0.74 µm²) constructs compared with the hMSC/EC group (p < 0.05) (Fig. 2C). Similarly, at day 7, the intercapillary distance was significantly increased in hMSC/EC/MP group in both aligned (111.31 ± 73.86 µm) (p < 0.0001) and random (85.60 ± 45.32 µm) (p < 0.01) constructs compared with the hMSC/EC group due to the reduction in microvascular network density (Fig. 2D). The self-assembled micro-vessels showed diameters ranging from 4–12 µm, which is at a similar scale as the native capillaries. At day 3, no significant difference in the micro-vessel diameter was observed between aligned or randomly organized micro-vessels without (aligned: 7.22 ± 3.48 µm, random: 8.13 ± 3.33 µm) or with macrophages (aligned: 8.28 ± 2.34 µm, random: 9.29 ± 4.77 µm) (Fig. 2E). However, at day 7, the hMSC/EC/MP groups (aligned: 10.13 ± 3.45 µm, random: 9.34 ± 2.39 µm) showed significantly increased vessel diameter compared with hMSC/EC (aligned: 8.09 ± 1.96 µm, random: 7.08 ± 3.22 µm) groups in both aligned (p < 0.01) and randomly (p < 0.0001) organized constructs (Fig. 2E).

Z-stacked confocal images revealed that both aligned and randomly organized micro-vessels have a tubular structure with an open lumen (Fig. 3). Pre-vascularized constructs without (hMSC/EC) or with macrophage (hMSC/EC/MP) co-culture showed similar tubular structure as observed from CD31 signal (green). A clear lumen was observed in the cross-sectional view from z-stacked images (white arrows) (Fig. 3A). Although no difference in micro-vessel structure was observed between hMSC/EC and hMSC/EC/MP groups at day 3, a reduction in micro-vessel length and integrity was observed at day 7 in constructs co-cultured with macrophages (hMSC/EC/MP) as compared with the hMSC/EC group (Fig. 3B).

Label-free assessment of macrophage response towards pre-vascularized constructs

Metabolic status of macrophages co-cultured with hMSCs only (hMSC/MP) or pre-vascularized constructs (hMSC/EC/MP) was evaluated via FLIM. FLIM based detection of NAD(P)H and FAD has been applied as a label-free approach to evaluate the metabolic perturbations of the cells and tissues [37–40]. FLIM measures the time a fluorophore remains in the excited state before returning to the ground state by emitting a photon [41]. The lifetime of NAD(P)H and FAD within the cells can be fitted to a two-component exponential decay curve, which resolves the lifetime and corresponding fractions of different conformations, protein-bound and free. Since the bound NAD(P)H lifetime is decided by the binding protein and affiliated with different metabolic pathways, the variations in the NAD(P)H bound-fraction and lifetime have been used to indicate shifts in the metabolic pathways [42–44]. Here, autofluorescence lifetime imaging reveals differences in fluorescence lifetimes of free and bound fractions of NAD(P)H and FAD of macrophages exclusively, co-cultured on aligned and random hMSCs/MP and hMSC/EC/MP constructs (Fig. 4, S3). Due to the spatial separation of macrophages from the hMSCs and ECs, the FLIM data is exclusively from macrophages. In representative autofluorescence lifetime images of NAD(P)H and FAD of macrophages, the nuclei are darker than the cytoplasm because both NAD(P)H and FAD mainly exist in the mitochondria and cytosol (Fig. 4A). The FLIM data revealed metabolic differences within the macrophages between the 3 and 7 day time points. On day 7, the macrophages have a lower fraction of free NAD(P)H (α₁) than on day 3 (Fig. 4B), consistently for both hMSC/MP and hMSC/EC/MP (aligned and random) constructs. The macrophages on aligned hMSC/MP constructs have a shorter lifetime of free NAD(P)H (τ₁) on day 7 compared with day 3 (Fig. 4C). Conversely, the lifetimes of free and bound NAD(P)H (τ₁, τ₂) in macrophages from aligned or random hMSC/EC/MP constructs were longer on day 7 compared to day 3 (Fig. 4C, D). Consequently, macrophages from hMSC/MP (random) and hMSC/EC/MP (aligned/random) exhibit a significantly longer mean fluorescence lifetime (τ_m) on day 7 compared to day 3 (Fig. 4E).

Additionally, FLIM revealed differences in macrophage autofluorescence lifetimes between macrophages seeded on prevascularized constructs and immunomodulatory hMSC-only constructs.. A lower fraction of free NAD(P)H (α₁) was observed for macrophages on random scaffolds with hMSC/EC/MP than random scaffolds with hMSC/MP on both day 3 and day 7 (Fig. 4B). Additionally, the lifetimes of free and bound NAD(P)H (τ₁, τ₂) are longer in macrophages on hMSC/EC/MP (random) compared to macrophages on hMSC/MP (random), which results in a longer mean NAD(P)H lifetime (τ_m) on day 3 and day 7 (Fig. 4C, D, E). Similar differences in NAD(P)H lifetime metrics, a lower fraction of free NAD(P)H (α₁) and longer lifetimes of free and bound NAD(P)H (τ₁, τ₂) were observed on day 7 for macrophages on hMSC/MP (aligned) compared with macrophages on hMSC/EC/MP (aligned) (Fig. 4C, D, E). The macrophages on all scaffolds on day 7 have an increased fraction of bound FAD (α₁) and a shorter lifetime of both free and bound FAD (τ₁, τ₂) than macrophages on day 3 (Fig. S3 A, B, C). These FAD lifetime variations lead to a shorter FAD mean lifetime (τ_m) of macrophages on day 7 compared to the mean FAD lifetime on day 3 (Fig. 4F).

Metabolic status of pre-vascularized constructs during macrophage co-culture

The overall inflammatory status of the entire pre-vascularized construct co-cultured with macrophages was evaluated via western blotting and human cytokine array. The immunogenicity of pre-vascularized constructs (hMSC/EC/MP) was compared with immunomodulatory hMSC-only constructs (hMSC/MP) lacking allogenic ECs, as a control. It is known that the inflammatory status of macrophages is mainly governed by glycolysis. To determine the immunogenic status of these constructs with macrophage co-culture, glycolysis associated (pro-inflammatory) markers (glucose transporter-1 (GLUT-1), hexokinase-2 (HK2)) and regenerative macrophage specific phenotypic markers (CD163, Arginase-1 (ARG1)) were evaluated (Fig. 5). It was observed that the hMSC/EC/MP showed increased expression of HK2 from day 3 to day 7 compared with hMSC/MP in both aligned and random groups (Fig. 5B). Similarly, aligned and random hMSC/EC/MP constructs showed increased expression of GLUT-1 receptor from day 3 to 7 compared to hMSC/MP constructs (Fig. 5C). Consistently, reduced expression of M2 macrophage-associated marker CD163 was observed from day 3 to day 7 in hMSC/EC/MP groups (aligned and random) compared with hMSC/MP group (Fig. 5D). No change in early growth response 2 (EGR2) expression was observed at day 3 between hMSC/EC/MP and hMSC/MP group. However, a reduced expression of EGR2 was observed in hMSC/EC/MP group compared with hMSC/MP group in both aligned and random constructs (Fig. 5E). Although ARG1 expression was higher in hMSC/EC/MP group compared with hMSC/MP group at day 3, it reduced to similar levels in both groups at day 7 (Fig. 5F). Both hMSC/MP and hMSC/EC/MP group showed similar expression of EGR2 at day 3 and day 7 (Fig. 5F). One of the well-studied immunomodulatory mechanisms by which hMSCs promote a macrophage shift from an inflammatory to regenerative phenotype is by secretion of prostaglandin E synthase 2 (PTGES2) [31]. Both aligned and random hMSC/EC/MP groups showed increased expression of PTGES2 from day 3 to day 7 compared with the hMSC/MP group, indicating an increased activation of this immunomodulatory mechanism over time (Fig. 5G).

Results obtained from micro-vessel characterization, FLIM and western blotting indicated that the pre-vascularized constructs co-cultured with macrophages (hMSC/EC/MP) for 7 days showed decreased density of microvascular networks and increased activation of pro-inflammatory markers. To further delineate the overall immunogenic status of hMSC/EC/MP constructs at day 7, the conditioned media and cell lysates were analyzed via a human cytokine array and compared with hMSC/MP constructs (Fig. 6). Fluorescent intensities of 49 cytokines and surface markers were detected and classified under 3 categories including pro-inflammatory cytokines (Fig. 6B), anti-inflammatory cytokines (Fig. 6C) and angiogenic cytokines (Fig. 6D). It was observed that hMSC/EC/MP constructs showed similar expression of proinflammatory cytokines interferon gamma (IFN-γ), interleukin (IL)-1α, IL-8, IL 17, granulocyte-macrophage colony-stimulating factor (GM-CSF), compared with the hMSC/MP group (Fig. 6E). However, hMSC/EC/MP showed increased expression of epithelial-derived neutrophil-activating protein 78 (ENA 78), intercellular adhesion molecule (ICAM)-I, macrophage migration inhibitory factor (MIF) compared to the hMSC/MP group (Fig. 6E). IL-6 was significantly reduced in hMSC/EC/MP compared to hMSC/MP (Fig. 6E). It has been shown that bone marrow derived hMSCs promote immune-suppression via secretion of IL-6 [45]. Correspondingly, hMSC co-cultured with macrophage (hMSC/MP) showed increased levels of IL-6 (Fig. 6E). Moreover, it was observed that the aligned constructs showed reduced levels of IL-8 and ICAM-I compared to random constructs in both hMSC/MP and hMSC/EC/MP groups (Fig. 6E), Overall, hMSC/EC/MP constructs showed a similar expression profile of pro-inflammatory cytokines as the hMSC/MP group (Fig. 6B), indicating the possible immunomodulatory effects of hMSCs in pre-vascularized group (hMSC/EC/MP). Aligned and random hMSC/EC/MP constructs had similar levels of anti-inflammatory cytokine IL-4, IL-10, IL-11, but higher levels of IL-22, macrophage inflammatory protein 3 alpha (MIP-3a), pentraxin-3 compared to hMSC/MP constructs (Fig. 6F). IL-10 and IL-22 have proven roles in preventing inflammatory responses and in promoting tissue regeneration [46]. hMSC/EC/MP constructs showed slightly increased expression of IL-10 and IL-22 compared to hMSC/MP constructs (Fig. 6F). It was observed that aligned hMSC/EC/MP constructs showed increased levels of pentraxin-3 and growth/differentiation factor (GDF)-15 compared to random constructs, while IL-22 levels were higher in random constructs compared to the aligned group (Fig. 6F). hMSC/EC/MP constructs showed higher secretion of angiogenic cytokines urokinase-type plasminogen activator receptor (uPAR), insulin-like growth factor-binding protein (IGFBP)-2, Serpin E1 and extracellular matrix metalloproteinase inducer (EMMPRIN) compared to hMSC/MP constructs (Fig. 6G). In pre-vascularized group (hMSC/EC/MP), increased levels of IGFBP-2 and vascular cell adhesion protein (VCAM)-1 were observed in aligned constructs, while increased levels of angiogenin, serpin E1 and EMMPRIN were observed in random constructs (Fig. 6G).

Assessment of anastomotic capability and immunogenicity of pre-vascularized constructs in vivo

hMSC-only or pre-vascularized constructs (hMSC/EC) developed on hDF-ECM were subcutaneously implanted in RNU nude rats and harvested at days 3, 7, 14 and 28 post implantation. Histological analysis of the implants and the recruitment of host cells surrounding the implants in the subcutaneous space were determined via H&E staining. (Figure S4). A continues reduction of cellular recruitment surrounding the implants in the subcutaneous space over 28 days (as observed by purple nuclei signal) indicated a possible resolution of inflammation and a lack of continuous active inflammation (Fig S4). The implanted cells remain viable until day 28 at the implant site as observed via hNA staining that co-localized with DAPI signal (Fig. 7A). The hNA signal was only present within the boundary of implant (indicated as white dotted line) and was absent elsewhere, clearly indicating the implanted allogenic cells survived for 28 days (Fig. 7A, bottom row). To determine the anastomotic capability and perfusability of engineered micro-vessels, biotinylated UEA-I, which specifically binds with functional human ECs was injected systemically in the left ventricle of rats prior to euthanization. Besides UEA-1 (green), vessels were co-stained with polyclonal antibody targeting CD31 (red) which can bind with rat/human ECs (Fig. 7B). The presence of UEA-1 signal in the subcutaneous implantation area clearly proves that the engineered human micro-vessels remained patent and were anastomosed with the host vasculature within one week of implantation (Fig. 7B). Magnified images of UEA-1 positive engineered micro-vessels with lumen surrounding a single EC were observed (Fig. S5 A), which clearly indicated anastomosis with host vasculature and a successful blood perfusion. In addition, remodeling of engineered micro-vessels was also observed with an increasing period of time post implantation. The significant increase in the diameter of UEA-1 positive micro-vessels were observed from day 3 (14.42 ± 8.30 µm) to day 7 (22.88 ± 9.47 µm) (p < 0.001), day 14 (21.71 ± 8.95 µm) (p < 0.001) and day 28 (23.96 ± 11.84 µm) (p < 0.001). The diameter of UEA-1 positive lumens (14.42 ± 8.30 µm) detected at day 3 was similar with the micro-vessel diameters measured in vitro (Fig. 2E). No significant change in UEA-1 positive vessel diameter was observed from day 7 to day 28 (Fig. 2E).

The immunogenicity of implanted constructs was evaluated by determining the recruitment of CD68 positive macrophage at the implant site. The subcutaneous tissues containing hDF-ECM scaffolds with only hMSCs (hMSC/hDF-ECM) (Fig. 8A) or micro-vessels (hMSC/EC/hDF-ECM) (Fig. 8B) were harvested to evaluate recruitment of macrophages. Macrophage infiltration from skin samples harvested from non-implanted regions far away from the hMSC/hDF-ECM and hMSC/EC/hDF-ECM implant site were used as a control (Fig. 8C). Macrophage recruitment surrounding the implant site was evaluated over the study period to understand the timeline for ongoing immune responses. Both hMSC/hDF-ECM and hMSC/EC/hDF-ECM showed a similar number of recruited macrophages over the 28 day period (Fig. S6). Although hMSC/EC/hDF-ECM group had slightly increased recruitment of macrophages compared to hMSC/hDF-ECM group at days 3, 14 and 28, the difference was not significant (Fig. S6). Both implants had a significantly increased number of recruited macrophages compared to the control skin (Fig. S6) (p < 0.0001). The significant increase in the number of recruited macrophages was observed from day 3 to day 7 in both constructs (Fig. 8D, E). Macrophage counts reached a maximum value at day 7 and then gradually reduced. A significant reduction in macrophage number was observed at day 14 compared to day 7 in both hMSC/hDF-ECM and hMSC/EC/hDF-ECM implants (Fig. 8D, E). The number of recruited macrophages was significantly reduced at day 28 compared with the other three time points for both of the implants (Fig. 8D, E), indicating a resolution of inflammation over time. No significant difference was observed for recruited macrophage number in the subcutaneous space of the control skin samples over the 28 day observation period (Fig. 8F).

One of the critical bottlenecks in engineered anatomical-scale tissues is a lack of built-in functional vasculature, which is essential to ensure exchange of gases, nutrients, and cell secreted toxins post implantation. Ideally, anatomical-scale built-in micro-vessels, that form a functional anastomosis with host vasculature within a few days, should be contained within the engineered tissue-construct to ensure graft survival. In addition, the engineered microvasculature should withstand physiological pressure without any leakage or elicit a negative immune response [47]. Microvascular networks in native tissues are organized in a hierarchical order to ensure optimal molecular exchange and function [3]. Most recent advances in the field of microfluidics have enabled self-assembly of microvascular networks within a hydrogel and tested their perfusability and potential of disease modelling and drug screening [6–8]. However, anatomical scale large pre-vascularized beds with capillary-sized micro-vessels that can be employed to engineer large, anatomical scale pre-vascularized tissues are currently lacking. More importantly, it is crucial to evaluate patency, perfusability and immunogenicity of these engineered pre-vascularized constructs in vivo. To address this challenge, we have previously developed a large hMSC-ECM sheet with dense, self-assembled microvascular networks under optimal physiological oxygen concentrations [15, 48]. Compared to the traditionally used ECM mimicking hydrogels, our hDF-ECM provides a superior compositional complexity as well as architectural tunability to guide micro-vessel growth in desired directions. To further validate its potential to engineer large tissue-constructs, the current study evaluated the patency, anastomotic capability, perfusability and the innate immune response toward these prevascularized hDF-ECM sheets in vitro and in vivo.

The aligned and randomly organized micro-vascular networks developed on the hDF-ECM scaffold exhibited a diameter similar to native capillary beds (Fig. 2) [49]. Consistent with our previous observation, aligned nano-fibrous hDF-ECM architecture showed increased micro-vessel length and density compared with micro-vessels developed on randomly organized ECM irrespective of macrophage-co-culture (Fig. 2). Importantly, self-assembled micro-vessels with diameters as small as < 20 µm showed a clear open lumen, confirming their tubular structures (Fig. 3). To evaluate immunogenicity of engineered constructs in vitro, macrophages were co-cultured with prevascularized constructs and compared with immunomodulatory hMSC-only constructs. The pre-vascularized constructs maintained micro-vascular density for 3 days, but exhibited significant degradation in vessel density after 7 days, indicating a macrophage-mediated destruction of micro-vessels (Fig. 2A). It is known that pro-inflammatory macrophage activation is dependent on anaerobic glycolysis, while anti-inflammatory macrophages rely on aerobic respiration, such as oxidative phosphorylation (OXPHOS), for their energy demands [50]. These metabolic perturbations in macrophages within the pre-vascularized or immunomodulatory hMSC-only constructs were revealed by FLIM-based imaging of NAD(P)H and FAD, important co-enzymes of glycolysis, OXPHOS, and glutaminolysis [23, 51–53]. FLIM can exclusively evaluate macrophages within the co-culture system due to the physical separation of macrophages from hMSCs and ECs at different depths within the constructs (Fig. 4, Fig S2C). In line with the in vitro micro-vascular network characterization (Fig. 2), significantly increased bound NAD(P)H lifetime (τ₂) was measured in macrophages that were co-cultured with prevascularized constructs for 7 days (Fig. 4). The difference in NAD(P)H lifetime components was exaggerated from day 3 to day 7 in pre-vascularized construct compared with immunomodulatory hMSC only constructs, possibly due to the activation of macrophages by the non-immunomodulatory HUVECs. Consistently, in the presence of macrophages, prevascularized constructs post 7 days exhibited increased expression of glycolysis associated pro-inflammatory proteins (GLUT-1 and HK2) and reduced expression of anti-inflammatory proteins (CD163, EGR2, ARG1) (Fig. 5). Whereas, owing to the immunomodulatory property of hMSCs, the hMSC-only groups (aligned and random) exhibited reduced expression of HK2 and GLUT-1 (Fig. 5B-C), and increased expression of CD163 and EGR2, proteins predominantly present in anti-inflammatory phenotypes of macrophage. EGR2 is a direct target of IL-4-activated STAT6 and has a broad functionality to further induce several transcription factors driving alternative macrophage polarization [54, 55]. ARG1 is a key enzyme involved in l-arginine metabolism, highly expressed in tumor-associated macrophages that have immunosuppressive and tumorigenic functions [56, 57]. Compared with hMSC-only group, pre-vascularized groups had increased ARG1 expression post day 3 of macrophage-culture, which further reduced on day 7, indicating a reduction in immunosuppressive activity of macrophages (Fig. 5E). One of the known mechanisms by which hMSCs promote immunomodulation is via secretion of PGE2, which activates production of anti-inflammatory cytokine IL-10 from macrophage to suppress inflammatory phenotype [31, 58]. The highest expressions of PGE2 (Fig. 5G) and IL-10 (Fig. 6F) were observed in day 7 prevascularized groups, indicating their maximum effort to suppress inflammatory response. Taken together, results obtained from IF-based characterization, FLIM, and western blotting indicated a macrophage maneuver towards the pro-inflammatory phenotype after 7 days of co-culture with prevascularized constructs, which was further verified in cytokine array analysis. Compared with the macrophage co-cultured hMSC only group, the day 7 pre-vascularized group had increased secretion of GM-CSF, IFN-γ, IL-1A, IL-8, IL-17A, MIP-3A which are key regulators that stimulate macrophage activation, due to the presence of non-immunomodulatory HUVECs in the constructs [59]. Furthermore, the prevacularized group improved expression of pentraxin-3 that is associated with vascular inflammation and endothelial dysfunction [60], which could be the reason why micro-vessels degraded (Fig. 2A). As expected, compared to the hMSC group, pre-vascularized constructs showed enhanced expression of angiogenic growth factors (CD31, VECAM-1, Angiogenin, IGFBP-2) [61] and proteolytic degradation of ECM (uPAR) [62], which are essential for self-assembly of micro-vascular formation (Fig. 6G). Aligned or random construct architecture also had effects on the metabolic perturbations in macrophages as observed via FLIM, and the immunogenicity of the entire constructs as revealed via western blot and cytokine array. However, the perturbations caused by aligned or random architectures were not as significant as those triggered by the presence or absence of non-immunomodulatory HUVECs within the constructs (Fig. 4–6, S3).

Although in vitro macrophage co-culture is a well-accepted model to mimic innate immune response, the immunogenicity of pre-vascularized constructs in vivo was further confirmed via subcutaneous implantation in athymic RNU nude rats, which have a robust innate immune system containing granulocytes, monocytes/macrophage, mast cells, natural killer cells, dendritic cells, and B cells. It has been reported that the RNU nude rats have an even stronger innate immune response compared to normal immune compatible rats [24–28]. An anticipated recruitment of host cells surrounding implant boundary was observed during the first week, which became less evident over time as observed by histological analysis (Fig. S4). After 4 weeks, the implant was well integrated in the subcutaneous space, without any signs of a pro-longed active inflammation (Fig S4). Macrophage recruitment was increased during the first week and gradually decreased overtime, with minimal levels of macrophages observed at day 28 (Fig. 8). Importantly, recruited macrophages did not show significant difference in numbers between immunomodulatory hMSC-only and pre-vascularized constructs over time (Fig S6), proving the hypo-immunogenic nature of these engineered constructs in vivo. Detecting perfusability of human-origin vessels in vivo via human EC-targeting UEA-1 lectin injection has been widely reported [63, 64]. UEA-1 positive lumens observed in tissues harvested post 3 days implantation indicated successful anastomosis of engineered micro-vessels within as early as 3 days of implantation. A slight increase in lumen diameter than the pre-implanted vessels was observed during the first week of implantation, suggesting a possible remodeling of the engineered micro-vasculature (Fig S5B). However, no further increase in UEA-1 positive vessel diameter was observed after the first week, indicating the vessels were stable and not significantly dilated post perfusion (Fig S5B). Although HUVECs were selected as a typical model EC-types in this proof-of-concept study, they can be replaced with more advanced and clinically relevant universal or hypoimmunogenic derivatives of induced pluripotent stem cells (hiPSC) derived ECs in future, to advance one step closer towards translational applications [65, 66]. In addition to maturing the self-assembled microvascular networks as pericytes, hMSCs are known to promote immunomodulation via secretion of interleukin-1 receptor antagonist (IL1RA), PGE2 and tumor necrosis factor-inducible gene 6 (TSG-6) [17, 31, 67] to mitigate the immune response and preserve microvascular integrity. hMSCs have been incorporated as bulk cells in the pre-vascularized construct. Their well proven multifaceted regenerative potential in several diseases [68] make the pre-vascularized construct versatile for various tissue-regeneration applications. The feasibility of creating large-prevascularized ECM sheets that can be used as building blocks will enable tissue-engineers to fabricate anatomical scale 3-D constructs to replace damaged or diseased tissues. The faster anastomotic capabilities and perfusability of these engineered microvessels will greatly improve the rate of implant survival by promptly restoring the oxygen and nutrient supply, and thereby ensuring the superior functionality or regenerative capability of implanted constructs.

A completely biological and native tissue-architecture mimicking pre-vascularized hMSC-based constructs was developed. The micron-scale vascular network remained patent during in vitro co-culture with macrophage and clearly showed an open lumen. Results obtained from label-free FLIM imaging and in vitro macrophage co-culture assays clearly revealed the micro-vessel patency and immunogenicity of engineered constructs via detecting metabolic perturbations in co-cultured macrophages. Subcutaneously implanted constructs remained viable during 28 days of the study. Pre-vascularized tissue constructs with self-assembled dense microvascular networks formed a functional anastomosis and showed successful perfusion and remodeling within 3 days of implantation. Immunomodulatory hMSC-containing pre-vascularized constructs did not stimulate prolonged inflammatory responses in vivo. The macrophage recruitment consistently alleviated post one week of implantation, indicating the hypoimmunogenic nature of the constructs. Overall, the engineered microvascular networks developed on completely biological ECM matrix scaffolds closely mimic the anatomy of native capillary beds and can be used as transplantable building blocks to fabricate anatomical scale large 3-D tissues.

Anisotropic and isotropic hDF-ECM fabrication

A silicon wafer with aligned grooves (width: 5 µm, Pitch: 8 µm, height: 270 nm) was prepared with a soft lithography technique at the microfabrication shared facility (MFF) at Michigan Technological University. Polydimethylsiloxane (PDMS) molds were cased from the silicon wafer using Sylgard 184 Silicone Elastomer Kit (Dow Corning, Midland, MI) by mixing base and cross-linker at 10:1 ratio followed by heating at 65⁰C for 4 hours. Flat PDMS was cast from a flat plastic petri dish. Micro-patterned and flat PDMS were coated with polydopamine and collagen-I prior to cell culture as described in the previous publication [29]. Briefly, PDMS were immersed in 0.01% W/V 3-hydroxytyramine hydrochloride (Dopamine-HCl) (ACROS Organics, Fisher scientific) for 24 hours followed by ethylene oxide sterilization. Polydopamine coated PDMS were immersed in bovine collagen (20 µg/mL) (Sigma Aldrich, St. Louis, MO) for 2 hours before cell seeding [29]. Neonatal human dermal fibroblasts (ATCC, Manassas, VA) (passage 3–5) were cultured on micropatterned and flat PDMS for 6 weeks to develop anisotropic or isotropic hDF sheets, which were then decellularized to fabricate anisotropic or isotropic hDF-ECM as described previously [14, 30]. Briefly, hDF sheets were decellularized using 1M NaCl, 0.5% Sodium dodecyl sulfate (SDS), 10 mM Tris and 5 mM ethylenediaminetetraacetate (EDTA) [14].

Prevascularization Of Hdf-ecm

Anisotropic or isotropic hDF-ECM were pre-vascularized using bone marrow derived hMSCs and human umbilical vein endothelial cells (HUVECs) as described previously [14]. Briefly, passage 3–5 hMSCs (1 X 10⁴ cells/cm²) (LONZA) were cultured on hDF-ECM for 7 days under physiological hypoxia (2% O₂) using a programmable hypoxia chamber (Biospherix, Inc., Parish, NY). After 7 days, passage 3–5 HUVECs (LONZA) (2 X 10⁴ cells/cm²) were seeded onto the hMSC-hDF-ECM and were cultured for another 7 days under physiological normoxia (21% O₂). Pre-vascularized hDF-ECM (hMSC/EC/ ECM) or hMSC/ECM (without HUVEC) were implanted in vivo or tested in vitro using macrophage co-culture. For in vivo experiment, only aligned constructs were implanted as proof of concept. Microvascular networks were characterized via measurement of vessel length, vessel diameter, intercapillary distance and percentage area covered by micro-vessels using EC-specific CD31 (PECAM1) marker as described previously [14].

Macrophage Co-culture On Tissue Constructs

THP-1 monocytes (ATCC) were cultured with RPMI-1640 media (ATCC) supplemented with 10% fetal bovine serum (FBS) (R&D systems) and 1% penicillin/streptomycin (Thermo Fisher). THP-1 monocytes were treated with freshly prepared Phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich) (50 ng/mL) supplemented RPMI-1640 media for 24 hours to promote their differentiation into macrophage. Upon 24 hours of PMA stimulation, almost all THP-1 cells become adherent. To study macrophage response in vitro, THP-1 derived macrophages were trypsinized and seeded onto the hMSC/ECM or hMSC/EC/ECM constructs with seeding density of 10⁵ cells/cm² to maintain macrophage to hMSC ratio 10:1 as suggested in a previously published study [31]. After seeding macrophage onto hMSC/ECM or hMSC/EC/ECM constructs, the co-cultures were maintained for another 7 days under physiological normoxia (21% O₂, 5% CO₂).

Evaluating Macrophage-metabolism Using Fluorescence-life Time (Flim) Imaging

Macrophages seeded onto anisotropic or isotropic hMSC-ECM as well as anisotropic or isotropic hMSC/EC/ECM were observed after 3 and 7 days by a custom-built multi-photon fluorescence lifetime microscope (Marianas, 3i). THP-1 derived macrophages co-cultured on hMSC/ECM or pre-vascularized ECM constructs were labeled as hMSC/macrophage or hMSC/EC/macrophage. A 40X water-immersion objective (1.1 NA) was used to couple the excitation and emitted light through an inverted microscope (ZEISS, Axio Observer 7). A tunable (680nm – 1080nm) Ti: sapphire femtosecond laser (COHERENT, Chameleon Ultra II) was tuned to 750nm with 12mW for Nicotinamide adenine dinucleotide phosphate (NAD(P)H) excitation and 890nm with 15mW for Flavin adenine dinucleotide (FAD) stimulation. A 447/60 nm bandpass filter isolated NAD(P)H fluorescence and a 550/88 nm bandpass filter isolated FAD fluorescence. Fluorescence lifetime images were acquired using photomultiplier tube (PMT) detectors (HAMAMATSU) attached to time-correlated single photon counting (TCSPC) electronics (SPC- 150N, Becker and Hickl). A pixel dwell time of 50 µs with 5 frame repeats was used, and each image of 256 x 256 pixels (270µm x 270µm) was obtained with an integration time of around 60 seconds. Both NAD(P)H and FAD images were acquired sequentially for five randomly selected positions for each sample, and three technical replicates were performed to ensure the reliability of the results. The instrument response function (IRF) was measured from the second harmonic signal generated from urea crystals excited at 900nm and captured in the PMT of the NAD(P)H channel. The fluorescence lifetime measurements were validated by imaging YG fluorescent beads (Fluoresbrite YG beads, 20 mm, Polysciences). The beads were measured to have an average fluorescence lifetime of 2.1ns, which is consistent with published studies [32]. Fluorescence lifetime decays of NAD(P)H and FAD were deconvolved from the IRF and fit to a two-component exponential decay (SPCImage; Becker & Hickl) to quantify the lifetimes of free and protein-bound NAD(P)H and FAD [33, 34]. A spatial bin of the center pixel and nine surrounding pixels was used. The fluorescence lifetime components of each pixel were computed by from a two-component exponential model, \(I\left(t\right)= {\alpha }_{1}{e}^{-t/{\tau }_{1}}+{\alpha }_{2}{e}^{-t/{\tau }_{2}}+C\), where I(t) is the fluorescence intensity at time t, α₁ and α₂ are the fractional contributions of the short and long lifetime components, respectively, τ₁ and τ₂ are their fluorescence lifetimes, and C accounts for background light. Images were then segmented into each cell based on the NAD(P)H intensity images by CellProlifer using a customized pipeline, and the mean values of the pixels in each cell region were calculated as the endpoints of each cell [35].

Reverse Transcription-quantitative Polymerase Chain Reaction (Rt-qpcr)

RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) and was treated with DNase I (Thermo Fisher) to remove genomic DNA contamination. Extracted RNA was reverse transcribed using VILO master mix (Thermo Fisher) and amplified by the QuantStudio3™ Real-Time PCR System (Applied Biosystems Waltham, MA) using SYBR green Real Time PCR Master Mixes (Life Technology) and commercially available primers (Sigma-Aldrich) (Table S1). GAPDH gene was used as an endogenous control and data were analyzed using the ΔΔCt method. Amplification measurements were performed in triplicate, and three individual samples were tested for statistical analysis.

Western Blotting

The tissue lysate from hMSC/macrophage and hMSC/EC/macrophage were prepared in radioimmunoprecipitation assay (RIPA) buffer supplemented with EDTA-free Protease Inhibitor (Sigma Aldrich) and Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) according to the manufacturer’s instructions. Denatured cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked using Intercept® (PBS) Blocking Buffer (LI-COR) followed by overnight incubation in primary antibody with 1:1000 dilution at 4°C. The membranes were stained using IRDye® 800CW goat anti-rabbit and IRDye® 680LT goat anti-mouse secondary antibodies (LI-COR) with 1:5000 dilution for 1 hour at room temperature (RT) and imaged with Odyssey® clx imaging system (LI-COR).

Cytokine Array Analysis

The tissue lysate from hMSC/macrophage and hMSC/EC/macrophage groups were prepared in lysis buffer 17 (R&D systems) supplemented with 10 µg/mL Aprotinin (Tocris Bioscience, Ellisville, MO), 10 µg/mL Leupeptin (Tocris Bioscience) and 10 µg/mL Pepstatin (Tocris Bioscience). 100 µg of tissue lysates as well as 250 µl of conditioned media from Day 7 hMSC/MP and hMSC/EC/MP groups were analyzed using Proteome Profiler human XL cytokine array kit (ARY022B, R&D systems) following manufacturer’s instructions. The membranes were stained using IRDye-800 CW streptavidin antibody (LI-COR) and imaged with Odyssey® clx imaging system (LI-COR).

Animal Experiments

Animal experiments were performed following protocols approved by the institutional committee for animal use and care regulations at Michigan Technological University and Texas A&M University. Athymic RNU nude rats (male, 8 weeks old, 200–250 grams weight) were purchased from Charles River Laboratories (Wilmington, MA). Rats were anesthetized with isoflurane and their backs were shaved with clippers. Each rat was surgically implanted with two constructs: (1) hMSC/ECM and (2) hMSC/EC/ECM, both with aligned architecture. A small incision was made on the right and left side of the rat to expose the subcutaneous space in which, hMSC/ECM or hMSC/EC/ECM constructs were implanted, respectively. Both incisions were kept at least 2–3 cm apart from each other and closed with suture clips. The rats were euthanized at day 3, 7, 14 and 28 post implantation and the skin was harvested from the right side and the left side of the back that contained hMSC/ECM and hMSC/EC/ECM implants. A region of skin from another random area from the back was also harvested to serve as a control. Therefore, a total of 3 samples were harvested from each rat and 5 rats were sacrificed at each time point (n = 5). Before euthanization, rats were injected with 100 µl of biotinylated Ulex Europaeus Agglutinin I (UEA I) (Vector Labs, Burlingame, CA) in the left ventricle of heart to allow UEA-I to enter the circulation to detect the anastomosis of engineered vessels. The harvested samples were submerged in OCT compound and were immersed in liquid nitrogen. Samples were stored at -80ºC until sectioning. Harvested tissue samples were sectioned at 6 µm thickness with the interval of ~ 50 µm using a cryostat (Thermo Fisher Scientific, HM 525NX). Tissue sections were placed on the Histobond slides (VWR International, Radnor, PA) and fixed with 4% paraformaldehyde prior to staining.

Histological Analysis, And Immunofluorescence Staining

Tissue sections were stained in the hematoxylin and eosin (H&E) solutions (Sigma-Aldrich) as described previously [36], to observe the overall histology of implants over the period of 28 days. Anastomosis of engineered micro-vessels with host vasculature was determined by using streptavidin Alexa Fluor™ 488 Conjugate secondary antibody (Invitrogen) that specifically binds with biotinylated UEA-I lectin using immunofluorescence (IF) staining. Besides lectin, engineered micro-vessels were also stained with polyclonal antibodies targeting endothelial markers CD31/PECAM-1. Implanted cells were observed via human nuclear antigen (hNA) staining. Recruitment of macrophage were observed via staining macrophage-specific marker CD68. Secondary antibodies goat-anti mouse Alexa Fluor™ 488 conjugate and goat-anti rabbit Alexa Fluor™ 555 conjugate were used for IF staining. Cell nuclei were stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI).

Statistical analysis

Statistical comparisons between experimental groups were performed by one-way ANOVA and Tukey's post hoc test using GraphPad Prism software. For FLIM analysis, statistical comparisons between experimental groups were performed by two-sided t-tests with the Bonferroni correction for multiple comparisons using R Studio software. Results displayed as mean ± standard deviation and were considered statistically significant for *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Acknowledgments

We Acknowledge Dr. Jonathan Bova and Dr. Weilue He for their contribution and help in the animal experiments. We also acknowledge Texas A and M University Microscopy and Imaging Center Core Facility (RRID:SCR_022128) and the Integrated Microscopy and Imaging Laboratory at Texas A&M College of Medicine (RRID:SCR_021637) for providing microscopy resources. This study was supported by the National Institutes of Health (R01HL146652) and the National Science Foundation (1703570) to FZ.

Author contributions

Conceptualization: DS, FZ

Methodology: DS, AS, LH, TC, SV

Supervision: FZ

Writing—original draft: DS, LH

Writing—review & editing: DS, KJB, AJW, JG, FZ

Competing interests

Authors declare that they have no competing interests.

Data and materials availability

All data are available in the main text or the supplementary materials.

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Perfusability and immunogenicity of implantable pre-vascularized tissues recapitulating native capillary network

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Abstract

Figures

Introduction

Results

Integrity of engineered microvascular networks upon macrophage co-culture

Label-free assessment of macrophage response towards pre-vascularized constructs

Metabolic status of pre-vascularized constructs during macrophage co-culture

Assessment of anastomotic capability and immunogenicity of pre-vascularized constructs in vivo

Discussion

Conclusions

Material And Methods

Anisotropic and isotropic hDF-ECM fabrication

Prevascularization Of Hdf-ecm

Macrophage Co-culture On Tissue Constructs

Evaluating Macrophage-metabolism Using Fluorescence-life Time (Flim) Imaging

Reverse Transcription-quantitative Polymerase Chain Reaction (Rt-qpcr)

Western Blotting

Cytokine Array Analysis

Animal Experiments

Histological Analysis, And Immunofluorescence Staining

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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