Angiogenic and Osteogenic Synergy of Human Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured on a Nanomatrix

To date, bone tissue regeneration strategies lack an approach that effectively provides an osteogenic and angiogenic environment conducive to bone growth. In the current study, we evaluated the osteogenic and angiogenic response of human mesenchymal stem cells (hMSCs) and green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) cocultured on a self-assembled, peptide amphiphile nanomatrix functionalized with the cell adhesive ligand RGDS (PA-RGDS). Analysis of alkaline phosphatase activity, von Kossa staining, Alizarin Red quantification, and osteogenic gene expression, indicates a significant synergistic effect between the PA-RGDS nanomatrix and coculture that promoted hMSC osteogenesis. In addition, coculturing on PA-RGDS resulted in enhanced HUVEC network formation and upregulated vascular endothelial growth factor gene and protein expression. Though PA-RGDS and coculturing hMSCs with HUVECs were each previously reported to individually enhance hMSC osteogenesis, this study is the first to demonstrate a synergistic promotion of HUVEC angiogenesis and hMSC osteogenesis by integrating coculturing with the PA-RGDS nanomatrix. We believe that using the combination of hMSC/HUVEC coculture and PA-RGDS substrate is an efficient method for promoting osteogenesis and angiogenesis, which has immense potential as an efficacious, engineered platform for bone tissue regeneration.


Results and Discussion
Alkaline phosphatase assay. ALP is considered an early marker of osteoblastic differentiation 31,32 . It is widely used to detect osteoblasts because an increase in ALP activity can signify the start of new bone formation by these cells. During the differentiation process, ALP activity within cell cultures will increase when organic phosphates are cleaved to create free, inorganic phosphates for use in mineralization 33 . Enhanced expression of ALP occurs during ECM maturation, which precedes mineralization 34,35 . Figure 2 shows the ALP activity measured in cultures at days 1, 7, 14, and 28. Because ALP is produced by osteogenic cells, observed ALP activity in cocultures is attributed to hMSCs and not HUVECs. Importantly, at days 7 and 14, hMSCs in cocultures generally showed slightly greater ALP activity compared to their monocultured counterparts. The respective ALP levels from hMSCs cocultured and monocultured on TCPs at day 7 were 0.011 ± 0.0006 and 0.008 ± 0.002 μg of ALP per μg of DNA. The ALP from hMSCs cocultured and monocultured on TCPs then increased at day 14 to 0.038 ± 0.005 and 0.032 ± 0.003 μg of ALP per μg of DNA, respectively. The enhanced osteogenesis that resulted from the coculture condition is in agreement with previous studies 22,36,37 . For instance, Hasirci's group showed that cocultures of rat bone marrow stem cells and rat aortic endothelial cells exhibited more ALP activity than either cell in monoculture 22 . The addition of PA-RGDS yet further amplified ALP activity; cocultured hMSCs on PA-RGDS exhibited ALP activity at day 7 (0.016 ± 0.002 μg ALP per μg of DNA) and day 14 (0.059 ± 0.004 μg of ALP per μg of DNA) that was significantly greater than the ALP activity of any other condition at the same timepoints. Interestingly, the ALP activity for cocultured hMSCs on PA-RGDS progressively increased from day 1 to day 14 but significantly decreased by day 28. A similar result was also demonstrated by Ma et al., in which the highest ALP activity in hMSC/HUVEC cocultures occurred on day 14 with a cell ratio of 50:50 17 . However, the ALP activity for monocultured hMSCs continuously increased throughout the incubation period, and the highest ALP activity (0.046 ± 0.00015 μg of ALP per μg of DNA) was observed at day 28. A promising result, the earlier and greater ALP activity in the PA-RGDS cocultures signifies not only increased osteogenesis, but a more rapid overall differentiation process 38,39 . In addition, at day 28, the ALP activity of hMSC monocultures on PA-RGDS was higher than that of hMSC monocultures on TCPs which also agrees with our previous reported data 28 . Overall, the data indicate that PA-RGDS combined with the coculture condition synergistically promotes greater osteogenic differentiation of hMSCs than other conditions in the current study.

Mineralization analysis via Alizarin Red quantification and von Kossa staining.
When osteogenic cells start to differentiate into osteoblasts, they begin to secrete mineral deposits 35 . Therefore, high mineral accumulation in the ECM usually indicates complete osteogenic differentiation of osteoblasts. In addition, ALP  activity has a significant role in initiating mineralization. Since the synergistic promotion of osteogenic differentiation of hMSCs by combining coculture and PA-RGDS was observed earlier, we next investigated the mineralization degree in the six groups of interest after a 21-day culture period using the Alizarin Red quantification kit. The Alizarin Red quantification for calcium deposition shows that mineralization induced by coculture and PA-RGDS was significantly higher than that of the other studied groups (Fig. 3a). The intensity of the absorbance of Alizarin Red in the coculture on PA-RGDS, coculture on TCP, monoculture on TCP and monoculture on PA-RGDS are 0.65 ± 0.04, 0.50 ± 0.03, 0.32 ± 0.014, and 0.42 ± 0.03, respectively (Fig. 3a). A comparable result was also observed in Alizarin Red quantification after 28-day culture: the mineralization resulting from the coculture and PA-RGDS achieved the highest calcium deposits among all the studied groups (see Supplementary Fig. S1). Moreover, to better visualize the mineralization degree, we characterized the extent of mineralization in the groups after a 28-day culture period using von Kossa staining. The reason we chose von Kossa staining is that calcium deposits stained by von Kossa staining are easier to observe than those stained by Alizarin red staining. As seen in Fig. 3b-g, the von Kossa staining results agree well with the result observed in Alizarin Red quantification. Specifically, von Kossa staining for calcium shows that cocultures contained a larger mineralized area with more mineral deposition than monocultures. In addition, hMSCs on PA-RGDS displayed greater mineralization than those on TCPs. This observation indicates the improved osteoconductive and osteoinductive potential of PA-RGDS cultures due to cell interactions with the RGDS ligand sequence (found in numerous ECM proteins), which may have triggered specific differentiation cues via cell-ECM interactions 29,40,41 . Moreover, the RGDS cell adhesion moiety has been shown to enhance osteoblast cell attachment and spreading, as well as induce mineralization [42][43][44] . Notably, cocultures on PA-RGDS exhibited the greatest degree of mineralization, as revealed by the larger stained area of mineral deposits. This result is consistent with the data collected from the ALP assay and further supports that osteogenesis is significantly enhanced when hMSCs are cocultured with HUVECs on PA-RGDS. Furthermore, Grellier et al. observed a similar result in an earlier study in which mineralization was more extensive when osteoprogenitors were cocultured with endothelial cells, rather than monocultured, and when both cells were immobilized on RGD-grafted alginate microspheres 45,46 . As previously mentioned, the peak ALP activity in cocultured hMSCs on PA-RGDS was observed at day 14, signifying that the differentiation process had begun prior to this day. During cell differentiation, peak ALP activity occurs before mineralized matrix formation, as ALP promotes mineralization 34,35 . Therefore, we speculate that mineralization started in PA-RGDS Osteogenic and angiogenic gene expression and protein secretion. In addition to ALP activity and mineralization, the gene expressions of several crucial osteogenic markers were quantified for all samples at 7, 14, and 21 days post cell-seeding. Analyzed osteogenic gene markers included runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), ALP, and osteocalcin (OCN). During the well-orchestrated process of hMSC differentiation into osteoblasts, these genes, which are indicative of osteogenic phenotypes, are upregulated in a specific sequence. Thus, they are considered established markers of osteoblastic development 47 . ALP gene expression was investigated first. As seen in Fig. 4a, on days 7 and 14, ALP gene expression was significantly upregulated in PA-RGDS cocultures as compared to other cultures. Upregulation of ALP in PA-RGDS cocultures helps explain the enhanced mineralization and ALP content in these cultures. As an early marker for osteogenesis, ALP gene expression peaked on day 14 in the PA-RGDS cocultures at a higher value than in all other cultures which signifies the heightened osteogenic potential of this test group. Though, on days 7 and 14, hMSC monocultures on PA-RGDS showed enhanced ALP gene expression compared to monocultures on TCPs gene expression was not as pronounced as that seen in PA-RGDS cocultures. In previous studies, endothelial cell and hMSC cocultures showed similar early upregulation of ALP expression compared to hMSC monocultures 16,36,48,49 . However, as seen in this study, PA-RGDS promotes even higher levels of early ALP gene expression. Moreover, a downregulation of ALP gene expression was observed on day 21 in cocultures on PA-RGDS nanomatrix, which further suggests that PA-RGDS nanomatrix combined with coculture can accelerate the osteogenic differentiation of hMSCs in coculture. This downregulation might be attributed to the development of an osteoid matrix around osteoblasts as mineralization progressed 47 .
Secondly, Runx2 gene expression was analyzed. Runx2 is the earliest transcriptional regulator necessary for bone formation and is also an essential mediator of osteoblast differentiation 50,51 . Seen in Fig. 4b, significant differences in Runx2 gene expression were not observed between any of the culture groups on day 7. A similar result was reported by Xue et al., in which coculturing hMSCs with HUVECs did not significantly increase their expression of Runx2 after 5 days of incubation compared with Runx2 gene expression in hMSC monocultures 16 . However, on day 14, coculturing substantially enhanced Runx2 gene expression as compared to that in hMSC monocultures. Similarly, Bidarra et al. also showed that Runx2 gene expression is upregulated in cocultures compared to monocultures on TCP 36 . Importantly, Runx2 gene expression in PA-RGDS cocultures was upregulated at day 14 more than all other cultures at that timepoint. This upregulation indicates that the RGDS cell adhesion ligand, coupled with the coculturing condition, can promote and accelerate Runx2 gene expression, leading to earlier phenotypic commitment of hMSCs to osteogenic differentiation. In addition, a decrease of Runx2 gene expression in PA-RGDS cocultures was seen on day 21. The interesting observation may be ascribed to the function and osteogenic cell source of Runx2; it is a transcription factor that increases the number of immature osteoblasts, which is strongly expressed by preosteoblasts and immature osteoblasts but downregulated by mature osteoblasts 52,53 . Thus, the observed upregulated expression of Runx2 on day 14 in cocultures on PA-RGDS indicates that a high number of hMSCs had differentiated into preosteoblasts and immature osteoblasts, but the subsequent downregulation supports the conclusion that most of these osteogenic cells had fully matured into osteoblasts by day 21.
Runx2 regulates the expression of bone matrix protein genes-including OCN-during osteoblast differentiation 52 . Since an upregulation of Runx2 in PA-RGDS cocultures was observed, next we investigated OCN gene expression, which is regarded as an established late marker for osteoblast differentiation that appears concomitantly with the mineralization phase of bone formation 47 . As seen in Fig. 5a, the OCN gene expression at day 14 for monocultures and cocultures on PA-RGDS was almost 3.5 times higher than its counterparts on TCPs, indicating that PA-RGDS can promote OCN gene expression However, on day 14, we did not see a synergistic effect on gene expression produced by the combination of coculturing and PA-RGDS. Moreover, on day 21, significant downregulations of OCN gene expression were observed in monocultures and cocultures on PA-RGDS; conversely, an upregulation in OCN gene expression was observed in TCP monocultures and cocultures on day 21, indicating a prolonged delay in osteoinduction compared to that of cultures on PA-RGDS. Thus, PA-RGDS appears to be the most potent factor that fosters accelerated OCN expression. However, on day 21, coculturing upregulated gene expression on TCPs compared to monoculturing. As OCN is highly expressed by mature osteoblasts, the accelerated upregulation of OCN gene expression observed on PA-RGDS cultures at day 14 suggests that more hMSCs had terminally differentiated by this timepoint than those on TCPs 47,52 . The subsequent downregulation of gene expression observed in the PA-RGDS cultures at day 21 may have been a result of mature osteoblasts becoming osteocytes and embedding in the ECM they secreted 47,52 . Compared to days 7 and 14, amplified OCN gene expression in TCP cultures at day 21 may be indicative of an increased number of mature osteoblasts, which had not yet become embedded osteocytes.
BMP-2 gene expression is shown in Fig. 5b. Compared with hMSC monocultures on TCPs, an earlier upregulation of BMP-2 was seen in response to the coculture condition and the PA-RGDS substrate. On day 7, the combination of coculture and PA-RGDS amplified the expression of the BMP-2 gene more than the PA substrate alone. However, at day 14, BMP-2 gene expression was the most pronounced in cocultures on TCPs. By day 21, cocultures on PA-RGDS displayed the most enhanced BMP-2 gene expression, which was, on average, more than 50 times higher than hMSC monocultures on TCPs at day 7 As one of the most readily detectable BMPs in osteogenic cultures, BMP-2 promotes the differentiation of mesenchymal progenitor cells into osteoblasts 47 . Moreover, BMP-2 is the most powerful inducer of bone formation in vivo. Studies have demonstrated that high doses of BMP-2 will initiate earlier bone formation in osteogenic cultures 54 . BMP-2 gene expression is upregulated early in the process of bone development and, as a signaling molecule, facilitates the expression of Runx2 and ALP in osteoprogenitor cells 47,55 . Therefore, the earlier upregulation at day 7 of BMP-2 gene expression in cocultures on PA-RGDS may signify an accelerated initiation of hMSC osteogenic differentiation in contrast with other cultures. The second upregulation of BMP-2 at day 21 suggests that there was higher potential for bone formation in cocultures on PA-RGDS than in other test groups.
The expression of vascular endothelial growth factor (VEGF) correlates with osteoblastic differentiation. VEGF gene expression is low when osteoblastogenesis begins, it increases in parallel with OCN expression during terminal differentiation, and it peaks during mineralization 56 . Thus, VEGF is an osteogenic marker. As shown in Fig. 6a, on day 14 and day 21, cocultures on PA-RGDS exhibited substantially greater expression than all other culture conditions. On day 21, VEGF gene expression in cocultures on PA-RGDS was almost 10 times higher than that in TCP cocultures, and it was almost 15 and 12 times higher than HUVEC and hMSC monocultures on PA-RGDS, respectively. In addition, on day 21, cocultures on TCPs had slightly higher VEGF gene expression than hMSC monocultures on TCPs, which agrees with a former study that demonstrated that coculturing osteoprogenitor cells with HUVECs can result in higher VEGF gene expression than that seen in monocultures 57 . Taken together, these data indicate that the coculture condition and PA-RGDS substrate can significantly promote the osteogenesis of hMSCs 46 . The enhancement of VEGF gene expression observed here may also explain the elevated level of ALP activity in the PA-RGDS cocultures on day 14, as observed using the ALP assay 58 .
We also investigated the degree of VEGF secretion using enzyme-linked immunosorbent assay. Significantly, on day 21, the synergistic enhancement of VEGF secretion resulting from PA-RGDS and coculture was prominent, as demonstrated by the result that the secreted VEGF amount in the coculture on PA-RGDS was the highest (Fig. 6b). The greatest VEGF production at day 21 in coculture on PA-RGDS compared to other groups may be ascribed to the greatest gene upregulation of VEGF in this group at day 14 an day 21 (Fig. 6a). More importantly, VEGF secreted by osteoblasts/osteocytes has high angiogenic power, as it serves as a paracrine factor that promotes endothelial cell proliferation. Indeed, a previous study reported that VEGF-secreting osteoblastic cells, derived from hMSCs, increased the proliferation of HUVECs 59 . Thus, VEGF is also used as an angiogenic marker. Therefore, the augmented VEGF gene expression and protein secretion in PA-RGDS cocultures at day 21 indicate that the hMSCs reached an elevated level of osteogenic differentiation and were expected to stimulate more angiogenesis in this culture group than in any other test groups.

Network formation imaging.
To check our hypothesis that increased VEGF gene expression and protein secretion would increase angiogenesis, we imaged the morphology of HUVECs and the extent of HUVEC network formation in each culture group. By day 7 post cell-seeding, HUVEC/hMSC cocultures on PA-RGDS showed aggregation and alignment of endothelial cells, as well as extensive pseudopodia formation characteristic of the vascular morphogenetic process (Fig. 7a). Cocultures on TCP did not display the same degree of extending pseudopodia as that seen in cocultures on PA-RGDS. By day 14, network formation, as indicated by further aggregation, spreading, and branching, was quite apparent in PA-RGDS cocultures (Fig. 7b). Cell elongation progressed slightly by day 21 in these cocultures (Fig. 7c). At day 14, cocultures on TCP showed considerate cell spreading but much less coalescence than cocultures on PA-RGDS (Fig. 7e). However, by day 21, the cell aggregation in TCP cocultures was much more reminiscent of that seen in PA-RGDS cocultures (Fig. 7f). In contrast to the cocultures, HUVEC monocultures showed much less vascular network formation. Nevertheless, PA-RGDS monocultures outperformed monocultures on TCPs. For instance, at day 14, HUVEC monocultures on PA-RGDS displayed some pseudopodia formation and cell clustering. However, from day 14 to day 21, vascular morphogenesis did not seem to progress. HUVECs in monocultures on TCPs showed little to no clustering and cell spreading throughout the duration of the study but remained relatively round and separated. It has previously been reported by Issaragrisil's group that the cytokines insulin-like growth factor 1, stromal cell-derived factor 1, and VEGF-all of which are secreted by hMSCs-can promote HUVEC vessel formation 60 . Thus, the accelerated endothelial cell spreading, aggregation, alignment and network formation in cocultures on PA-RGDS indicates that cell-cell interactions between osteogenic and endothelial cells are vital to promote vascular network formation, which may be further enhanced through interactions with the cell adhesion ligand RGDS provided by the functionalized PA.
In conclusion, we used ALP assay, von Kossa staining, Alizarin Red staining, analysis of osteogenic and angiogenic gene expression and related protein content, and characterization of endothelial cell network formation to evaluate the synergistic effects of PA-RGDS nanomatrix and coculturing hMSCs with HUVECs on hMSC osteogenesis and HUVEC angiogenesis. As compared to monocultures and all cultures on TCPs, the PA-RGDS nanomatrix and coculture condition synergistically enhanced ALP activity, mineralization character, and most of the studied osteogenic gene expression by hMSCs. Moreover, upregulated VEGF gene expression and increased VEGF secretion as well as network-like structures were observed in cocultures on PA-RGDS, hinting at a synergy between hMSCs and HUVECs and PA-RGDS that improved angiogenesis.
The results of the current study clearly suggest that the PA-RGDS nanomatrix, combined with the beneficial cell-cell crosstalk in HUVEC/hMSC cocultures, offers a promising biomimetic solution for bone tissue engineering that provides an angiogenic and osteogenic environment with the potential to stimulate, and even accelerate, bone healing and growth. humidity, and 5% carbon dioxide. When confluent, the cells were passaged using 0.05% trypsin and then seeded in TCPs. The seeded cells were cultured at the previously described standard conditions. During both cell expansion and plate culture, media was replaced every 2 to 3 days. For all experiments, the following conditions were used: PA-RGDS nanomatrix substrates, used for experimental groups, and TCPs, used for control groups. On each substrate, 3 cell-seeding conditions were used: 1) hMSCs in monoculture, 2) GFP-HUVECs in monoculture, and 3) hMSCs cocultured with GFP-HUVECs. In monocultures used for the alkaline phosphatase assay and von Kossa staining, 13,500 GFP-HUVECs or hMSCs were seeded per well. In cocultures, 13,500 hMSCs and HUVECs were each seeded per well, yielding a combined total of 27,000 cells per well of the 48-well plates. For Alizarin Red quantification as well as VEGF secretion study, 25,000 GFP-HUVECs or hMSCs were seeded per well. In cocultures, 25,000 hMSCs and HUVECs were each seeded per well, yielding a combined total of 50,000 cells per well of the 24-well plates. Six repetitions were used for each condition.
In the GFP-HUVEC and hMSC monocultures used for qRT-PCR experiments and network formation imaging, 80,000 cells were seeded per well. For cocultures, 80,000 hMSCs and GFP-HUVECs were each seeded per well, equaling a total of 160,000 cells per well of the 6-well plates. Each condition was repeated in quadruplicate. After cell seeding, a 1:1 volume ratio of osteogenic differentiation medium and endothelial cell growth medium was added to each well for cell culture. The osteogenic differentiation medium was made of a basal medium and an hMSC Osteogenic SingleQuots kit (Lonza, Walkersville, MD) that contained the following supplements: 0.5% dexamethasone, 0.5% ascorbate, 10% meningeal cell growth supplement, 2% L-glutamine, 1% penicillin-streptomycin, and 1% B-glycerophosphate. The endothelial cell growth medium consisted of EBM supplemented with the EGM-2 SingleQuots kit, which contained the following supplements: 2% fetal bovine serum, 0.04% hydrocortisone, 0.4% human fibroblast growth factor-B, 0.1% VEGF, 0.1% R3 insulin like growth factor-1, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% GA-1000 (gentamicin, amphotericin-B), and 0.1% heparin (Lonza, Walkersville, MD).
Alkaline phosphatase assay. Cells were harvested with trypsin at 1, 7, 14, and 28 days and stored in Eppendorf tubes at −80 °C until they were retrieved for analysis. An ALP fluorometric assay (Abcam, Cambridge, MA) was used to measure the amount of ALP from each sample. Specifically, 60 μL of cell lysate, 60 μL of alkaline buffer, and 100 μL of phosphatase substrate solution were added to each well of a 96-well plate and incubated for 1 hour at 37 °C. Standards in known concentrations ranging from 0 μM to 1,000 μM were prepared using p-nitrophenol and added to designated wells in the same plate. After incubating for 1 hour, the kinase reaction was stopped by adding 100 μL of 0.3 M NaOH to each well. Absorbances were measured at 405 nm using a microplate reader (Synergy HT, BIO-TEK Instruments, Winooski, VT) and then compared to standards with known ALP content. Results were normalized to the total cell number at each timepoint, as measured by the PicoGreen DNA Assay (Molecular Probes, Eugene, OR). More precisely, Picogreen Dye from the assay kit was added to prepared samples, which were incubated in the dark for 15 minutes. The amount of DNA was then measured using a fluorescence microplate reader and was compared to prepared standards with known DNA content. Finally, ALP results were normalized to measured DNA content.
Von Kossa staining. After 28 days, cells were fixed with 10% formalin according to the manufacturer's protocol (Abcam, San Francisco, CA). Stained cells were imaged using color brightfield microscopy.
Alizarin Red staining and quantification. After 21 and 28 day culture, Alizarin Red quantification was conducted through the Alizarin Red Quantification Assay (ScienCell, Carlsbad, CA) according to the manufacturer's protocol using a microplate reader (Synergy HT, BIO-TEK Instruments, Winooski, VT).The absorbance was measured at 405 nm.
qRT-PCR. At days 7, 14, and 21, GFP-HUVECs and hMSCs were collected and lysed using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). RNA from the cells was isolated according to the manufacturer's protocol. RNA was suspended in nuclease free water, and an ND-1000 UV spectrophotometer (Nanodrop, Wilmington, DE) was used to quantify the concentration of RNA for each sample. Complementary DNA was then synthesized using 500 ng of RNA, which was reverse transcribed in a 2720 Thermo Cycler (Applied Biosystems, Foster City, CA) using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. Samples were prepared in a 96-well PCR plate using the TaqMan Master Mix protocol. Each sample consisted of 2 µL of cDNA solution, 10 µL of 2x master mix, 7 µL of RNA-free water, and 1 µL of gene primer (Runx2, BMP-2, ALP, OCN, VEGF, or GAPDH gene primer) from a TaqMan Gene Expression Assay kit (Applied Biosytems, Foster City, CA). The PCR plate was run in a LightCycler 480 (Roche Life Science, Indianapolis, IN) for the following cycles: pre-incubation at 50 °C for 2 minutes and 95 °C for 10 min; amplification for 45 cycles, at 95 °C for 15 seconds and 60 °C for 1 minute during each cycle; melting at 95°C for 5 seconds and 65 °C for 1 minute; and cooling at 40 o C for 30 seconds. Runx2, BMP-2, ALP, and OCN gene amplifications were used to evaluate osteogenic gene expression. VEGF pathway gene amplification was used to evaluate angiogenic gene expression. Gene expression was normalized against the housekeeping gene, GAPDH. As described in a previous study, the 2 −ΔΔCT method was used to assess gene expression 29 . For Runx2, BMP-2, ALP, and OCN gene expression, data at each timepoint is expressed as a fold ratio relative to data acquired for the hMSC monoculture on TCP at day 7. VEGF gene expression at each timepoint is shown as a fold ratio relative to the GFP-HUVEC monoculture on TCP at day 7.
Evaluation of VEGF secretion. To measure the amount of secreted VEGF from the six groups of interests, enzyme-linked immunosorbent assay (ELISA; R&D system, MN, USA) was conducted. Supernatant from the samples was collected every other day, and the accumulative secretion of VEGF on day 7, 14 and 21 were analyzed by sandwich ELISA method using a microplate reader (Synergy HT, BIO-TEK Instruments, Winooski, VT). The absorbance was measured at 450 nm.
Network formation imaging. Using fluorescence microscopy, GFP-HUVECs were imaged at 7, 14, and 21 days post cell-seeding to assess the extent of cell spreading, alignment, and aggregation. Statistical analyses. Presented data are representative of experimental results. Each experiment was performed at least three independent times with conditions repeated in quadruplicate at each timepoint. Values are expressed as mean ± standard deviation. To assess significance between data, Student's t test or one-way analysis of variance (ANOVA) and post hoc analysis using Tukey's range test for multiple comparisons were conducted. For all tests, p < 0.05 is considered statistically significant. p < 0.005 is considered extremely statistically significant. Moreover, the p values for Fig. 3a

Data Availability
The datasets generated during the current study are available from the corresponding author on reasonable request.