*2.9. Cell Proliferation Assay*

For the proliferation evaluation, HUVEC were seeded into the well containing HepBPS specimen with a density of 3 <sup>×</sup> <sup>10</sup><sup>4</sup> cells. HUVEC proliferation was examined by a Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, USA) on day 1, 4, and 7. At each time point, medium containing 10% CCK-8 was added to each well and incubated for 4 h at 37 ◦C. The medium was transferred into a new 96-well plate for measuring the optical absorbance at 450 nm with ELISA Microplate Reader.

## *2.10. Statistical Analysis*

All data sets were analyzed by Student's *t*-test for comparison between two groups using the GraphPad 8.0 software. Data were expressed as mean ± standard division of the mean (SD), and statistical significance was set at *p* < 0.05. All experiments were conducted in triplicate.

### **3. Results**

### *3.1. Characterization of the Heparinized Bovine Pericardial Scaffold (HepBPS)*

Macroscopical observation presented a significant difference between the BPS (Figure 1A) and HepBPS (Figure 1C), in which HepBPS adopted a diffuse brown appearance. After incubation with the TBO solution, the BPS sample obtained a light blue color (Figure 1B), whereas a homogeneous presence of purple crystals was clearly detected on the HepBPS sample (Figure 1D). This result indicated a successful deposition of heparin on the scaffold using the LbL assemble technique. Quantitatively, heparin content in the HepPBS was determined as 169.5 <sup>±</sup> 17.31 mg/cm<sup>2</sup> (Figure 1E).

The pericardium derived scaffold was composed of extracellular matrix fibers, which are basic and was stained with eosin as an acid dye (Figure 2A). After heparin modification, the outer layer of sections was stained dark blue (Figure 2B). Heparin is highly acidic because of sulfate and carboxylic acid groups [16,17], which could generate selective reactions with hematoxylin as a basic dye. SEM images confirmed this variation in surface morphology between these surfaces before and after heparin modification (Figure 2C,D). After modification with heparin by the LbL technique, SEM illustration showed the deposition of the DHI/heparin complex as coatings around the fibrils of the HepBPS structure (Figure 2D).

There was the apparent formation of a blood clot on the surface of the BPS sample compared with the thrombus-free surface of HepBPS sample. On the BPS membrane, some adhered platelets were found (Figure 2E), which corresponded to its thrombus formation. HepBPS performed better hemocompatibility, with nearly no platelets attached (Figure 2F).

(Figure 2D).

(Figure 2F).

After modification with heparin by the LbL technique, SEM illustration showed the deposition of the DHI/heparin complex as coatings around the fibrils of the HepBPS structure

There was the apparent formation of a blood clot on the surface of the BPS sample compared with the thrombus-free surface of HepBPS sample. On the BPS membrane, some adhered platelets were found (Figure 2E), which corresponded to its thrombus formation. HepBPS performed better hemocompatibility, with nearly no platelets attached

**Figure 1.** Preparation of heparinized bovine pericardial scaffold. (**A**)—Macroscopic observation of Bovine pericardial scaffold (BPS). (**B**)—Stereo microscope image of BPS after toluidine blue incubation. (**C**)—Heparinized bovine pericardial scaffold (HepBPS). (**D**)—Stereo microscope image of HepBPS after toluidine blue incubation. (**E**)—Heparin quantification of the scaffolds. \*\*\*\*: p value < 0.0001. **Figure 1.** Preparation of heparinized bovine pericardial scaffold. (**A**)—Macroscopic observation of Bovine pericardial scaffold (BPS). (**B**)—Stereo microscope image of BPS after toluidine blue incubation. (**C**)—Heparinized bovine pericardial scaffold (HepBPS). (**D**)—Stereo microscope image of HepBPS after toluidine blue incubation. (**E**)—Heparin quantification of the scaffolds. \*\*\*\*: *p* value < 0.0001. *Polymers* **2022**, *14*, x FOR PEER REVIEW 7 of 12

**Figure 2.** Characterization of heparinized bovine pericardial scaffold. (**A**)—Hematoxylin and Eosin (H&E) staining of Bovine pericardial scaffold (BPS). (**B**)—H&E staining of Heparinized bovine pericardial scaffold (HepPBS). (**C**)—Scanning electron microscope (SEM) of BPS. (**D**)—SEM of HepPBS. (**E**)—Platelet attachment and SEM examination of BPS. (**F**)—Platelet attachment and SEM examination of HepBPS. H&E staining: All scale bars are 10 μm. SEM: All scale bars are 50 μm. Red arrows indicate platelets. White arrows indicate white blood cells. *3.2. In-Vitro Cytotoxicity Tests* In-vitro cytotoxicity tests aimed to provide predictive evidence of biocompatibility. **Figure 2.** Characterization of heparinized bovine pericardial scaffold. (**A**)—Hematoxylin and Eosin (H&E) staining of Bovine pericardial scaffold (BPS). (**B**)—H&E staining of Heparinized bovine pericardial scaffold (HepPBS). (**C**)—Scanning electron microscope (SEM) of BPS. (**D**)—SEM of HepPBS. (**E**)—Platelet attachment and SEM examination of BPS. (**F**)—Platelet attachment and SEM examination of HepBPS. H&E staining: All scale bars are 10 µm. SEM: All scale bars are 50 µm. Red arrows indicate platelets. White arrows indicate white blood cells.

HUVEC were cultured in the condition of the HepBPS liquid extract. After 24 h incubation, no cell death and changes in cell pattern were observed in the complete medium as a negative control (Figure 3A). Meanwhile, 20% DMSO solution as a positive control was highly toxic to the cells, resulting in cell death and detachment (Figure 3B). For the non-

### *3.2. In-Vitro Cytotoxicity Tests*

In-vitro cytotoxicity tests aimed to provide predictive evidence of biocompatibility. HUVEC were cultured in the condition of the HepBPS liquid extract. After 24 h incubation, no cell death and changes in cell pattern were observed in the complete medium as a negative control (Figure 3A). Meanwhile, 20% DMSO solution as a positive control was highly toxic to the cells, resulting in cell death and detachment (Figure 3B). For the nonwashed sample, which imitated the immediate effect of HepBPS when implanted, there was a proper negative effect on cell viability indicated by the decrease in cell adherence density (Figure 3C) and relatively low RGR percentage (75.83%). After 24 h washing in PBS 1X, the cytotoxicity of the HepBPS liquid extract was pretty low, showing as high a level of RGR as 96.56% (Figure 3E). The liquid extract of DMSO caused harsh affects on cell viability, with RGR values at 3%. *Polymers* **2022**, *14*, x FOR PEER REVIEW 8 of 12 washed sample, which imitated the immediate effect of HepBPS when implanted, there was a proper negative effect on cell viability indicated by the decrease in cell adherence density (Figure 3C) and relatively low RGR percentage (75.83%). After 24 h washing in PBS 1X, the cytotoxicity of the HepBPS liquid extract was pretty low, showing as high a level of RGR as 96.56% (Figure 3E). The liquid extract of DMSO caused harsh affects on cell viability, with RGR values at 3%.

**Figure 3.** Observation of HUVEC cultured in different solutions. (**A**)—Culture medium as a negative control. (**B**)—Liquid extract from latex as a positive control. (**C**)—Liquid extract from a nonwashed HepBPS. (**D**)—Liquid extract from HepBPS after 24 h wash in PBS 1X. (**E**) Cytotoxicity determination (Magnification 10×). All scale bars are 100 μm. **Figure 3.** Observation of HUVEC cultured in different solutions. (**A**)—Culture medium as a negative control. (**B**)—Liquid extract from latex as a positive control. (**C**)—Liquid extract from a non-washed HepBPS. (**D**)—Liquid extract from HepBPS after 24 h wash in PBS 1X. (**E**) Cytotoxicity determination (Magnification 10×). All scale bars are 100 µm.

### *3.3. Cell Attachment on the Scaffolds 3.3. Cell Attachment on the Scaffolds*

HUVEC were used to examine the cell attachment support of the scaffolds. HUVEC were seeded onto either BPS or HepBPS and visualized by calcein staining (Figure 4) and SEM (Figure 5). HUVEC attachment on both scaffolds was detected after culturing for 24 h. Somehow, the density of endothelial cells on the HepBPS membrane was similar to that on the surface of the BPS membrane (Figure 4B,D). SEM images also exhibited HUVEC spreading morphology with cellular projections interacting with scaffold components. HUVEC were used to examine the cell attachment support of the scaffolds. HUVEC were seeded onto either BPS or HepBPS and visualized by calcein staining (Figure 4) and SEM (Figure 5). HUVEC attachment on both scaffolds was detected after culturing for 24 h. Somehow, the density of endothelial cells on the HepBPS membrane was similar to that on the surface of the BPS membrane (Figure 4B,D). SEM images also exhibited HUVEC spreading morphology with cellular projections interacting with scaffold components.

### *3.4. Cell Proliferation on the Scaffolds*

Cell proliferation was determined by CCK8 assay at different time points, on day 1, 4, and 7, as shown in the chart (Figure 5E). In both types of scaffolds, the proliferation rate of HUVEC within the first four days was recorded. During the next 4 and 7 days, the optical absorbance of the incubated solution did not significantly increase, indicating a limited growth rate after 7 days. However, data indicated a slower cell growth rate in the scaffolds after heparinization, which was shown as a significantly higher in OD value of the BPS group compared to the HepBPS group.

*Polymers* **2022**, *14*, x FOR PEER REVIEW 9 of 12

**Figure 4.** Calcein staining of BPS and HepBPS after HUVEC seeding. (**A**)—BPS without cell seeding. (**B**)—BPS seeded with HUVEC. (**C**)—HepBPS without cell seeding. (**D**)—HepBPS seeded with HU-VEC. All scale bars represent 100 μm. **Figure 4.** Calcein staining of BPS and HepBPS after HUVEC seeding. (**A**)—BPS without cell seeding. (**B**)—BPS seeded with HUVEC. (**C**)—HepBPS without cell seeding. (**D**)—HepBPS seeded with HUVEC. All scale bars represent 100 µm. **Figure 4.** Calcein staining of BPS and HepBPS after HUVEC seeding. (**A**)—BPS without cell seeding. (**B**)—BPS seeded with HUVEC. (**C**)—HepBPS without cell seeding. (**D**)—HepBPS seeded with HU-VEC. All scale bars represent 100 μm.

seeded with HUVEC. (**E**)—CCK8 assay for cell proliferation. All scale bars represent 50 μm *3.4. Cell Proliferation on the Scaffolds* Cell proliferation was determined by CCK8 assay at different time points, on day 1, **Figure 5.** Assessment of cell attachment by SEM and cell proliferation on scaffolds. (**A**)—BPS without cell seeding. (**B**)—BPS seeded with HUVEC. (**C**)—HepBPS without cell seeding. (**D**)—HepBPS seeded with HUVEC. (**E**)—CCK8 assay for cell proliferation. All scale bars represent 50 μm **Figure 5.** Assessment of cell attachment by SEM and cell proliferation on scaffolds. (**A**)—BPS without cell seeding. (**B**)—BPS seeded with HUVEC. (**C**)—HepBPS without cell seeding. (**D**)—HepBPS seeded with HUVEC. (**E**)—CCK8 assay for cell proliferation. All scale bars represent 50 µm.

### 4, and 7, as shown in the chart (Figure 5E). In both types of scaffolds, the proliferation rate *3.4. Cell Proliferation on the Scaffolds* **4. Discussion**

of HUVEC within the first four days was recorded. During the next 4 and 7 days, the optical absorbance of the incubated solution did not significantly increase, indicating a limited growth rate after 7 days. However, data indicated a slower cell growth rate in the scaffolds after heparinization, which was shown as a significantly higher in OD value of the BPS group compared to the HepBPS group. Cell proliferation was determined by CCK8 assay at different time points, on day 1, 4, and 7, as shown in the chart (Figure 5E). In both types of scaffolds, the proliferation rate of HUVEC within the first four days was recorded. During the next 4 and 7 days, the optical absorbance of the incubated solution did not significantly increase, indicating a Acellular-tissue-matrix-derived biomaterials provide extensive applications in clinical surgery due to their ready availability and potential tissue regeneration. In the case of cardiovascular patch fabrication, bovine pericardium is the most well-known material for its suitable thickness, low rate of suture bleeding, good biocompatibility, and mechanical

limited growth rate after 7 days. However, data indicated a slower cell growth rate in the

properties. As the cardiovascular patch is always in direct contact with the bloodstream, besides the mentioned advantages, this type of material still has a drawback regarding its hemocompatibility. A hemocompatible material should not cause any adverse interactions with blood components, especially not activating blood coagulation or blood clots. However, the pericardium is a biologic tissue; it is mainly composed of collagen fibers, which create an attractive surface for the absorption of plasma proteins and platelet attachment. Therefore, blood clots or thrombosis on the material surface is an obvious consequence. Therefore, there is a demand to improve the hemocompatibility of this material.

To prevent early thrombosis after implantation, surface modification strategies have been developed to improve blood compatibility, including the immobilization of bioactive anticoagulants. In this case, heparin is the most commonly used in systemic anticoagulant therapy. Our previous study successfully established a heparinization of the bovine pericardial scaffold using the layer-by-layer assembly technique [9]. The results showed that heparin could be incorporated into the bovine pericardial scaffold, as proven via SEM images, histological analysis, and heparin amount assay. The seven cycles selected for the LbL technique was determined due to the high level of heparin accumulation, which indicated seven assembly cycles as the heparin immobilization threshold.

In this current study, we also used this technique to immobilize heparin to the scaffolds and further tested for their anti-thrombotic activity and endothelialization support. The results confirmed that the preparation technique effectively deposited heparin in the bovine pericardial scaffold. The macroscopic analysis demonstrated adequate uniform coverage over the surface of the scaffold. Compared with the untreated scaffolds, heparinization resulted in a visually clot-free surface and empty platelet attachment. This observation was similar to the present studies on adopting heparin for surface modification, including decellularized vascular graft and live matrix [8,9,18,19]. Accordingly, surface modification with heparin could provide anti-thrombosis with two effects. In a direct impact, a surface with heparin would prevent the adsorption of plasma proteins and platelet adherence, therefore successfully creating an anti-thrombosis surface. In the other way, after modification, the material can release heparin which, in turn, suppresses the activity of thrombin, thus creating an anti-thrombosis microenviroment within the material and keeping the material surface in a free-clotting condition [9,18].

Endothelial cells form a consensus layer in the blood vessel and maintain the normal physiology condition of blood vessel. One of the most essential functions of the endothelial cell is to prevent thrombosis. Therefore, from a material perspective, endothelialization is a process in which the endothelial cells can form a layer on the material surface. If the cardiovascular patch could achieve this ideal stage, long-term anti-thrombosis could be guaranteed. Our published data showed that the heparinized bovine pericardial scaffolds could release heparin and maintain the anti-thrombus stage for 30 days [9]. This result indicated that the blood clotting on the scaffold surface possibly happens as a consequence of complete heparin release. Therefore, the ability to support endothelialization could, in turn, ensure the blood compatibility of the scaffold. Overall, our study demonstrated that HepBPS could provide an appropriate attachment and proliferation of human endothelial cells. However, before this performance, the HepBPS should undergo 24 h washing in PBS 1X solution; otherwise, potential cytotoxicity could be a certain (as shown in cytotoxicity assay, Figure 3). Although heparin is frequently used as an anticoagulant, there were findings of the toxic effects of heparin in cell cultures [20]. Additionally, there was the presence of DHI ions in the HepBPS, which was also unloaded during incubation and possibly caused a decrease in cell viability. Meanwhile, the in-vitro cytotoxicity of the releasing heparin and DHI ions in our specific case and present publications [9,10,20] remains unsolved, thus demanding a detailed investigation in further study. Endothelialization was detected on HepBPS via endothelial cell attachment and proliferation on the scaffold (as shown in Figures 4 and 5). This implied the two-phase anti-thrombotic activity of the HepBPS when implanted. In the early time of implantation, heparin release and heparin on the graft surface would take the main role in anti-thrombus. Then, the scaffold itself could

support endothelialization, which benefits the long-term thromboresistance by preventing the sub-endothelial matrix from blood contact.

As mentioned earlier, endothelial cells were confirmed to attach to a heparin-modified material surface [11–13,17], which was also found in our current result. The results also showed that the endothelialization effect on the heparinized scaffolds was not comparable to the un-modified one, which might raise a contradiction on the initial purpose of employing heparin on the bovine pericardial scaffolds. In fact, SEM images revealed completely coated microfibers with the heparin/DHI complex after the LbL assembly procedure. Consequently, cell attachment would be dismissed due to reduced or a lack of cell–matrix interaction. Endothelial cells were shown not to express direct receptors for heparin. The positive effects of the heparin immobilized scaffold on endothelial cells were described via "bridging" molecules, e.g., chitosan, gelatin [11,17], or binding and stabilizing cell growth factors, e.g., VEGF [12,13]. Therefore, the decrease in cell adherence or proliferation on HepBPS could be explainable. Another mechanism for endothelialization relates to the protein adsorption level on the scaffold. In-vivo transplantation clearly demonstrates that protein adsorption is the first event that happens at the interface, leading to a later material associated with cell attachment and growth [11,13]. The heparinized scaffold is negatively charged, possibly minimizing negatively charged protein adsorption [18,21] and endothelialization. Taken together, our in-vitro results on HepBPS remained the predictive evidence for the scaffold's ability to support endothelial cell attachment and proliferation in both cases, including the later period of heparin release in vitro/in vivo, long-term interaction, and uptaking of plasma proteins and growth factors in vivo. Therefore, the mechanisms for interactions between the bovine pericardium, heparin and cells need to be performed. Further investigations on these undefined behaviors of the heparinized bovine pericardial scaffold in vitro and in vivo are also recommended to provide a better understanding of the clinical application.

## **5. Conclusions**

In this study, heparinized bovine pericardial scaffolds were constructed via LbL assembly successfully. The results demonstrated that the use of the heparinization technique provided anti-thrombosis and prevented platelet adhesion. After an adequate heparin release by immersing in PBS 1X, the scaffolds could support the attachment and proliferation of endothelial cells, which exhibited a promising effect on endothelialization and long-term hemocompatibility. However, in order to achieve complete understanding of its behaviors, additional investigation should be performed.

**Author Contributions:** M.T.N.N. prepared the material, conducted experimental work, wrote the draft paper, and performed the theatrical analysis; H.L.B.T. mentored, reviewed, and revised the papers. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is funded by Vietnam National University Ho Chi Minh City (VNU-HCM) under grant number B2021-18-03.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data presented in this study are available in the article.

**Acknowledgments:** All authors acknowledge funding received for this project from Vietnam National University Ho Chi Minh City (VNU-HCM) under grant number B2021-18-03. We acknowledge the contributing advice on the blood-contacting test and cardiovascular applications from Thang Quoc BUI, Pediatric Cardiovascular Surgery, Doctor of Medicine: Cho Ray Hospital, Ho Chi Minh City, Vietnam.

**Conflicts of Interest:** The authors declare no conflict of interest.
