*3.6. Evaluation of Cell Proliferation of Fibroblast Cells on Scaffolds*

In general, a functional scaffold requires the ability to support attachment and promote proliferation of cultured cells [67]. In line with it, the L929 fibroblasts cells behavior towards SCCC and SBCC scaffolds with different collagen concentrations was investigated as shown in Figure 7. Cells adhered well with progressive growth and by day three, the scaffold surfaces supported high cell density. The cell proliferation was spotted to increase significantly on scaffold coated with 2.5 wt.% until it reaches 10 wt.% as compared to the collagen free scaffold. However, the number of fibroblast cells decreased (10.6 × <sup>10</sup><sup>5</sup> cells/mL) at the highest collagen peptide concentration (12.5 wt.%). This may be attributed to the reduction of pore size, which caused less pore accessibility and proliferation [8,30,68,69].

In the current study, 10 wt.% collagen coated scaffold with pore size around 108.6 ± 8.7 μm demonstrated highest proliferation rate (12.4 × <sup>10</sup><sup>5</sup> cells/ mL), as shown in Figure 8, in comparison to control, as well as 2.5 wt.%, 5 wt.% and 7.5 wt.% collagen coated scaffolds. In short, scaffolds fabricated using combined techniques displayed the highest cell proliferation. These findings clearly implied the enhancement of cell proliferation attributes to the effects of collagen on cell viability. In short, these findings clearly demonstrated the process of incorporating collagen layer on the scaffold is an efficient way to initiate cell attachment and supports cell growth [63,64].


**Table 3.** Water contact angle of scaffolds with various collagen peptide concentration.

**Figure 6.** Water uptake analysis of SSD coated/collagen coated P(3HB-*co*-4HB); SCCC and SSD blend/collagen coated P(3HB-*co*-4HB); SBCC scaffolds. Data represent means ± SD (*n* = 3). Mean data accompanied by different alphabets as of SCCC scaffolds (a–d) and SBCC scaffolds (A–D) indicates significant difference within each respective group (Tukey's HSD test, *p* < 0.05).

**Figure 7.** Proliferation of L929 cells on the SSD coated/collagen coated P(3HB-*co*-4HB); SCCC and SSD blend/collagen coated P(3HB-*co*-4HB); SBCC scaffolds. Data represent means ± SD (*n* = 3). Mean data accompanied by different alphabets as of SCCC scaffolds (A–F) and SBCC scaffolds (a–g) indicates significant difference within each respective group (Tukey's HSD test, *p* < 0.05).

**Figure 8.** Micrograph of proliferation of L929 cells on (**a**) control-P(3HB-*co*-4HB), SCCC scaffolds (**b**) SCCC 10 wt.%. Data represent means ± SD (*n* = 5).

#### *3.7. Antimicrobial Analysis of SCCC and SBCC Scaffolds*

Antimicrobial analysis was carried out using the colonization test as summarised in Table 4. Antimicrobial substance, silver sulfadiazine (SSD), was incorporated in the scaffolds. Silver compounds, especially (SSD), has been widely used as an antibacterial agent in various biomedical applications [69,70]. Based on the results obtained, both SCCC and SBCC scaffolds revealed desirable antimicrobial effects. However, SBCC scaffolds required 48 h to inhibit certain pathogenic microorganisms which was due to the elution of silver sulfurdiazine impregnated with SSD possessed, whereby Ag ions were physically entrapped in the scaffolds where controlled release of antimicrobial agent occurred [70]. Meanwhile, the results revealed that in SCCC with scaffolds, the silver ion was continuously released directly leading to almost 100% inhibition for most of the microorganism within 12 h. Both scaffolds showed different functionality according to the releasing rate of silver ion. The schematic of the antimicrobial release of both the scaffolds is illustrated in Figure 9. The SCCC scaffolds, which rapidly release SSD, are thus appropriate for further work towards dermal application, especially skin damage to the epidermis and the upper dermis that can be regenerated spontaneously and healed in relatively shorter periods [71–74]. On the condition of chronic wounds, such as diabetic ulcers, long-term release of antimicrobials is highly suggested since regeneration occurs at the edges of injuries [75]. Therefore, the SBCC scaffold can be beneficial for such cases. The antimicrobial effect of SBCC scaffold is effective by the significantly prolonged release of silver ion, which continues to kill microbes after the release system is exhausted. The release of silver ions is accompanied by the contact killing of the layer that contains silver ion gradually released by diffusion and scaffold degradation [69]. Furthermore, according to Heo and coworkers [73], silver sulfadiazine binds with microbial DNA and releases the sulfonamide, interfering with the intermediary metabolic pathway [76].


**Table 4.** Antimicrobial test of SCCC and SBCC scaffolds against various microorganisms.

Values are mean ± SD of three replicates; NA denotes not applicable.

**Figure 9.** Schematic represents the releasing rate of silver ion from (**a**) SCCC scaffolds which rapidly release SSD and (**b**) the slow release of silver ion impregnated in the SBCC scaffolds.

#### **4. Conclusions**

In this study, we demonstrated that a combination of a simple and green approach to fabricate collagen and SSD incorporated P(3HB-*co*-4HB) scaffolds using porogen leaching and freeze-drying techniques. In comparing the SCCC and SBCC scaffolds, both the scaffolds differed in the incorporation of antimicrobial agent. Biomaterial based microbial infections pose serious concerns in the biomedical field. This study focuses on the development of highly efficient potential biomaterials that release the antimicrobial agents. This is in response to the limitations caused by some biomaterials with antimicrobial properties that inhibit microbial infections but slow down the cell seeding and tissue integration. Here, both the SCCC and SBCC scaffolds enhanced cell seeding and proliferation of L929 cells. Nonetheless, SCCC has higher antibacterial efficiency within the first 24 h, whereby the antibiotic is rapidly released as compared to the controlled release of the antimicrobial properties in SBCC scaffolds. Entrapment of SSD in P(3HB-*co*-4HB), as in SBCC, resulted in a reduced burst release of SSD as compared to SCCC. Nonetheless, both the SCCC and SBCC scaffolds could be an excellent candidate to inhibit microbial colonization based on

the biomaterial application without causing antibiotic resistance. The study provides evidence and elucidates the surface interface-cell interactions of the modified P(3HB-*co*-4HB) scaffolds and release of the antimicrobial agent from the scaffolds, thus paving the way in developing infection-resistance biomaterials in the biomedical field in the future.

**Author Contributions:** Conceptualization, A.-A.A.A. and S.V.; methodology, T.P.G.; validation, T.P.G., A.-A.A.A., S.V. and W.M.K.; formal analysis, T.P.G. and S.V.; investigation, T.P.G.; resources, A.-A.A.A.; data curation, T.P.G., W.M.K., S.V.; writing—original draft preparation, S.V.; writing review and editing, S.V. and S.R.; visualization, A.-A.A.A., A.K.H.P.S. and S.R.; supervision, A.-A.A.A.; project administration, T.P.G.; funding acquisition, A.-A.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** We would like to acknowledge Universiti Sains Malaysia (USM) for the research fund provided (311.PCCB.411954, 'USM-Strategic Initiative-Ten Q1-Q2').

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study is openly available.

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

### **References**

