Layer-by-Layer Fabrication of PAH/PAMAM/Nano-CaCO3 Composite Films and Characterization for Enhanced Biocompatibility

Nanoparticle production and functionalization for various biomedical uses are still challenging. Polymer composites constituted of poly(amidoamine) (PAMAM), polyallylamine hydrochloride (PAH), and calcium carbonate (CaCO3) nanoparticles have good biocompatibility with physiological tissue and fluids, making them excellent candidates for biomedical applications. This study investigated the characteristics of polymeric/nano-CaCO3 composite films based on a PAH/PAMAM matrix, which were fabricated through layer-by-layer synthesis on quartz glass substrates. It was found that the as-prepared elastic moduli of the resultant (PAH/PAMAM)n-CaCO3 (where n represents the number of bilayers) composite films varied from 1.40 to 23.70 GPa for different degrees of cross-linking when 0.1 M nano-CaCO3 particles were incorporated into the polymer matrix. The highly cross-linked (PAH/PAMAM)15-CaCO3 composite film had the highest recorded elastic modulus of 23.70 GPa, while it was observed that for all the composite films fabricated for the present study, the addition of the nano-CaCO3 particles approximately doubled the elastic modulus regardless of the degree of polymerization. Live/Dead assays were used to determine whether the produced composite films were compatible with human lung fibroblast cells. The findings indicate that the (PAH/PAMAM)7.5-CaCO3 composite film had the most positive effect on cell growth and proliferation, with the (PAH/PAMAM)15-CaCO3 composite film demonstrating significant ion transport behavior with low impedance, which was considered good for in vivo rapid cell-to-cell communication. Therefore, the (PAH/PAMAM)7.5-CaCO3 and (PAH/PAMAM)15-CaCO3 composite films are potential tissue engineering biomaterials, but further studies are essential to generate more data to evaluate the suitability of these composites for this and other biomedical functions.


Introduction
Over the past few years, bioinspired and biomimetic techniques have been used to generate functional materials that promote tissue engineering [1][2][3]. Compared with other surface synthesis methods, layer-by-layer (LbL) fabrication technology has garnered great interest due to its inherent simplicity, adaptability, and nanoscale control, among other characteristics. In recent years, there has been a great deal of research on LbL organic multilayer composite films that have been infiltrated with calcium carbonate [4,5]. Various driving forces and assembly processes for LbL self-assembly have led to many LbL synthesized biomaterials, such as polyelectrolytes, biomolecules, and colloidal particles. ese synthetic biomaterials have got physicochemical properties and functions that are useful in tissue engineering [6].
Biocompatibility is a significant issue when selecting or engineering biomaterials to circumvent adverse reactions in the host body, such as inflammation during tissue engineering [7][8][9][10][11]. Mechanical and structural properties are frequently required for tissue engineering to work well. Enhanced biocompatibility has become increasingly important in wound healing, bone repair, and spinal cord injury therapy [12][13][14][15]. Up to this point, significant effort has been directed toward enhancing the surface attributes such as stiffness, roughness, and hydrophilicity of LbL fabricated films [16]. Layer constituents cross-linking is the most extensively employed strategy among these techniques. Hillberg investigated the influence of genipin cross-linking on cell adhesion to LbL polyelectrolyte films using a cell linkage assay and discovered that cell adherence and spread on polymeric films were boosted by the cross-linking [17]. However, no comprehensive investigations on the film's surface chemistry and the respective impact on cell multiplication were conducted in the following years. Silva et al. [18] developed a method for adjusting cell adherence of LbL assembly films by using chitosan with or without crosslinking and coated with alginate. Cross-linking of multilayer films causes significant cell adhesion, spreading, and proliferation changes, which are linked to the alteration of surface chemistry and mechanical characteristics resulting from this process [18].
Coral shells and other organisms are rich sources of calcium carbonate, a vital biological mineral for life. Hydrated metastable forms of calcium carbonate exist in monohydrocalcite (the most stable form), calcium carbonate hexahydrate (the second most stable form), and the unstable amorphous calcium carbonate (ACC) phases. ese phases are known by their mineral names, which are calcite, aragonite, and vaterite [19,20], respectively, as shown in Figure 1. When water is dissolved in the vaterite polymorph, it forms nonporous calcite (the most stable polymorph). CaCO 3 mineralization and biomimetic synthesis have attracted research interest due to their prevalent use in various industries. In recent years, the biocompatibility and biodegradability of CaCO 3 in pharmaceutical and biological applications have been extensively studied [21,22]. Several studies have enhanced the mechanical properties of polymeric/nano-CaCO 3 [23][24][25][26][27], with the underlying variables being the film architecture and fabrication methods employed.
e influence of covalent crosslinking on retarded release in LbL PAMAM multilayers needs additional study [10,15]. is study combined covalent interlayer connections with drug reservoirs to retain model drug molecules. Multilayers and the drug reservoir are made from PAMAM dendrimers with carboxylic terminal groups. First, standard LbL multilayers are made, and then post-infiltration is followed by photochemical crosslinking to form covalent interlayer links.
In numerous labs, poly (allylamine hydrochloride) (PAH) has been examined separately and as a combination of polyelectrolyte connecting mechanisms, including the usage of PAMAM dendrimers [10]. Researchers employed concentrations, molecular weight, and ionic strength to modify the film thickness of polymeric multilayer films generated by successive self-assembly of aqueous nanoparticles with PAH and poly (ethyleneimine).
PAMAM dendrimers are widely employed as scaffolds for biomedical applications, particularly in tissue engineering and drug delivery systems, due to their good biocompatibility, biodegradability to harmless chemicals, and bioactivity [28]. Despite their high biocompatibility, these biopolymers are challenging to manufacture and have poor mechanical characteristics [29]. In this work, PAH/ PAMAM/nano-CaCO 3 composite films were successfully synthesized, characterized, and then evaluated using Live/ Dead assays for enhanced biocompatibility with human lung fibroblast cells (HLFCs). HLFCs are isolated from fully developed lung tissue and are the most prevalent type of cell in lung interstices. HLFCs are important for controlling inflammation and repair of pulmonary tissue, making these cells very useful in studying and treating respiratory diseases or disorders like asthma, tuberculosis, and lung cancer, and for pulmonary wound repair.
e PVDF-HFP solution was made by dissolving 1 g PVDF-HFP and 1 g PVDF-HFP copolymer in 7 mL dimethylformamide (DMF) and placed in the oven for 1.5 h at 80°C. e solution was annealed on a freshly cleaned glass plate, and the necessary thickness was achieved by spreading the film on a glass stick to control the thickness and allowing it to cool at room temperature. DMF was evaporated by placing the substrate in an oven for 6 h at 80°C. e resultant films were cut into 3 cm × 3 cm squares and placed in plasma cleaner to improve the membranes' hydrophilicity. e PVDF-HFP membrane was used for the LbL method of PAH/PAMAM 15 , which was followed by the biomineralization of CaCO 3 .

Fabrication of Cross-Linked PAH/PAMAM Multilayers on Quartz Glass/Indium Tin Oxide (ITO) Substrates and Piezoelectric Film.
e piezoelectric film substrates were first cleaned for 10 min with a plasma cleaner for hydrophilicity.   e LbL PAH/PAMAM multilayers were then assembled as previously described by [28][29][30]. To obtain the low, medium, and high cross-linking multilayers, the (PAH/PAMAM) n asprepared multilayers sample were submerged in 5 mg·mL −1 (pH 3.8) of DAS aqueous solution for 10 s, 3 min, and 40 min (note that the subscript n denotes the number of bilayers whereby each substitute represents 0.5 layers). Noncrosslinking samples were not immersed in DAS and were used as a control. e multilayer film substrates were illuminated for 5 min with a 400 W mercury lamp at a distance of 20 cm after being dried by nitrogen flow. To test the stability of the composite film multilayers, the substrates were submerged into an aqueous NaOH solution (pH 12) for 3 min after photochemical cross-linking to assess the multilayer stability. Both quartz and ITO glass were used as substrates for multilayer preparation and processed similarly.

Mineralization of Calcium Carbonate Nanoparticles in the LbL PAH/PAMAM Matrices.
e multilayered films were immersed in 0.1 M CaCl 2 solution for 30 min before being immersed in 0.1 M Na 2 CO 3 solution for a further 30 min to produce CaCO 3 nanoparticles and then air-dried at ambient temperature; this method was adopted from our previous work [30,31].

Sample Preparation Procedure.
e polyelectrolytes PAH (pH 9.5) and PAMAM (pH 7) with carboxyl-terminal groups were utilized as a prototype for incorporating multilayer film in a layer-by-layer method to demonstrate the methodology (Scheme 1). A previous method of photochemical cross-linking was used to produce the multilayer film samples [29,30], and the researchers believe that hydrogen bonding and electrostatic interactions were the driving forces behind multilayer synthesis. Table 2 lists the major instruments used in this study for materials characterization purposes.

Physiochemical Properties.
A Dimension 3100 atomic force microscope (AFM) obtained from Veeco, USA, was used to analyze the polyelectrolyte multilayers' structure and surface morphology under a Tapping Mode probe with constant amplitude. Peak Mode QNM tapping was used to measure the nanoscopic Elastic's moduli on the AFM. e AFM images of the nanocomposites samples were then used for digital scan image processing with the commercial software Sigma Scan Pro (Sigma Co., US). e surface morphology of calcium carbonate and the multilayer morphology were both studied using a scanning electron microscope (SEM), JSM 7401. D/MAX-RC diffractometer (Rigaku, Japan) patterns of powder X-ray diffraction (XRD) were acquired at an 8°/min scanning rate (40 kV, 100 mA) to analyze the crystal structure of the prepared composite film materials, identify the crystalline phases present in a composite material, and reveal information about its chemical composition.
e Raman spectroscopy and mapping were conducted on a Micro-Raman spectrometer (Horiba), mapping area � 5 μm × 5 μm, to generate detailed chemical images based on a sample's Raman spectrum. An electrochemical workstation was used to measure the electric potential (PGSTAT 302 N, Metrohm Autolab B. V.). Ahead of measurement, silver paint (Agar No. 0443) was used to engineer silver electrodes on all sides of the composite film substrates to ensure precise electrode cutting from the samples.

Electrical Characteristics.
A CHI760 E electrochemical workstation was used to perform and measure the cyclic voltammetry scanning (CV) and electrochemical impedance (EIS) (Chenhua, Shanghai, China) of the nanocomposites.
e studies were carried out in a three-electrode configuration with phosphate-buffered saline (PBS) pH 7.4, 0.1 M as the backup electrolyte. e multilayers built on the ITOglass substrate served as the working electrodes. At the same time, the measurements with a platinum counter electrode in addition to an Ag/AgCl reference electrode were utilized. Different electrical potentials of 5 mV amplitude were applied to the electrochemical workstation for the EIS measurements.  four (PAH/PAMAM) 7.5 -nano-CaCO 3 composite film samples, namely, noncross-linked, low cross-linked, medium cross-linked, and highly cross-linked. It took 60 min for the adsorbates to attain equilibrium concentration on the composites. In order to eliminate molecules that were only superficially adsorbed during the loading process, allcomposite film samples were briefly washed in deionized water before determining the absorbance of the multilayer films. ereafter, the films were submerged in PBS solution in order to conduct the molecular release trials and pressed during this release process to induce piezoelectricity. A specified time interval was chosen to measure the absorbance of the release solution by taking 6 mL of the solution and returning it after the measurement. (PAH/PAMAM) 7.5 and (PAH/PAMAM) 7.5 -nano-CaCO 3 medium cross-linked composite film samples were further subjected to molecular desorption tests using the same test procedures but only at pH 5.4 and 7.4 to determine the impact of the calcium carbonate mineral on the desorption characteristics of the composite films.

Cell Culture.
A Zeiss Leica-440 inverted reflectance laser scanning confocal microscope (Zeiss, Jena, Germany) was used to examine cell morphology. HLFC layers were grown on highglucose Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine Serum and an antibiotic-antimycotic solution comprising 100 μgL −1 penicillin and streptomycin sulfate as previously described by [31]. e culture cells were grown in a moistened atmosphere with 5% CO 2 and 95% air at 37°C. Quartz glass 1 cm × 1 cm square quartz glass-LbL films were disinfected for 30 min using a Co 60 (c) laser and then inserted in a 24-well plate cavity for subsequent cell seeding. e cultivated cell density of the samples was 1 × 105 per well. After incubation for 24 h, cells were stained for 10 min through the LIVE/DEAD Viability/Cytotoxicity Kit, then rinsed three times using PBS solution to eradicate free methylene dye before observing cell morphologies. In addition to the as-prepared composite nanoparticle test, the LIVE/DEAD assay determines cellular membrane stability and cytoplasmic enzyme performance. e assay's effectiveness is predicated on the capability of healthy cells. e results show the characteristics of columnar crystals of spherical vaterite and rhombohedral calcite [28,[32][33][34]. e SEM results clearly show that as the degree of cross-linking increases, the shape of the crystals changes. e small smooth vaterite crystals are transformed into rough, larger, more porous, irregular spherical aggregates, as shown in Figure 2.

Results and Discussion
On the other hand, calcite's smooth well-faceted rhombohedral morphology was transformed into larger irregular aggregates with rough faces. is is because there was nucleation on the existing crystal. erefore, the nanocomposite morphology was greatly influenced by crosslinking degrees, as shown in SEM-EDX analysis (Figures 3(a)-3(d)) and the S1 morphological characterization of CaCO 3 on the PVDF-HFP membrane. Elemental mapping of calcium, carbon, and oxygen was detected from all components on the cross section multilayers as shown in S1(c) and S1(d).
e crystallographic direction of the nanocrystals was examined with a TEM (Figures 4(a), 4(c), and 4(e)) and selected area electron diffraction (SAED) analysis (Figures 4(b), 4(d), and 4(f )). e TEM and SAED images (Figures 4(a) and 4(b)) attest that an ACC structure was dispersed within the matrix. e spherical with irregular, rough surface structures are confirmed to be vaterite, and the irregular are rhombohedral calcites. e SAED images (Figures 4(d) and 4(f )) show single crystals of calcite and vaterite, respectively.

XRD and FT-IR.
e X-ray diffraction measurements ( Figure 5(a)) showed that the obtained crystals are all mixtures of calcite and a small amount of vaterite. e XRD patterns of the nanocomposite films exhibited high-intensity peaks at 2θ values at 28.1°, and 29.4°corresponds to (104, 006) planes in the calcite (JCPDS no. 88-1807) [35]. e 2θ values at 25.4°correspond to (112) plane in the vaterite according to (JCPDS no. 33-0268) to vaterite [36,37]. ere is a small peak at 2θ values 27°corresponding to vaterite.
In the mid-FT-IR spectrum (examined from 4000-500 cm −1 ), the absorption bands of amorphous calcium carbonate (carbonate ion) can be split into four regions: v1 � 1080 cm −1 , v2 � 870 cm −1 , v3 � 1400 cm −1 , and  ) from calcium carbonate crystals. Similar findings have previously been observed for calcium carbonate crystals [27,33,34]. e stretching vibrations of C-H groups in the PAH/PAMAM multilayers cause the distinctive peaks to shift. e formation of hydrogen bonds between the C-H group in PAH and the C�O functional group in PAMAM was linked to the redshift of the C-H bond, which resulted in a drop in the electron cloud density of the C-H bond. e redshifts of N-H and C�O were supposed to be caused by charge transfer and hydrogen bonding between PAH and PAMAM [38]. Kim and Park reported that the presence of C-N stretching (amine) at 1088 cm −1 in a composite sample showed that polymerization happened early in the CaCO 3 mineralization [39]. e intensities of these peaks improved as the number of bilayers increased from 0 to 7, demonstrating the successful production of the PAH/PAMAM multilayers. Figure 6(a), the Raman spectra show a sharp peak at 520 nm for all the film samples under consideration. is is the laser excitation line for a quartz glass substrate, which was used as the substrate material to fabricate all the samples for this study. Additional peaks are located between 1085 cm −1 and 1087 cm −1 . e peak at 1085 cm −1 for the cross-linked composite film suggests the formation of ACC, and the peak at 1087 cm −1 is assigned to carbonates v1 symmetric stretch modes of calcite and vaterite, respectively [26]. e film was only immersed in Na 2 CO 3 solution for the control sample, and thus the results only show a broad peak at 950 cm −1 , with no peak for carbonate observed. Figures 6(b)-6(f )) illustrate the results of the Raman color mapping for noncross-linked composite films, which show a fair intensity with a maximum peak of 1087 cm −1 Raman peak (v1 CO 3 ) [40]. e CO 3 2− content mapping results illustrate the history of the carbonation process and the development of carbonation in specific areas. Carbonation appears to have begun at the exposed surface (blue area), with the carbonation front steadily increasing with time.

AFM.
Cross-linked (PAH/PAMAM) 7.5 multilayers film thickness data (Figures 7(a)(i)-7(a)(iv)) show thickness measurements of 17.449 nm, 18.736 nm, 53.342 nm, and 33.351 nm for non, low, medium, and high crosslinking films, respectively. e stability of the multilayers during the fabrication of cross-linked PAH/PAMAM multilayers confirmed the significant variation in film thickness before mineralization. After immersion of the PAH/PAMAM multilayers in the basic solution (NaOH) for 3 min, noncrossed-linked films disintegrated completely, resulting in low film thickness compared to other prepared films [30]. After mineralization, the four-film samples were not smooth. Hence, AFM could not measure the thickness of the multilayer films, and SEM was therefore used to measure the thickness after the growth of calcium carbonate. e results (Figures 7(b)(v)-7(b)(viii)) show a drastic increase in the film of 781.8 nm, 1.787 μm, and 2.912 μm, 2.669 μm for non-, low-, medium-, and highly cross-linked composite films, respectively. e fully crossed-linked film exhibited a decrease in film thickness as previously reported [30] because the film became more  compact, leading to confined spaces within the multilayer structure [41].

Mechanical Properties.
Quartz glass substrates were used to prepare all samples for characterization with the AFM. e peak force tapping AFM was applied to probe the nano-indentation of (PAH/PAMAM) 7.5 to determine the elastic modulus before and after mineralization. Elastic modulus data in Table 3 show that (PAH/ PAMAM) 7.5 film and(PAH/PAMAM) 7.5 -nano-CaCO 3 film stiffnesses rise by 10 and 17 times, respectively, with an increasing degree of cross-linking. Before mineralization, the average elastic modulus for (PAH/ PAMAM) 7.5 was 5.2 GPa, and after mineralization, the average elastic modulus (PAH/PAMAM) 7.5 -CaCO 3 was determined to be 10.6 GPa. As a result, the modulus increases by nearly 2 times. e results demonstrate a significant improvement in mechanical characteristics and are equivalent to natural materials such as bones and nacres. Unmineralized bone collagen has a modulus of nearly 1-2 GPa, which increases to 10-20 GPa during mineralization [5].
In vivo, HLFCs experience stress and strain due to the rhythmic nature of the respiratory cycle, thus certain mechanical properties are important for their optimal function. Tensile strength and elastic modulus are good indicators of whether a synthesized biomaterial can perform to expectations under stress-strain conditions in vivo. e elastic moduli for tissue culture plastic and glass are in the 2 to 4 GPa range [42], validating the stiffness achieved for the composites in this study, factors such as composition and degree of mineralization as determinants of the elastic modulus of different body tissues and organs. HLFCs in vivo are likely to experience an elastic modulus in the 1 kPa range, meaning these composites should be expected to perform mechanically for pulmonary functions, but because of the extreme stiffness for the cross-linked samples, they are probably best suited for other tissue engineering applications where high stiffness and strength are required [43]. But the volume fraction of the nano-CaCO 3 and the degree of polymerization can be regulated in a series of experiments until the desired elastic moduli and strength are obtained.

Piezoelectric Properties.
e piezoelectric capabilities of the prepared films were investigated by creating electric signals by pushing the film with a finger to generate the piezoelectric effect, which caused film deformation and resulted in combined piezoelectric and dielectric production [31]. PVDF-HFP/(PAH/PAMAM) 7.5 generated a voltage of 1.75 V in response to the finger push force of 5 N, which decreased to 0 V after 19 s, as shown in Figure 8. In comparison, pure PVDF-HFP produced an electric potential of 0.5 V, which dropped to 0 V after 12 s. PVDF-HFP/PAH/PAMAM) 7.5 -CaCO 3 nanocomposite film, on the other hand, developed a voltage of 1.5 V that decreased to 0 V after just 25 s.

Electrochemical Properties: Impedance and Cyclic Voltage.
e electrical characteristics of medium cross-linked polymeric/nano-CaCO 3 films coated on ITO [44] were examined using CV scanning (Figure 9 is suggests that the (PAH/PAMAM) 15.5 -CaCO 3 is more conductive in PBS than the other three composite films. EIS in PBS was used to examine further the conductivity and electro-activity of the polymeric/ nano-CaCO 3 layer. In the high-frequency range, the Nyquist plots for both (PAH/PAMAM) 15.5 , (PAH/PAMAM) 7.5 -CaCO 3, and (PAH/ PAMAM) 15.5 -CaCO 3 films revealed a quasi-semicircular arc, indicating a strong redox activity (Figure 9(b)). e (PAH/PAMAM) 15.5 -CaCO 3 composite film samples demonstrated significant ion transport behavior with low impedance. e diameter of the semicircles matches with the RCT, supporting the CV data. e low electrical resistance is advantageous for in vivo rapid cell-to-cell transmission using spontaneous bioelectrical signals generated by ion and pump activity among nearby cells [45].

Molecular Adsorption and Release.
e adsorption of protein molecules on the surfaces of biomaterials is an indispensable feature of the chemical and physiological reactions at the biomaterial and biological environment interface. Synthetic biomaterials implanted in the living body are expected to be able to release different functional molecules for protein signaling to nearby tissues or fluids [46]. us, evaluation of the biomaterials/proteins interaction and molecular adsorption/desorption of proteins on synthetic biomaterial surfaces is critical for many biomedical functions, such as drug delivery and tissue engineering [47]. Figure 10 illustrates the impact of the CaCO 3 nanoparticles on MB release from the medium cross-linked (PAH/ PAMAM) 7.5 -nano-CaCO 3 and (PAH/PAMAM) 7.5 composite films at pH 5.4 and 7.4. e CaCO 3 nanoparticles lower the MB desorption at either pH, but pH 5.4 consistently results in much improved molecular release, as shown in Figure 10. us, the absence of the mineral and low pH promotes MB release. e results show that MB was completely released during the first 30 min at both pH values for either film before mineralization, whereas the release was sustained after mineralization. e graphs in S2 show that increasing the degree of cross-linking increases the MB adsorption capacity of the (PAH/PAMAM) 7.5 -nano-CaCO 3 composite film, while the optimum pH for MB molecular desorption is 4.5. Antibiotic drugs and gentamycin sulfate were added to the calcium carbonate-loaded multilayers.
erefore, S3 illustrates that low pH and a high degree of cross-linking favor adsorption and desorption of gentamycin sulfate molecules from the (PAH/PAMAM) 7    and 5.4) from calcium carbonate-loaded multilayers is also pH-dependent, as shown in S3. is was due to the pHdependent solubility of CaCO 3 , which causes the matrix to disintegrate in an acidic environment, releasing most of the adsorbed drugs [48]. However, since the pH of human blood is generally in the 7.35-7.45 range, the (PAH/PAMAM) 7.5 film can be considered ideal for fast in vivo drug delivery applications. Still, if retarded drug delivery is the goal, then the (PAH/PAMAM) 7.5 -nano-CaCO 3 film is the better biomaterial.

Cell
Culture. e medium cross-linked composites films were used to test the biocompatibility for cell culture of HLFCs using LIVE/DEAD assays. Two parameters were considered: the organic content and the presence/absence of CaCO 3 . Composites with 7.5 and 15.5 multilayers of PAH/ PAMAM but no CaCO 3 served as substrates for the control experiments. Films with CaCO 3 were used as test experiments, and as shown in the early stage of the 24-cell culture, the cells seeded onto the four samples attached well to the substrates and showed elongated morphology, which corresponds with healthy features of the fibroblast cells (Figures 11(a1)-11(a4)). However, as shown in Figure 11(a2), the number of cells on (PAH/PAMAM) 7.5 -CaCO 3 films was higher than that of the three other samples. After 72 h, the difference in cell morphology and cell density was even clearly distinct. e cells on (PAH/PAMAM) 7.5 -CaCO 3 remained healthy and exhibited elongated shapes and increased cell density, illustrating the effect of cell proliferation, as shown in Figures 11(b2) and 11(c2), while the cells on the other films exhibited shrunken round morphology with irregular aggregates [49] as well as a decrease in cell density due to dead cells that were no longer attached to the substrate Figures 11(b1), 11(c1), 11(b3), 11(c3), 11(b4), and 11(c4). Nevertheless, both film composites were biocompatible and favored cell growth and proliferation.
Furthermore, the morphology of the cells on the (PAH/ PAMAM) 7.5 -CaCO 3 was more elongated and stretched than the other films because of the positive effect of the organicinorganic content. e hard film, the film with low organic matter content, favors cell attachment and proliferation over softer films with high organic content (PAH/PAMAM) 15.5 -CaCO 3 . Because of the little toxicity and high-tissue compatibility [28], the results show that this polymeric/nano-CaCO 3 film can potentially be used for tissue engineering. films, some with and others without nano-CaCO 3 particles. e stiffness of the (PAH/PAMAM) 7.5 film and the (PAH/ PAMAM) 7.5 -nano-CaCO 3 film rose by 10 and 17 times, respectively, with an increasing degree of cross-linking from noncross-linked to fully cross-linked, and with the addition of nano-CaCO 3 particles. Fluorescent images showed increased proliferation of HLFCs, especially for the PAH/(PAMAM) 7.5 -CaCO 3 films. (PAH/PAMAM) 15.5 , (PAH/PAMAM) 7.5 -CaCO 3, and (PAH/PAMAM) 15.5 -CaCO 3 films exhibited strong redox activity, while the highest current response was observed for (PAH/PAMAM) 15.5 -CaCO 3 composite film sample. In the biocompatibility test, the cells on (PAH/PAMAM) 7.5 -CaCO 3 remained healthier and exhibited better-elongated shapes and increased cell density compared to the (PAH/PAMAM) 15.5 -CaCO 3 composite film sample. us, lower polymeric content in the composite films favors cell attachment and proliferation but reduces the current responsiveness. e presence of nano-CaCO 3 particles in the synthetic composite films had a negative effect on molecular adsorption and release, implying potential unsatisfactory performance in drug delivery applications. Overall, the results suggest that the composite films have a potential application in tissue engineering to stimulate cell growth and proliferation. Furthermore, the nano-CaCO 3 particles can be utilized as filling agents in organic multilayered films to improve their mechanical properties for biomedical functions.

Future Work.
e synthesized nanomaterial composites have high tissue compatibility and biomineralization, which could be exploited for biomedical functions in the future. e slow molecular release kinetics can be exploited for controlled application of medication in certain circumstances or to avoid squandering expensive treatments by optimizing the release of treatment from a matrix. To better understand how LbL nanoparticles interact with biological systems, further studies are needed to investigate factors like strength, elasticity, and shape and how these influence biological responses in tissue engineering. Extensive biocompatibility tests over prolonged periods are required in order to determine the suitability of these composites for biomedical applications. Computeraided nanomaterial preparation is crucial to the reproducibility and speed of preparation of tissue engineering upgrades.
us, the application of Computer-Aided Design (CAD) systems in the design and modeling of the composites is recommended, while other specialized modeling systems can be deployed to study their in vivo behavior.

Data Availability
On request, the data used to support the findings of this study can be obtained from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest. cationic dye (MB) into multilayers of (PAH/PAMAM) 7.5 -CaCO 3 with different cross-linking degrees. S3: performance characterization loading and release of a cationic antibiotic (GS) into multilayers of (PAH/PAMAM) 7.5 -CaCO 3 with different cross-linking degrees. (Supplementary Materials)