In Situ and Ex Situ Designed Hydroxyapatite: Bacterial Cellulose Materials with Biomedical Applications

Hydroxyapatite (HAp) and bacterial cellulose (BC) composite materials represent a promising approach for tissue engineering due to their excellent biocompatibility and bioactivity. This paper presents the synthesis and characterization of two types of materials based on HAp and BC, with antibacterial properties provided by silver nanoparticles (AgNPs). The composite materials were obtained following two routes: (1) HAp was obtained in situ directly in the BC matrix containing different amounts of AgNPs by the coprecipitation method, and (2) HAp was first obtained separately using the coprecipitation method, then combined with BC containing different amounts of AgNPs by ultrasound exposure. The obtained materials were characterized by means of XRD, SEM, and FT-IR, while their antimicrobial effect was evaluated against Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Staphylococcus aureus), and yeast (Candida albicans). The results demonstrated that the obtained composite materials were characterized by a homogenous porous structure and high water absorption capacity (more than 1000% w/w). These materials also possessed low degradation rates (<5% in simulated body fluid (SBF) at 37 °C) and considerable antimicrobial effect due to silver nanoparticles (10–70 nm) embedded in the polymer matrix. These properties could be finetuned by adjusting the content of AgNPs and the synthesis route. The samples prepared using the in situ route had a wider porosity range and better homogeneity.


Introduction
One of the most significant advances in the field of tissue engineering is the development of a porous three-dimensional matrix [1]. In order to act as an optimal bone support, the synthetic matrix must have a series of properties, including biocompatibility, biodegradability, appropriate porosity (similar to the replaced tissue), antimicrobial activity, and production reproducibility [2,3]. In addition to these requirements, it is also recommended that they have mechanical properties similar to natural bone, such as compressive strength, fatigue resistance, and high Young's modulus [4].
((C 6 H 9 NO) n ), and sodium borohydride (NaBH 4 , >99%), purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvents were American Chemical Society (ACS, Washington, DC, USA) purity. Bacterial cellulose membrane was produced in the laboratory by Gluconacetobacter sp. strain isolated from traditionally fermented apple vinegar in the Microbiology Laboratory of the Chemical and Biochemical Engineering Department, University Politehnica of Bucharest, based on a protocol previously described [40].
In order to obtain 500 mL colloidal silver (100 ppm concentration), an aqueous silver nitrate solution (AgNO 3 ) was used as silver precursor, to which 30 mL sodium citrate (0.3 M) was added. After 12 min, 30 mL of Polyvinylpyrrolidone (PVP, 0.007 M) and 5 mL NaBH 4 (1 M) were added to reduce Ag + to Ag 0 nanoparticles. Finally, 5 mL of oxygenated water (30%) was added, and stirring was maintained for another 10 min approximately, until a light blue color (due to the size of the nanoparticles) was obtained ( Figure 1) [41].
In order to obtain 500 mL colloidal silver (100 ppm concentration), an aqueous silver nitrate solution (AgNO3) was used as silver precursor, to which 30 mL sodium citrate (0.3 M) was added. After 12 min, 30 mL of Polyvinylpyrrolidone (PVP, 0.007 M) and 5 mL NaBH4 (1 M) were added to reduce Ag + to Ag 0 nanoparticles. Finally, 5 mL of oxygenated water (30%) was added, and stirring was maintained for another 10 min approximately, until a light blue color (due to the size of the nanoparticles) was obtained ( Figure 1) [41]. The bacterial cellulose synthesized by the Gram-negative bacteria (Gluconacetobacter sp.) was boiled at 80 °C in water alkalized with sodium hydroxide (pH 14, measured by colorimetric method). After purification, BC was washed in distilled water until it reached neutral pH. Afterward, it was minced using a blender (Silvercrest, Neckarsulm, Germany) and weighed according to the recipe. Previously, the amount of dry matter was determined on a quantity of BC by eliminating the humidity, and it was found that 0.25 g of dry BC can be obtained from 10.62 g of wet BC [17].
Two synthesis methods were used to obtain the bacterial cellulose and HAp-based composites. The first method involved obtaining in situ hydroxyapatite nanoparticles directly on cellulose fibers and subsequently adding the AgNPs solution, followed by homogenization using an ultrasound probe (composites further referenced as BC1, BC2, BC3, and BC4).
For the synthesis of 2 g HAp, the amount of precursors required to obtain the material with different concentrations of AgNPs (0, 1, 2, and 5 wt %) was calculated.
The Ca 2+ and PO4 3− precursors, Ca(NO3)2•4H2O, and (NH4)2HPO4 were solubilized in distilled water, and bacterial cellulose was added in the calcium nitrate solution (see Figure 2). The mixtures were homogenized by magnetic stirring, and the ammonium phosphate solution was added The bacterial cellulose synthesized by the Gram-negative bacteria (Gluconacetobacter sp.) was boiled at 80 • C in water alkalized with sodium hydroxide (pH 14, measured by colorimetric method). After purification, BC was washed in distilled water until it reached neutral pH. Afterward, it was minced using a blender (Silvercrest, Neckarsulm, Germany) and weighed according to the recipe. Previously, the amount of dry matter was determined on a quantity of BC by eliminating the humidity, and it was found that 0.25 g of dry BC can be obtained from 10.62 g of wet BC [17].
Two synthesis methods were used to obtain the bacterial cellulose and HAp-based composites. The first method involved obtaining in situ hydroxyapatite nanoparticles directly on cellulose fibers and subsequently adding the AgNPs solution, followed by homogenization using an ultrasound probe (composites further referenced as BC 1 , BC 2 , BC 3 , and BC 4 ).
For the synthesis of 2 g HAp, the amount of precursors required to obtain the material with different concentrations of AgNPs (0, 1, 2, and 5 wt %) was calculated.
The Ca 2+ and PO 4 3− precursors, Ca(NO 3 ) 2 •4H 2 O, and (NH 4 ) 2 HPO 4 were solubilized in distilled water, and bacterial cellulose was added in the calcium nitrate solution (see Figure 2). The mixtures were homogenized by magnetic stirring, and the ammonium phosphate solution was added dropwise. After homogenization, the pH was adjusted to 10.5 with an ammonium hydroxide solution. The obtained precipitates were aged for 24 h, then washed with distilled water until pH 7 was achieved. After washing, the appropriate amount of silver colloidal solution that had been previously obtained was added to each composition according to the centralizing table (Table 1). The obtained mixture was mixed for 3 min in the presence of ultrasound to ensure the best possible homogeneity dropwise. After homogenization, the pH was adjusted to 10.5 with an ammonium hydroxide solution. The obtained precipitates were aged for 24 h, then washed with distilled water until pH 7 was achieved. After washing, the appropriate amount of silver colloidal solution that had been previously obtained was added to each composition according to the centralizing table (Table 1). The obtained mixture was mixed for 3 min in the presence of ultrasound to ensure the best possible homogeneity and then poured into Petri dishes (d = 54 mm), frozen, and subsequently subjected to the freeze-drying process (freezing at −55 °C for 12 h, vacuum at 0.001 mbar for 12 h, and heating under vacuum for 24 h to 35 °C) in order to obtain porous composite materials [42].

Sample Name Bacterial Cellulose Content (%) HAp Content (%) AgNP Content (%)
The second method of synthesis involved the separate synthesis of HAp by the coprecipitation method, followed by its addition to bacterial cellulose gel in the presence of ultrasound for 3 min, as described in Figure 3.  After homogenization, the required amount of silver colloidal solution was added, followed by the steps previously described in the in situ method. The composites thus obtained by the ex situ method were noted as BC5, BC6, BC7, and BC8, and their composition is presented in Table 1.

Physicochemical Characterization
Investigation of the crystallinity of the powders was performed by means of X-ray diffraction After homogenization, the required amount of silver colloidal solution was added, followed by the steps previously described in the in situ method. The composites thus obtained by the ex situ method were noted as BC 5 , BC 6 , BC 7 , and BC 8 , and their composition is presented in Table 1.

Physicochemical Characterization
Investigation of the crystallinity of the powders was performed by means of X-ray diffraction (XRD) technique using the PANalytrical Empyrean (Malvern, Bruno, the Netherlands) equipment in Bragg-Brentano geometry equipped with a Cu anode (λCuKα = 1.541874 Å) X-ray tube. The spectra were acquired in the range of 10-80 • 2θ angles (Bragg angle) with an acquisition step of 0.02 • and an acquisition time of 100 s. The scanning electron microscopy (SEM) images were performed with a FEI Inspect F50 microscope coupled with an energy-dispersive spectrometer (EDS) (FEI, Eindhoven, the Netherlands). Both secondary electron and backscattered electron detectors were used at 30 kV accelerating voltage. The TEM images of AgNPs were obtained using the high-resolution transmission electron microscope TecnaiTM G2 F30 S-TWIN equipped with selected-area electron diffraction (SAED) detector, purchased from the company FEI. This microscope operates in transmission mode at a voltage of 300 kV with a resolution of 2 Å. Research conducted by Fourier transform infrared spectroscopy (FT-IR) involved the analysis of a small amount of samples using the Nicolet iS50R spectrometer (Thermo Fisher Waltham, MA, USA). The measurements were performed at room temperature utilizing the total reflection attenuation module (ATR), and 32 scans of the samples between 4000 and 400 cm −1 were performed using a resolution of 4 cm −1 . The differential thermal analysis (ATD-DSC) were performed using a Shimadzu DTG-TA-50H equipment (Shimadsu, Sanjo, Japan) at 25-700 • C with a heating rate of 10 • C/min.
The open porosity of the freeze-dried composite materials was calculated with Equation (1) for each material prepared in order to observe the porosity level according to the chosen manufacturing method, while the water absorption was calculated with Equation (2): where M we is the wet sample weight, M d is the dry sample weight, and M w is the sample weight in water.

Degradability
To test their biodegradability, the samples were placed in a 12-well plate in which phosphate-buffered saline (PBS) and simulated body fluid (SBF) were added, similar to the processes involved in the human body. After immersion of the samples in fluid, their integrity was monitored for 7 days. The degradation rate was calculated with Equation (3) for each material: where M 7days is the sample weight in SBF after 7 days of immersion in SBF, and M initial is the sample weight after immersion in SBF. All the weight values were obtained at room temperature using a hydrostatic analytic balance.

Antimicrobial Efficiency
The antimicrobial behavior of the freeze-dried composite materials was qualitatively assessed by an adapted growth inhibition assay [43]. To cover a wide spectrum of clinically relevant model microbial species, one Gram-positive (Staphylococcus aureus ATCC 23235), one Gram-negative bacteria (Escherichia coli ATCC 25922), and one yeast (Candida albicans ATCC 10231) laboratory strain were used. The standard work protocol for the adapted version of the disc diffusion method implies the preparation of microbial suspensions of 0.5 McFarland standard density (1.5 × 10 8 colony forming units (CFU)/mL), prepared in sterile buffered saline solution. The obtained microbial suspensions were afterward used to swab inoculate the entire surface of the nutrient agar Petri dishes. After inoculation, identical size samples of the sterile coatings were aseptically placed on the inoculated agar surface, and the plates were incubated at 37 • C for 24 h to allow the growth of bacteria. After incubation, the growth inhibition zone diameter (mm) was measured. A wider inhibition zone suggests a higher antimicrobial effect of the fibrous dressing, reflecting the ability of AgNPs contained into the composite material to diffuse within the agar.

Results and Discussions
The thermal analysis corresponding to the composite samples are presented in Figure 4. The two minor weight losses that occurred at temperatures below 200 • C were probably related to the volatilization of solvents and physical water. The main mass loss was observed in the range 250-450 • C, with the corresponding exothermic effect being strong and intense and indicating burning of the organic component of the composite (bacterial cellulose). Regarding the compositional aspects, the thermal analysis allowed an accurate assessment of the loading degree depending on the material deposited on the surface or between the fibers of the bacterial cellulose. It was observed that certain changes associated with endothermic processes occurred in the thermogravimetric (TG) curve with the addition of silver nanoparticles. Hence, in the 450-600 • C interval, exothermic effects generated by the combustion of BC were observed (see Figure 4a), while in the 600-700 • C interval, it can be assumed that the oxidation of silver nanoparticles and dehydroxylation of HAp occurred [15].  In order to demonstrate the composition, hydroxyapatite was analyzed by XRD technique. The diffractograms are presented in Figure 5.
Due to the fact that BC5-BC8 samples were made by direct mixing of bacterial cellulose with hydroxyapatite (ex situ), the composition of this sample was not expected to change; therefore, the XRD analysis was only performed for bacterial cellulose, simple hydroxyapatite, and BC3 and BC4 composites (in situ) [44,45]. We observed (Figure 4) significant differences regarding mass loss between the samples obtained by in situ vs. ex situ method. The total weight loss in the temperature range of 30-700 • C was 45% for BC 1 , 65% for BC 4 , 68% for BC 5 , and 70% for BC 8 . The composites obtained in situ had a lower weight loss, which suggests good loading of BC with calcium phosphate phases (HAp).
In order to demonstrate the composition, hydroxyapatite was analyzed by XRD technique. The diffractograms are presented in Figure 5. In order to demonstrate the composition, hydroxyapatite was analyzed by XRD technique. The diffractograms are presented in Figure 5.
Due to the fact that BC5-BC8 samples were made by direct mixing of bacterial cellulose with hydroxyapatite (ex situ), the composition of this sample was not expected to change; therefore, the XRD analysis was only performed for bacterial cellulose, simple hydroxyapatite, and BC3 and BC4 composites (in situ) [44,45].  Due to the fact that BC 5 -BC 8 samples were made by direct mixing of bacterial cellulose with hydroxyapatite (ex situ), the composition of this sample was not expected to change; therefore, the XRD analysis was only performed for bacterial cellulose, simple hydroxyapatite, and BC 3 and BC 4 composites (in situ) [44,45].
It was observed that, in all the analyzed samples, the existence of bacterial cellulose and HAp was obvious. In addition, the low-intensity peak around 2θ = 38 • , which can be assigned to the (111) crystalline plane, indicated the presence of silver nanoparticles in the composite structure. Investigation of the composites BC 3 and BC 4 revealed peaks located at 2θ values of 15, 16, and 23 • , which can be attributed to bacterial cellulose according to ICDD 00-056-1718.
As the HAp peaks were poorly visible in XRD analysis, FT-IR analyses were used to better highlight hydroxyapatite formation. The results are presented in Figure 6.
The vibrational frequencies characteristic of bacterial cellulose were observed at 3500-3200 cm −1 (OH stretch vibrations) and 2958 cm −1 (CH 2 and CH 3 stretch vibrations). The wide band observed in the region of 3500-3200 cm −1 , attributed to the hydroxyl groups within the bacterial cellulose, increased in absorbance with higher silver content. This behavior suggested that the presence of HAp crystals affected cellulose hydroxyl groups, probably by covering them at the surface. Furthermore, the change observed for the band attributed to intramolecular hydrogen bonding (~3500 cm −1 ) confirmed a strong interaction between the OH groups and calcium phosphate. The chemical interaction between HAp and BC stabilized the composite so that it could maintain its mechanical integrity, an aspect required for bone substituents [46].
The FT-IR bands observed at 1020 cm −1 and 570-600 cm −1 were attributed to the vibrational modes of PO 4 3− . Because the stretching vibration of CO 3 2− also appeared (at 1418 cm −1 ), absorption of CO 2 from the air is suggested [47]. This is mainly a result of the affinity for carbonate of HAp as well as the lack of heat treatment during the in situ synthesis (which favors the release of CO 2 ). Carbonated hydroxyapatite contributes to the biomimetism increase of the obtained composites, which can promote the process of osteoregeneration. It was observed that the in situ method accelerated the nucleation of HAp crystals onto BC fibers instead of crystallization as higher absorbance values were registered for BC 5 -BC 8 samples, which contained highly crystalized HAp. The bands observed at 1641 and 643 cm −1 correspond to the stretching and deformation vibrations of AgO, respectively, thus confirming the presence of silver in the obtained materials [48]. This result supports the idea that the composite material developed here possesses essential physicochemical properties and could be very useful for biomedical applications, especially hard tissue engineering.
Materials 2020, 13, 8 of 17 It was observed that, in all the analyzed samples, the existence of bacterial cellulose and HAp was obvious. In addition, the low-intensity peak around 2θ = 38°, which can be assigned to the (111) crystalline plane, indicated the presence of silver nanoparticles in the composite structure. Investigation of the composites BC3 and BC4 revealed peaks located at 2θ values of 15, 16, and 23°, which can be attributed to bacterial cellulose according to ICDD 00-056-1718.
As the HAp peaks were poorly visible in XRD analysis, FT-IR analyses were used to better highlight hydroxyapatite formation. The results are presented in Figure 6. The vibrational frequencies characteristic of bacterial cellulose were observed at 3500-3200 cm −1 (OH stretch vibrations) and 2958 cm −1 (CH2 and CH3 stretch vibrations). The wide band observed in the region of 3500-3200 cm −1 , attributed to the hydroxyl groups within the bacterial cellulose, increased in absorbance with higher silver content. This behavior suggested that the presence of HAp crystals affected cellulose hydroxyl groups, probably by covering them at the surface. Furthermore, the change observed for the band attributed to intramolecular hydrogen bonding (~3500 cm −1 ) confirmed a strong interaction between the OH groups and calcium phosphate. The chemical interaction between HAp and BC stabilized the composite so that it could maintain its mechanical integrity, an aspect required for bone substituents [46].
The FT-IR bands observed at 1020 cm −1 and 570-600 cm −1 were attributed to the vibrational modes of PO4 3− . Because the stretching vibration of CO3 2− also appeared (at 1418 cm −1 ), absorption of CO2 from the air is suggested [47]. This is mainly a result of the affinity for carbonate of HAp as well as the lack of heat treatment during the in situ synthesis (which favors the release of CO2). Carbonated hydroxyapatite contributes to the biomimetism increase of the obtained composites, which can promote the process of osteoregeneration. It was observed that the in situ method accelerated the nucleation of HAp crystals onto BC fibers instead of crystallization as higher absorbance values were registered for BC5-BC8 samples, which contained highly crystalized HAp. The bands observed at 1641 and 643 cm −1 correspond to the stretching and deformation vibrations of AgO, respectively, thus confirming the presence of silver in the obtained materials [48]. This result supports the idea that the composite material developed here possesses essential physicochemical properties and could be very useful for biomedical applications, especially hard tissue engineering.
Through the two analyses performed, it was possible to notice the elemental composition of materials (EDS) as well as the homogeneity of hydroxyapatite particle dispersion.
The SEM image highlighted the fibrous structure of BC (see green arrow), which were decorated with inorganic particles (see blue arrow). In the SEM images performed on the composites in which HAp was obtained in situ (Figure 7a-d), a better homogeneity was observed compared to the cases in which HAp was obtained separately and subsequently mixed with BC (ex situ) (  Through the two analyses performed, it was possible to notice the elemental composition of materials (EDS) as well as the homogeneity of hydroxyapatite particle dispersion.
The SEM image highlighted the fibrous structure of BC (see green arrow), which were decorated with inorganic particles (see blue arrow). In the SEM images performed on the composites in which HAp was obtained in situ (Figure 7a-d), a better homogeneity was observed compared to the cases in which HAp was obtained separately and subsequently mixed with BC (ex situ) (Figure 8a-d. The interaction between HAp nanoparticles distributed in the 3D network of BC stabilized the composite so that it could maintain its mechanical integrity, an aspect required for bone substituents. In addition, EDS analysis confirmed the presence of the elements specific for hydroxyapatite (Ca, P, and O) as well as the presence of silver for the samples in which it was added (Figure 7(b 2 -d 2 ) and Figure 8(b 2 -d 2 )).
Transmission electron microscopy images showed the silver nanostructure (Figure 9a,b), with the dimensions of the silver particles being in the range 3-60 nm. It could be observed that the quasi-spherical morphology of nanosilver and some areas were darker while others were brighter; the darker areas indicate a higher degree of crystallinity of the material. Figure 9d shows a SAED image with information on the crystallinity of the analyzed material. The presence of diffraction rings with higher light intensity shows a high degree of crystallinity.
The calculated open porosity for each prepared material is presented in Figure 10a, and the calculated water absorption is presented in Figure 9b. Figure 10a shows that the composites obtained by in situ approach had a large porosity compared to samples obtained by ex situ. This suggests that the in situ route will provide a biodegradable polymer with excellent water retention and, possibly, good osteoinductivity, which can be used as an artificial substitute for hard tissue.
Materials 2020, 13, 9 of 17 composite so that it could maintain its mechanical integrity, an aspect required for bone substituents. In addition, EDS analysis confirmed the presence of the elements specific for hydroxyapatite (Ca, P, and O) as well as the presence of silver for the samples in which it was added (Figure 7(b2-d2) and Figure 8(b2-d2)).
(a) (a1) (a2)   Transmission electron microscopy images showed the silver nanostructure (Figure 9a,b), with the dimensions of the silver particles being in the range 3-60 nm. It could be observed that the quasi-spherical morphology of nanosilver and some areas were darker while others were brighter; the darker areas indicate a higher degree of crystallinity of the material.   Figure 9d shows a SAED image with information on the crystallinity of the analyzed material. The presence of diffraction rings with higher light intensity shows a high degree of crystallinity.
The calculated open porosity for each prepared material is presented in Figure 10a, and the calculated water absorption is presented in Figure 9b. Figure 10a shows that the composites obtained by in situ approach had a large porosity compared to samples obtained by ex situ. This suggests that the in situ route will provide a biodegradable polymer with excellent water retention and, possibly, good osteoinductivity, which can be used as an artificial substitute for hard tissue. As can be observed, when the silver nanoparticle concentration increased, the composite porosity decreased for susceptible types of composite, which resulted in lower absorption capacity. Even though high porosity, which is associated with increased absorption capacity, is an important structural parameter for bone substituents, the registered decrease due to Ag addition is not significant in this case as the water absorption was still greater than 1000-1500%.
Visual inspection (Figure 11) is an efficient technique for investigation of simulated in vitro degradation of BC samples, and it has also been used in recent literature [49] in order to investigate As can be observed, when the silver nanoparticle concentration increased, the composite porosity decreased for susceptible types of composite, which resulted in lower absorption capacity. Even though high porosity, which is associated with increased absorption capacity, is an important structural parameter for bone substituents, the registered decrease due to Ag addition is not significant in this case as the water absorption was still greater than 1000-1500%.
Visual inspection (Figure 11) is an efficient technique for investigation of simulated in vitro degradation of BC samples, and it has also been used in recent literature [49] in order to investigate the degradation of cellulose-based materials. Generally, visual inspection implies macroscopic pictures of the immersed samples while observing, in a qualitative manner, the presence of detached fragments, the apparition of denser fragments that may provide additional mechanical integrity for cell growth, and so on. As expected, the composite materials did not show major changes after immersion in PBS and SBF.
(a) (b) Figure 10. Porosity (a) and water absorption (b) results for the obtained composites (presented as mean ± S.D. of three replicates and * p < 0.005 obtained by single-factor ANOVA test.
As can be observed, when the silver nanoparticle concentration increased, the composite porosity decreased for susceptible types of composite, which resulted in lower absorption capacity. Even though high porosity, which is associated with increased absorption capacity, is an important structural parameter for bone substituents, the registered decrease due to Ag addition is not significant in this case as the water absorption was still greater than 1000-1500%.
Visual inspection ( Figure 11) is an efficient technique for investigation of simulated in vitro degradation of BC samples, and it has also been used in recent literature [49] in order to investigate the degradation of cellulose-based materials. Generally, visual inspection implies macroscopic pictures of the immersed samples while observing, in a qualitative manner, the presence of detached fragments, the apparition of denser fragments that may provide additional mechanical integrity for cell growth, and so on. As expected, the composite materials did not show major changes after immersion in PBS and SBF.
After seven days, no major visual changes were observed, and the degradation was below 5% (see Figure 12), a sign that bacterial cellulose prevented the disintegration of the composite. A rapid biodegradation of the implanted material is not desired because it takes time for it to integrate better into the host tissue. Another problem is that by biodegradation, remnants/fragments of the material can reach the level of sensitive areas, which would be fatal.
Due to the higher homogeneity and better interactions between the phosphate phase (HAp) and BC, the composites obtained in situ had a lower degradation compared to those obtained using ex situ methods. After seven days, no major visual changes were observed, and the degradation was below 5% (see Figure 12), a sign that bacterial cellulose prevented the disintegration of the composite. A rapid biodegradation of the implanted material is not desired because it takes time for it to integrate better into the host tissue. Another problem is that by biodegradation, remnants/fragments of the material can reach the level of sensitive areas, which would be fatal.
Due to the higher homogeneity and better interactions between the phosphate phase (HAp) and BC, the composites obtained in situ had a lower degradation compared to those obtained using ex situ methods.

Antimicrobial Potential
The antimicrobial effect of the obtained composite materials was different among the tested samples, being influenced by the synthesis approach, AgNP content, and microbial species. Figure  13 shows the diameters of the inhibition area of the microorganisms grown in the presence of the tested materials.
Because composites BC1 and BC5 did not contain antibacterial AgNPs, they were used as control samples for evaluation of the other samples. Figure 13. The diameter of the growth inhibition zone of the tested microorganisms grown in the presence of the obtained BC samples containing various amounts of AgNPs. The plain AgNP control is represented by 10 uL of AgNPs (used at maximum equivalent amount contained in BC samples), which was added to a commercial filter paper disc of a similar size as the obtained BC samples.
The antimicrobial characteristic of the obtained composites was clearly influenced by the concentration of AgNPs, with samples with higher content of silver exhibiting the greatest microbial growth inhibition, regardless of the synthesis approach. It was observed that the materials obtained

Antimicrobial Potential
The antimicrobial effect of the obtained composite materials was different among the tested samples, being influenced by the synthesis approach, AgNP content, and microbial species. Figure 13 shows the diameters of the inhibition area of the microorganisms grown in the presence of the tested materials. The antimicrobial characteristic of the obtained composites was clearly influenced by the concentration of AgNPs, with samples with higher content of silver exhibiting the greatest microbial growth inhibition, regardless of the synthesis approach. It was observed that the materials obtained by the in situ method had a more pronounced overall antibacterial characteristic (growth inhibition zones ranging 5-11 mm) compared to the samples obtained by the ex situ method (diameter of inhibition zones ranging 5-9 mm). Figure 13. The diameter of the growth inhibition zone of the tested microorganisms grown in the presence of the obtained BC samples containing various amounts of AgNPs. The plain AgNP control is represented by 10 uL of AgNPs (used at maximum equivalent amount contained in BC samples), which was added to a commercial filter paper disc of a similar size as the obtained BC samples.
Because composites BC 1 and BC 5 did not contain antibacterial AgNPs, they were used as control samples for evaluation of the other samples.
The antimicrobial characteristic of the obtained composites was clearly influenced by the concentration of AgNPs, with samples with higher content of silver exhibiting the greatest microbial growth inhibition, regardless of the synthesis approach. It was observed that the materials obtained by the in situ method had a more pronounced overall antibacterial characteristic (growth inhibition zones ranging 5-11 mm) compared to the samples obtained by the ex situ method (diameter of inhibition zones ranging 5-9 mm).
The different antimicrobial effects of the materials obtained by in situ and ex situ routes correlated with their physicochemical properties. Samples obtained by the in situ route showed a larger porosity, suggesting that the bioactive compounds (i.e., AgNPs) may be absorbed more efficiently in pores. Moreover, a higher porosity degree can be directly associated with an easier release of the bioactive agent, therefore inducing an increase in the antimicrobial activity of the final composite. This idea is supported by the results obtained with the control AgNPs utilized in equivalent amounts and added to sterile commercial filter paper, which demonstrated lower inhibition zones.
The most efficient growth inhibition was observed against the yeast strain C. albicans ATCC 10231, with the result being relevant for samples obtained by both in situ and ex situ methods. However, the composites obtained in situ also showed increased antimicrobial activity against the Gram-positive S. aureus strain. This result suggests that the obtained composite materials may act differently on microbial cells, depending on the particularities of their cellular wall. Such differences were observed before with silver nanoparticles [50][51][52][53].

Conclusions
In this study, we report the synthesis of hydroxyapatite-bacterial cellulose silver nanocomposites obtained by two routes using coprecipitation, namely, in situ and ex situ assembly. These materials contained an organic part (bacterial cellulose), an inorganic part (hydroxyapatite), and an antimicrobial agent (AgNPs) contained in various amounts, thereby conferring new bioactive properties on the composite materials. Physicochemical and antimicrobial studies demonstrated that the most efficient in terms of potential biomedical applications were the samples obtained by the in situ approach. The porosity range of the in situ materials was greater than the porosity of ex situ composites, while the best antimicrobial activity was observed for the material coded BC 4 , which had a content of 5 wt % AgNPs. Due to the physicochemical structure, together with the already demonstrated great antimicrobial properties and low biodegradability of these materials, they have potential applications as successful candidates for biomedical applications, especially in hard tissue engineering. Their current limitation relates to the fact that further tests performed on osteoblast differentiation and mineralization (e.g., alkaline phosphatase and alizarin red S) are needed.