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Nanomaterials 13 (2): 321 (2023)
Casillas-Santana, Miguel A.
Slavin, Yael N.
Zhang, Peng
Niño-Martínez, Nereyda
Bach, Horacio
Martínez-Castañón, Gabriel A.
Bone is a natural nanocomposite composed of proteins and minerals that can regenerate itself. However, there are conditions in which this process is impaired, such as extensive bone defects and infections of the bone or surrounding tissue. This study evaluates the osteoregenerative capacity of bone grafting materials in animals with induced bone defects. Colloidal chitosan dispersion nanocomposites, nanohydroxyapatite–chitosan (NHAP-Q) and nanosilver–chitosan (AgNP-Q), were synthesized and characterized. Non-critical-size defects in Wistar rats were used to evaluate the material’s biocompatibility, and critical-size defects in the calvarias of guinea pigs were used to evaluate the regenerative capacity of the bones. Moreover, the toxicity of the nanocomposites was evaluated in the heart, liver, spleen, kidneys, and skin. Histological, radiographic, and electron microscopy tests were also performed. The results showed that neither material produced pathological changes. Radiographic examination showed a significant reduction in defects (75.1% for NHAP-Q and 79.3% for AgNP-Q), angiogenesis, and trabecular formation. A toxicological assessment of all the organs did not show changes in the ultrastructure of tissues, and the distribution of silver was different for different organs (spleen > skin > heart > kidney > liver). The results suggest that both materials are highly biocompatible, and AgNP-Q achieved similar bone regeneration to that reported with autologous bone. The main research outcome of the present study was the combination of two types of NPs to enhance antimicrobial and osteoregeneration activities. These colloidal chitosan dispersions show promise as future biomaterials in the medical field for applications in fast-healing fractures, including broken bones in the oral cavity and hip replacement infections.
Citation: Casillas-Santana, M.A.;Slavin, Y.N.; Zhang, P.;Niño-Martínez, N.; Bach, H.;Martínez-Castañón, G.A.Osteoregeneration of Critical-SizeDefects UsingHydroxyapatite–Chitosan andSilver–Chitosan Nanocomposites.Nanomaterials 2023, 13, 321. https://doi.org/10.3390/nano13020321Academic Editor: Miguel GamaReceived: 21 December 2022Revised: 8 January 2023Accepted: 9 January 2023Published: 12 January 2023Copyright: © 2023 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).nanomaterialsArticleOsteoregeneration of Critical-Size Defects UsingHydroxyapatite–Chitosan and Silver–Chitosan NanocompositesMiguel A. Casillas-Santana 1 , Yael N. Slavin 2, Peng Zhang 2, Nereyda Niño-Martínez 3, Horacio Bach 2,*and Gabriel A. Martínez-Castañón 1,*1 Laboratorio de Nanobiomateriales, Facultad de Estomatología, Universidad Autónoma de San Luis Potosí,San Luis Potosí 78290, Mexico2 Division of Infectious Diseases, Faculty of Medicine, University of British Columbia,Vancouver, BC V6G 3Z6, Canada3 Facultad de Ciencias, Universidad Autónoma de San Luis Potosi, San Luis Potosí 78295, Mexico* Correspondence: hbach@mail.ubc.ca (H.B.); mtzcastanon@fciencias.uaslp.mx (G.A.M.-C.)Abstract: Bone is a natural nanocomposite composed of proteins and minerals that can regenerateitself. However, there are conditions in which this process is impaired, such as extensive bone defectsand infections of the bone or surrounding tissue. This study evaluates the osteoregenerative capacityof bone grafting materials in animals with induced bone defects. Colloidal chitosan dispersionnanocomposites, nanohydroxyapatite–chitosan (NHAP-Q) and nanosilver–chitosan (AgNP-Q), weresynthesized and characterized. Non-critical-size defects in Wistar rats were used to evaluate the ma-terial’s biocompatibility, and critical-size defects in the calvarias of guinea pigs were used to evaluatethe regenerative capacity of the bones. Moreover, the toxicity of the nanocomposites was evaluatedin the heart, liver, spleen, kidneys, and skin. Histological, radiographic, and electron microscopytests were also performed. The results showed that neither material produced pathological changes.Radiographic examination showed a significant reduction in defects (75.1% for NHAP-Q and 79.3%for AgNP-Q), angiogenesis, and trabecular formation. A toxicological assessment of all the organsdid not show changes in the ultrastructure of tissues, and the distribution of silver was differentfor different organs (spleen > skin > heart > kidney > liver). The results suggest that both materialsare highly biocompatible, and AgNP-Q achieved similar bone regeneration to that reported withautologous bone. The main research outcome of the present study was the combination of two typesof NPs to enhance antimicrobial and osteoregeneration activities. These colloidal chitosan dispersionsshow promise as future biomaterials in the medical field for applications in fast-healing fractures,including broken bones in the oral cavity and hip replacement infections.Keywords: silver nanoparticles; hydroxyapatite particles; chitosan; critical-size defect; animal model1. IntroductionBone is a natural nanocomposite composed of proteins and minerals, which has themechanisms and the intrinsic capacity to regenerate by itself [1]. However, there areconditions in which this process is impaired, such as extensive bone defects and boneor surrounding tissue infections. Furthermore, bone tissue may be lost due to trauma orperiodontal disease, representing a challenge for orthopedists, maxillofacial surgeons, andreconstructive specialists.In adults, only minor bone defects can heal spontaneously [2]. The deterioration inwound repair is related to the rapid internal growth of soft tissue towards the bone defect,thus inhibiting bone proliferation and formation at the edges of the bone defect [3]. One ofthe significant challenges oral and maxillofacial surgeons face is the restoration of defectsin the maxillofacial bones.Autogenous bone grafts are the preferred material when craniofacial reconstructionis required [4]. Although highly effective, many problems are associated with their use.Nanomaterials 2023, 13, 321. https://doi.org/10.3390/nano13020321 https://www.mdpi.com/journal/nanomaterialsNanomaterials 2023, 13, 321 2 of 18For example, autogenous grafts can increase the operative time by increasing the risks ofinfection, donor site pain, and morbidity [5,6], and they are often mechanically unstableduring surgery and might be insufficient for reconstructing extensive defects. These graftsmay also undergo resorption, requiring subsequent surgical procedures. As a result, therehas been broad interest in generating biomaterials that provide optimum conditions forcells with the osteogenic potential to minimize these problems and angiogenesis.Hydroxyapatite (HA) is a calcium phosphate ceramic material that is the main mineralcomponent of vertebrates. Depending on its source, it may have a structure similar tobone tissue. HA possesses the characteristic of osteoconductivity, allowing the connectivetissue to penetrate the surrounding bone, inducing a process of ossification. In addition,calcium phosphates can bind and concentrate endogenous bone morphogenic proteins,leading to osteoinduction (cell recruitment and stimulation leading to pre-osteoblast de-velopment) [7]. Combining HA with chitosan (deacetylated chitin) improves the poormechanical properties of HA, making it a promising material for biomedical applicationsdue to its biocompatibility. It also improves its ability to facilitate the regenerative processin wounds. For example, chitosan combined with nano-HA in the form of spheres andimplanted in calvarial (skullcap) defects promoted bone regeneration in 12 weeks [8,9].Chitosan alone has also been shown to enhance cellular activity, inducing proliferation(reaching a plateau at 0.1 mg/mL) in human periodontal ligament fibroblasts, as well asupregulating alkaline phosphatase (ALP) activity (known to be a marker of osteogenicdifferentiation) and increasing type I collagen production [10].Silver nanoparticles (AgNPs) have been widely used for their bactericidal effect andtheir ability to reduce bacterial skin infections [11–13]. Moreover, it was reported thatAgNPs promoted regeneration tissue in a rat model of thermal damage to the back [14].However, in this study, the partial-thickness wound typically healed after approximately35 days. Still, in animals treated topically with AgNPs, the wounds healed in 26 days,indicating that AgNPs could modulate the local and systemic inflammatory response afterburn injury by modulating the cytokine profile [14].Studies have shown that colloidal chitosan dispersions have a high potential to mimicthe extracellular matrix [15–17]. This is because cells and tissues are immersed within a3D lattice in vivo, constituting a complex extracellular environment with high porosity. Incontrast, a 2D culture system is too simple to parameterize the native environment [18,19].This study aimed to evaluate the biocompatibility of colloidal nano-HA-chitosan(NHAP-Q) and AgNP-chitosan (AgNP-Q) dispersions and their capacity to improve bonerepair processes in critical-size defects using animal models. This study is novel because itshowed antibacterial and osteoregenerative activities when a colloidal chitosan dispersionwas prepared with AgNPs and NHAPs.2. Methods2.1. Synthesis of NPsAgNPs were synthesized by dissolving 0.169 g of AgNO3 (Fermont, Monterrey, Mex-ico) in 100 mL of deionized water. Then, 0.1 g of gallic acid (Sigma-Aldrich, St. Louis,MO, USA) previously dissolved in 10 mL of deionized water was added, and the pH wasimmediately adjusted to 11 using 1.0 M NaOH solution. This dispersion was kept in thedark at room temperature.The synthesis of NHAP was carried out in an aqueous solution using a precipitationtechnique. First, a reaction was prepared by briefly mixing 50 mL of a 1 M aqueous solutionof Ca(NO3)2·4H2O with 50 mL of 0.6 M KH2PO4, adjusting the pH to 10 with dropwiseaddition of NH4OH. The reaction was kept under magnetic stirring for 1 h and allowed tosettle for 24 h. The next day, the formed nanoparticles were washed with deionized water(×4) and then heated at 900 ◦C for 1 h. The powders were kept at room temperature.Nanomaterials 2023, 13, 321 3 of 182.2. Colloidal Chitosan Dispersion SynthesisThe AgNP-Q and NHAP-Q colloidal chitosan dispersions were prepared with a minormodification of published protocols [20]. Briefly, 2 mL of acetic acid was mixed with48 mL of the AgNP or NHAP solutions, after which 1.95 g of chitosan (310–375 kDa,Sigma-Aldrich) and 0.2 mL of glycerol were added under magnetic stirring until the gelwas formed.2.3. Characterization of the NPsThe NPs used in this study were characterized by transmission electron microscopy(TEM, JEOL, JEM-1230, Tokyo, Japan) operated at 100 kV. In addition, dynamic lightscattering (DLS) was used to measure the size distribution of the particles, and theirzeta potential was measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern,Worcestershire, United Kingdom) operating at 25 ◦C with a He-Ne laser at a wavelength of633 nm and a detection angle of 90◦.To confirm the composition of NPs, X-ray diffraction analysis was performed usingdried powders of the NPs. X-ray diffraction patterns were recorded on a GBC-DifftechMMA model, GBC, Braeside, Victoria, Australia), with Cu Kα irradiation at λ = 1.54 Å. Inaddition, the Vis-NIR absorption spectra of AgNPs were obtained using a CHEMUSB4-VIS-NIR (Ocean Optics, Dunedin, FL, USA) spectrophotometer.2.4. Rheological Characterization of the Colloidal Chitosan DispersionsThe colloidal chitosan dispersions were characterized using a rheometry test (RT) andenvironmental scanning electron microscopy (ESEM). The rheometry test was performedwith a U.S. 200 Paar Physica Rheometer equipped with Peltier temperature control. Thedetermination was carried out using a cone–plate geometry (MK31, 50 mm in diameter,1◦ tilt, and 0.49-micron truncation). An aliquot of the sample was deposited at the base ofthe plate to secure it further. The distribution of the NPs in the gels was observed using anenvironmental scanning electron microscope (ESEM Quanta 200, FEI, Hillsboro, OR, USA),and no special sample preparation was needed for this observation.2.5. Bactericidal ActivityA panel of four multidrug-resistant pathogenic bacteria was used to determine the an-tibacterial activity of the colloidal chitosan dispersions. Bacterial strains clinically isolatedfrom the Hospital Central Dr. Ignacio Morones Prieto (San Luis Potosí, SLP, México) weretested and identified as previously described [21]. Strains tested in this study includedthe Gram-positive Staphylococcus aureus 700 (resistance: ciprofloxacin, clindamycin, ery-thromycin, oxacillin, and penicillin), Staphylococcus epidermidis 710 (resistance: penicillin,oxacillin, gentamicin), and Staphylococcus epidermidis 6 (resistance: penicillin, erythromycin),and the Gram-negative Acinetobacter baumannii 808 (resistance: meropenem). Strains werecultured in Mueller–Hinton broth (BD) and were maintained on solidified broth with 1.5%agar at 4 ◦C. Bacterial strains were exposed to concentrations of 2.5 to 25 µg/mL. Theminimal inhibitory concentration (MIC) was measured following published protocols [22],defined as the concentration at which no bacterial growth was observed.2.6. Cytotoxic Activity of Osteoblasts and MacrophagesPrimary osteoblast cells were obtained from mice in the in vivo study (see below).First, mouse femurs were processed according to published protocols [23]. Briefly, femurswere scraped with a scalpel (#11) to clean the remainder of the flesh, and the bone marrowwas flushed with PBS using a syringe (5 mL) and a needle (27G). Next, femurs were cutinto 1–2 cm pieces using sterile scissors and washed in PBS (×4) for 5 min each. Then, 4 mLof a solution of collagenase II (Sigma-Aldrich) was added, and the bones were vigorouslyshaken at 37 ◦C for 2 h to remove any debris. Next, the bone pieces were transferred to tissueculture dishes (20 pieces/dish) and cultured with a medium containing DMEM (Sigma-Aldrich) supplemented with antibiotics (100 U/mL penicillin, 50 µg/mL streptomycin,Nanomaterials 2023, 13, 321 4 of 18and 50 µg/mL gentamycin, Invitrogen, Waltham, MA, USA), amphotericin B (1.25 µg/mL,Fungizone), 100 µg/mL ascorbate (Sigma-Aldrich), and 10% fetal calf serum (Invitrogen).An alkaline phosphatase (ALP) test [24] was performed to confirm the identity ofthe osteoblast: 3 × 105 cells/well were dispensed on a 96-well plate overnight at 37 ◦Csupplemented with 5% CO2. The next day, cells were carefully washed with PBS (×2) andfixed with 10% formalin for 1 min. Then, the formalin solution was aspirated, and cellswere washed with DMEM supplemented with 0.05% Tween-20. The staining of the cellswas completed after the addition of the substrate 5-bromo-4-chloro-3-indolyl-phosphate(BCIP) (175 µg/mL from a stock of 50 mg/mL in 70% dimethylformamide (DMF)) and4-nitro blue tetrazolium chloride (NBT) (225 µg/mL from a stock solution of 50 mg/mL in70% dimethyl formamide). Finally, cells were incubated at room temperature in the darkfor ~10 min and analyzed under a microscope.All the experiments involving cells were cultured at 37 ◦C in an atmosphere supple-mented with 5% CO2. The human-derived monocyte cell line THP-1 (ATCC TIB-202) wasused in this study to measure the cytotoxic effects of the colloidal chitosan dispersions.Cells were grown in tissue culture flasks using RPMI 1640 (Sigma-Aldrich), supplementedwith 10% fetal calf serum (FCS) (Invitrogen), 2 mM L-glutamine, and amphotericin B(2 µg/mL, Fungizone). THP-1 cells (3 × 105/well in a 96-well plate) became adherent uponsupplementation of 40 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich).Osteoblasts and macrophages were exposed to the colloidal chitosan dispersions atthe following concentrations: 1.25, 2.5, 5, 10, and 20 µg/mL for 18 h. Each treatment wasperformed in triplicate. Untreated cells were used as a negative control, whereas cellstreated with 0.1% Tween-20 (Fisher) were used as a positive control.The cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assay as published previously [25].2.7. Osteoblast Proliferation AssayTo assess osteoblast proliferation, the 5-bromo-2′-deoxyuridine (BrdU) assay wasused (BrdU Cell Proliferation Assay Kit, Cell Signaling Technologies, Danvers, MA, USA)according to the manufacturer’s instructions. Briefly, osteoblast cells were cultured asdescribed above, and 2× 104 cells were dispensed in a 96-well plate. Once the cells adheredto the bottom of the well, a BrdU solution was added, and the plates were incubated at37 ◦C supplemented with 5% CO2 for 48 h. At 24 h, the medium of one plate was carefullydisposed of, and 100 µL/well of the fixing/denaturing solution was added. The plate wasleft at room temperature for 30 min, and 100 µL/well of the detection antibody solutionwas added after removing the previous solution. After 1 h incubation at room temperature,the solution was removed, and the plate was washed (×3) with the wash buffer. Then,100 µL/well of HRP-conjugated secondary antibody solution was added, and the platewas kept at room temperature for 30 min. Lastly, the wells were rewashed with the washbuffer (×3). To develop the test, 100 µL of the TMB substrate was added and incubatedfor 30 min at room temperature. To finalize the reaction, 100 µL of the stop solution wasadded, and the absorbance at 450 nm was measured using a plate reader (Epoch, Biotek,Winooski, VT, USA).2.8. Anti-Inflammatory AssayTHP-1 cells at a concentration of 5 × 104 cells/well were dispensed on a 96-well plateand activated after adding 100 ng/mL PMA (Sigma-Aldrich). Plates were incubated at37 ◦C in a humidified atmosphere of 5% CO2 for 24 h. Then, adherent cells were gentlywashed with fresh medium (×3) and incubated in the presence of the compounds for 6 h.After removing the compounds and washing the cells, an immunological response wasinitiated after adding 500 ng/mL of E. coli lipopolysaccharide (LPS) (Sigma-Aldrich). After12 h, supernatants were transferred to a new 96-well plate and kept at −20 ◦C for furtheranalysis. The pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and the anti-inflammatory interleukin-10 (IL-10) were used to evaluateNanomaterials 2023, 13, 321 5 of 18the inflammatory response using a kit (R&D) and according to the instructions of themanufacturer. DMSO alone and DMSO combined with LPS were used as negative controls,whereas prednisone (1 mM, Sigma-Aldrich) was used as a positive control.2.9. Animal ModelsThis study was evaluated and approved by the ethics committee of the U.A.S.L.P. un-der the protocol CIS-FE-028-014. Furthermore, the experiments were conducted followingthe Mexican Legislation Standard of NOM-063-SSA1-1993 and the General Health Law(Guide for the Care and Use of Laboratory Animals, The National Academies Press).Three protocols were used in this study: Protocol A evaluated the biocompatibilityof the materials. In this protocol, three defects of non-critical size were performed on thetibial axis of the limb in Wistar rats (n = 5), weighing between 400–500 g, and performedin duplicate. Colloidal NHAP-Q chitosan dispersion was implanted in defect 1 (Group 1),colloidal AgNP-Q chitosan dispersion in defect 2 (Group 2), and a blood clot in defect 3 asa control group (Group 3). Protocol B evaluated the regenerative capacity of the materials.In this protocol, two critical defects (8 mm each) were made on the calvaria of guinea pigs(n = 10) weighing between 700 and 900 g. Colloidal NHAP-Q chitosan dispersion wasimplanted in the right defect (Group 1), and colloidal AgNP-Q chitosan dispersion wasimplanted in the left defect (Group 2). Protocol C was used to measure the distributionof Ag in different organs. In this protocol, colloidal AgNP-Q chitosan dispersions weresub-dermally implanted in the dorsal region of guinea pigs (n = 5). Another group ofguinea pigs was sub-dermally injected with saline as a control.According to the experimental protocol, the animals were randomly assigned to twoor three groups with food and water access ad libitum.2.10. Surgical ProcedureIn protocol A, all rats were anesthetized with an intraperitoneal injection of ketamine(33 mg/kg, Anesket, ketamine hydrochloride 1000 mg/10 mL). Then, the right limb wasshaved, and the skin surface was disinfected with iodopovidone solution (DermoDineIodopovidone 11 g/100 mL). Before surgery, the bone was exposed through an incisionapproximately 2 cm in length using a scalpel blade (#15). Three defects with a diameter of2 mm were made on the tibial axis of the right limb using a carbide burr # 3 under constantirrigation with saline solution to prevent overheating. The first defect was implantedwith colloidal NHAP-Q chitosan dispersion to fill the cavity evenly, the second defect wasimplanted with colloidal AgNP-Q chitosan dispersion, and the third defect was implantedwith a blood clot to be used as a control (Figure S1). After 2 weeks, the same procedurewas performed on the animal’s left limb. After an evolution period of 2 weeks, the rightlimb had a repair process of 4 weeks (Figure S2).In protocol B, guinea pigs were sedated with an intraperitoneal injection of ketamine(40 mg/kg, Anesket, ketamine hydrochloride 1000 mg/10 mL). Then, the parietal–frontalskin was disinfected with iodopovidone (DermoDine). Before surgery, an incision ofapproximately 3 cm was made with a scalpel blade (#15) from the nasofrontal suture tothe external occipital protuberance to expose the parietal bone after raising the flaps witha periosteal elevator. Two defects of critical size (8 mm diameter each) were made inthe calvaria using a carbide cutter (#8) at low speed (maximum speed of 500 rpm) withcontinuous and copious irrigation with saline solution to avoid overheating. Special carewas taken to prevent damage to the dura mater, and the diameter of the defects wasconfirmed with a periodontal probe. The defect in the right parietal was implanted with acolloidal NHAP-Q chitosan dispersion and the left parietal defect with a colloidal AgNP-Qchitosan dispersion. Guinea pigs were sacrificed at 4 or 8 weeks, and the calvarias wereremoved with a diamond disk (600 grit) at low speed.In protocol C, the guinea pigs were sedated with ether. After complete disinfectionof the interscapular zone (between the shoulder blades) with iodopovidone (DermoDine),two incisions (left and right) of approximately 2 cm were performed, and 100 mg ofNanomaterials 2023, 13, 321 6 of 18colloidal AgNP-Q chitosan dispersion was sub-dermally injected in the left dorsal incision.In contrast, the right incision was maintained as control. Animals were sutured andeuthanized after a postoperative period of 5 weeks. The targeted organs, heart, liver, spleen,kidneys, and the skin surrounding the implantation zone, were extracted.In all the protocols mentioned above, once the materials were implanted, the openwounds were sutured with black silk 4-0. The animals were recovered with light and darkcycles of 12/12 h and the antibiotic ceftriaxone (140 mg/kg) and the analgesic metamizole(570 mg/kg) for 3 d. Weight fluctuations were monitored daily.2.11. Histological and Radiographic AnalysesSamples obtained in all the protocols above were stored in 10% formaldehyde andrefrigerated at 4 ◦C. For protocols A and B, the pieces were decalcified with 10% HNO3under constant agitation for 72 h and 7 days, respectively, and histological staining wasperformed with hematoxylin-eosin. For protocols A and B, three random sections of eachdefect were taken for histological examination. The de novo bone formation, residualmaterial, pathological changes, and inflammatory response were investigated in eachsection. In protocol C, ultrastructural changes and the presence of inflammatory filtrateswere examined by a pathologist under a light microscope with a magnification of 10×, 20×,and 40×. In addition, each image was digitally captured with a camera attached to themicroscope for further analysis.For protocol B, before histological processing, a conventional X-ray of the calvaria wastaken with an angle of 90◦, a distance of 10 cm, and an exposure of 14 milliseconds usinga 5 MP Samsung camera (2592 × 1944 pixels, autofocus, LED flash). Then, radiographicanalysis was performed with the program ImageJ.2.12. Blood ParametersBlood samples were collected by a direct puncture in the auricle or descending aortain the heart after sedation with ether. A sample of 2 mL of blood was drained per guineapig and divided into two portions. The first portion (250 µL) was collected in a Vacutainersupplemented with EDTA, and the rest of the sample was placed in a centrifuge tube.A complete blood count was performed, and the number of lymphocytes (LYM),neutrophils (GRAN), erythrocytes (RBC), hemoglobin (HGB), hematocrit (HCT), meancorpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscularhemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets(PLT), and mean platelet volume (MPV) were measured using a blood cell counter (AbbottDiagnostics Cell-Dyn 1700 and Alcyon 300, Ramsey, Minnesota, USA). In addition, the renaland hepatic functions were assessed by measuring AST, ALT, urea, and creatinine usinga biochemical blood analyzer (Biosystems A15, Barcelona, Spain). The assays followedinternational standards for the clinic and hematology [26].2.13. Ag Content in TissuesThe heart, kidney, liver, skin, and spleen were collected and analyzed for their Agcontent (n = 5). Each organ (1 g) was homogenized and digested with an aqueous solutionof HNO3 (32%, 10 mL) for 48 h at 50 ◦C. After complete disintegration, 1 mL of the organdigestion was mixed with 9 mL of deionized water, and the Ag analysis was performed byatomic absorption (AA-7000, Shimadzu, Kyoto, Japan).2.14. Histopathology Analysis of the DefectsSamples were stored in formaldehyde (10%) and kept at 4 ◦C. The samples weredecalcified with HNO3 (10%) under constant agitation for 72 h (protocol A) or 7 d (protocolB). The histological analysis was performed in three random sections after staining withhematoxylin–eosin. For protocols A and B, each section was analyzed for de novo ossifica-tion, pathological changes, and the presence of immune cells. In protocol C, ultrastructuralNanomaterials 2023, 13, 321 7 of 18changes and white cell infiltration were assessed. The analyses were performed by apathologist, who was not exposed to the identity of each sample.2.15. Analysis of CalvariasFor protocol B, guinea pig calvarias were dissected and removed at the end of the8 weeks and prepared conventionally for histology but were not stained. Histologicalsections were placed on the object holders without the protection of the resin; they werecovered with gold (fine-coat ion sputter JFC 1100) and taken to a scanning electron micro-scope (JEOL, JSM-6610) for histomorphometric interpretation at 10 kV, at 10 mm of workingdistance, and using a secondary electron detector.The guinea pig calvarias were removed and prepared conventionally for their histolog-ical analysis. However, they were not stained and covered with resin. Instead, they werecovered with gold before the last step for their proper observation. For this observation, atleast five random points of each calvaria were assessed.2.16. Statistical AnalysisA student t-test was used for statistical analysis. A p-value < 0.05 was consideredsignificant. GraphPad Prism version 6.00 for Mac (San Diego, CA, USA) was used for thestatistical calculations.3. Results3.1. NP CharacterizationTEM revealed that the average size of the AgNP was 13 nm with a spherical shape(Figure 1A), whereas the NHAP ranged from 19 to 32 nm with a rod-like shape (Figure 1B).The NHAP synthesized in this study resembled the bone matrix of 70% crystalline HA,typically measured at 20–80 nm in length and 2–5 nm in thickness [27]. The zeta potentialresults were−48.4 and−26.8 mV for the AgNPs and NHAP, respectively (Figure S3). Basedon the characterization results, both NPs will not agglomerate because of the high negativezeta potential.Nanomaterials 2023, 13, x FOR PEER REVIEW 8 of 19 Figure 1. Characterization of the NPs used in this study by TEM analysis of (A) AgNPs, scale bar: 20 nm and (B) HANPs, scale bar: 100 nm. Figure 2. X-ray diffraction patterns of the NPs synthesized in this study. (A) AgNPs. (B) NHAPs. (C) Surface plasmon resonance of AgNPs.3.2. Rheological Characterization of the Colloidal Chitosan Dispersions ESEM images showed that once AgNPs and NHAP agglomerated, they were incor-porated into the matrix (Figure 3). However, ESEM images showed good distribution of these NPs in the gel matrix. In addition, the colloidal chitosan dispersions showed a pseu-doplastic or shear-thinning behavior (viscosity decreases when the velocity increases). This property could be used when colloidal chitosan dispersions are used. This would be advantageous since the dispersion would become more fluid while it is manipulated, leading to an easier distribution on the surface. In addition, the viscosity would probably increase when the stress ceased, preventing the dispersion from flowing from the appli-cation site. A BA B CFigure 1. Ch racterization of the sed in this tudy by TEM analysis of (A) AgNPs, scale bar:20 nm and (B) HANPs, scale : .The AgNPs were identified as elemental Ag after comparing the diffraction patternwith the published data (JCPDS card no. 04-0783). The three most intense peaks correspondto the planes (111), (200), and (220) (Figure 2A). These results indicate that there is nopreferential growth of the crystals. In the case of the NHAP, it can be indexed as hexagonalHA compared with JCPDS card no. 09-0432; no other phases or compounds were detected,Nanomaterials 2023, 13, 321 8 of 18indicating that the prepared samples were pure NHAP (Figure 2B). By observing thebroadening of the diffraction peaks, it seems that AgNPs present excellent crystallinity,whereas NHAPs are slightly less crystalline.Nanomaterials 2023, 13, x FOR PEER REVIEW 8 of 19 Figure 1. Characterization of the NPs used in this study by TEM analysis of (A) AgNPs, scale bar: 20 nm and (B) HANPs, scale bar: 100 nm. Figure 2. X-ray diffraction patterns of the NPs synthesized in this study. (A) AgNPs. (B) NHAPs. (C) Surface plasmon resonance of AgNPs. 3.2. Rheological Characterization of the Colloidal Chitosan Dispersions ESEM images showed that once AgNPs and NHAP agglomerated, they were incorporated into the matrix (Figure 3). However, ESEM images showed good distribution of these NPs in the gel matrix. In addition, the colloidal chitosan dispersions showed a pseudoplastic or shear-thinning behavior (viscosity decreases when the velocity increases). This property could be used when colloidal chitosan dispersions are used. This would be advantageous since the dispersion would become more fluid while it is manipulated, leading to an easier distribution on the surface. In addition, the viscosity would probably increase when the stress ceased, preventing the dispersion from flowing from the application site. A BA B CFigure 2. X-ray diffraction patterns f Ps synthesized in this study. (A) gNPs. (B) NHAPs.(C) Surface plasmon resonance of s.3.2. Rheological Characterization of the Colloidal Chitosan DispersionsESEM images showed that once AgNPs and NHAP agglomerated, they were incor-porated into the matrix (Figure 3). However, ESEM images showed good distributionof these NPs in the gel matrix. In addition, the colloidal chitosan dispersions showed apse doplastic or shear-thi ning behavior (viscosity decreases when the velocity i creases).This prop rty could be used when colloidal chitosan dispersions ar used. This wouldbe advantageous since the dispersion would become more fluid while it is manipulate ,leading to an easier distribution on the surface. In addition, the viscosity would prob-ably increase when the stress ceased, preventing the dispersion from flowing from theapplication site.Nanomaterials 2023, 13, x FOR PEER REVIEW 9 of 19 Figure 3. Rheological characterization of the colloidal chitosan dispersions. ESEM was used to char-acterize the (A) AgNP-Q (scale bar: 50 μm) and (B) colloidal NHAP-Q chitosan dispersions (scale bar: 50 μm). (C) The viscosity of both colloidal chitosan dispersions was measured. 3.3. Bactericidal Activity of the Colloidal Chitosan Dispersions A potent antibacterial activity was measured when the colloidal chitosan dispersions were tested against four Gram-positive and Gram-negative multidrug-resistant bacterial strains (Table 1). A range of MICs between 7.5 and 20 μg/mL was measured. Table 1. Antibacterial activity of colloidal chitosan dispersions against multidrug-resistant bacteria expressed as MIC (μg/mL). Bacterial Strain Colloidal AgNP-Q Chitosan Dispersion NPs Colloidal Chitosan Dispersions Control Acinetobacter baumannii 12.5 R Staphylococcus epidermidis 6 7.5 R Staphylococcus epidermidis 710 12.5 R Staphylococcus aureus 700 20 R R = resistant. 3.4. Cytotoxicity and Proliferation Assays The cytotoxic effects of the colloidal NHAP-Q/AgNP-Q chitosan dispersions on pri-mary osteoblasts and the human monocyte cell line THP-1 were assessed. The results showed toxicity at 5 and 12.5 μg/mL when incubated with osteoblasts and macrophages, respectively (Figure 4A,B). Different concentrations of the colloidal NHAP-Q and AgNP-Q chitosan dispersions were exposed to osteoblasts to determine whether the colloidal chitosan dispersions were detrimental to cellular proliferation. The colloidal chitosan dispersion lacking the AgNPs was used as a negative control. The results showed that the colloidal NHAP-Q and AgNP-Q chitosan dispersions did not affect the proliferation of the cells at concentrations of <5 μg/mL as measured by BrdU incorporation into the DNA (Figure 4C). However, at con-centrations of >5 μg/mL, a significant decrease in DNA incorporation was measured com-pared with the negative control, suggesting inhibition of osteoblast growth. Interestingly, at concentrations of 20 μg/mL, both colloidal NHAP-Q and AgNP-Q chitosan dispersions and the colloidal chitosan dispersion inhibited osteoblast proliferation. A B CA B CFigure 3. Rheological characterization of the colloidal chitosan dispersions. ESEM was used tocharact riz the ( ) AgNP-Q (scale bar: 50 µm) and (B) colloidal NHAP-Q chit s (scalebar: 5 µ ). ( ) e isc sit f t c lloi al c itosan dispersions as easured.3.3. Bactericidal Activity of the Colloidal Chitosan DispersionsA potent antibacterial activity was measured when the colloidal chitosan dispersionswere tested against four Gram-positive and Gram-negative multidrug-resistant bacterialstrains (Table 1). A range of MICs between 7.5 and 20 µg/mL was measured.Nanomaterials 2023, 13, 321 9 of 18Table 1. Antibacterial activity of colloidal chitosan dispersions against multidrug-resistant bacteriaexpressed as MIC (µg/mL).Bacterial Strain Colloidal AgNP-Q ChitosanDispersion NPsColloidal ChitosanDispersions ControlAcinetobacter baumannii 12.5 RStaphylococcus epidermidis 6 7.5 RStaphylococcus epidermidis 710 12.5 RStaphylococcus aureus 700 20 RR = resistant.3.4. Cytotoxicity and Proliferation AssaysThe cytotoxic effects of the colloidal NHAP-Q/AgNP-Q chitosan dispersions onprimary osteoblasts and the human monocyte cell line THP-1 were assessed. The resultsshowed toxicity at 5 and 12.5 µg/mL when incubated with osteoblasts and macrophages,respectively (Figure 4A,B).Nanomaterials 2023, 13, x FOR PEER REVIEW 10 of 19 Figure 4. Effect of the colloidal chitosan dispersions on cytotoxicity and the immune response. Mac-rophages (A) and osteoblasts (B) were exposed to different concentrations of colloidal AgNP-Q chi-tosan dispersions, and the cytotoxicity was measured using the MTT assay and according to the experimental methodology. Staurosporine was used as a positive control, whereas untreated cells were used as a negative control. (C) A proliferation assay was performed in osteoblasts using the BrdU test over 48 h and according to the methodology. (D) An immunological response was meas-ured using the pro-inflammatory cytokine IL-6 measured in the supernatant of the culture. LPS (lip-opolysaccharide) and untreated cells were used as positive and negative controls, respectively. Col-loidal NP-chitosan dispersion, colloidal AgNP-Q chitosan dispersion. All the experiments were per-formed in triplicate. The asterisk represents a p-value of <0.05. Data shown are the mean ± S.D. 3.5. Immunological Response To determine whether the exposure of macrophages to the colloidal chitosan disper-sions would modulate an immunological response, the levels of the pro-inflammatory cy-tokines IL-6 and TNF-α and the anti-inflammatory IL-10 cytokine were measured. A sig-nificant increase of ~60% in the concentration of IL-6 was measured compared with the untreated control. This increase was observed when macrophages were exposed to AgNP- colloidal chitosan dispersion of ≥5 μg/mL (Figure 4D). Interestingly, TNF-α and IL-10 were not significantly changed when macrophages were exposed to colloidal NP-chitosan dispersion concentrations ranging between 5 and 50 μg/mL (data not shown). 3.6. Postoperative Assessment The repair process progressed in protocols A and B without any sign of infection. No changes in animal behavior, paralysis, shortness of breath, or signs of pain were recorded. Furthermore, the weight curves were normal in all animals with a progressive increase in weight, which indirectly reflected that neither the grafted material, colloidal NHAP-Q or AgNP-Q chitosan dispersions, nor the surgical procedure produced any changes in the animal that modified its behavior for food and drink intake (data not shown). A BC DFigure 4. Effect of the colloidal chitosan dispersions on cytotoxicity and the immune response.Macrophages (A) and osteoblasts (B) were exposed to different concentrations of colloidal AgNP-Qchitosan dispersions, and the cytotoxicity was measured using the MTT assay and according tothe experimental methodology. Staurosporine was used as a positive control, whereas untreatedcells were used as a negative control. (C) A proliferation assay was performed in osteoblasts usingthe BrdU test over 48 h and according to the methodology. (D) An immunological response wasmeasured using the pro-infl mmatory cytokin IL-6 measured in the superna ant of the culture. LPS(lipopolysaccharide) and untreated cel s were used as positive and negativ cont ols, respectively.Colloidal NP-chitosan dispersion, colloidal AgNP-Q chitosan dispersion. All the experiments wereperformed in triplicate. The asterisk represents a p-value of <0.05. Data shown are the mean ± S.D.Nanomaterials 2023, 13, 321 10 of 18Different concentrations of the colloidal NHAP-Q and AgNP-Q chitosan dispersionswere exposed to osteoblasts to determine whether the colloidal chitosan dispersions weredetrimental to cellular proliferation. The colloidal chitosan dispersion lacking the Ag-NPs was used as a negative control. The results showed that the colloidal NHAP-Q andAgNP-Q chitosan dispersions did not affect the proliferation of the cells at concentrationsof <5 µg/mL as measured by BrdU incorporation into the DNA (Figure 4C). However, atconcentrations of >5 µg/mL, a significant decrease in DNA incorporation was measuredcompared with the negative control, suggesting inhibition of osteoblast growth. Inter-estingly, at concentrations of 20 µg/mL, both colloidal NHAP-Q and AgNP-Q chitosandispersions and the colloidal chitosan dispersion inhibited osteoblast proliferation.3.5. Immunological ResponseTo determine whether the exposure of macrophages to the colloidal chitosan disper-sions would modulate an immunological response, the levels of the pro-inflammatorycytokines IL-6 and TNF-α and the anti-inflammatory IL-10 cytokine were measured. Asignificant increase of ~60% in the concentration of IL-6 was measured compared with theuntreated control. This increase was observed when macrophages were exposed to AgNP-colloidal chitosan dispersion of ≥5 µg/mL (Figure 4D). Interestingly, TNF-α and IL-10were not significantly changed when macrophages were exposed to colloidal NP-chitosandispersion concentrations ranging between 5 and 50 µg/mL.3.6. Postoperative AssessmentThe repair process progressed in protocols A and B without any sign of infection. Nochanges in animal behavior, paralysis, shortness of breath, or signs of pain were recorded.Furthermore, the weight curves were normal in all animals with a progressive increase inweight, which indirectly reflected that neither the grafted material, colloidal NHAP-Q orAgNP-Q chitosan dispersions, nor the surgical procedure produced any changes in theanimal that modified its behavior for food and drink intake.3.7. Histological AnalysisFor a qualitative evaluation of the inflammatory response in the tissues, the levels ofresponse observed were defined as: (1) when the grafts do not stimulate any inflammatoryresponse; (2) when there is a presence of macrophages and plasma cells, which means thatthe material has been detected in the local tissue and presented to B lymphocytes; (3) amoderate immune response with the presence of giant cells; and (4) a severe inflammatoryresponse accompanied by necrosis [20]. In our study, the histological evaluation in protocolA (biocompatibility) showed that the repair process was normal with no changes such aspro-inflammatory cells, bone resorption, any degree of inflammation, or necrosis. However,for Group 1 (colloidal NHAP-Q chitosan dispersion), the histological results revealeda slight bone growth on the margins toward the central part of the defect, presumablybecause the implanted material was uniformly distributed throughout the defect. Still,some particles remained present after 2 weeks and 4 weeks with few signs of resorption(Figure 5A).For Group 2 (colloidal AgNP-Q chitosan dispersion), the histological observationshowed small amounts of residual material at 2 weeks with improved resorption of theimplanted material at 4 weeks, as well as the formation of trabecular bone very similarto basal bone (Figure 5B). It is important to mention that none of the materials tested inthis study stimulated the implanted materials’ pro-inflammatory or rejection process. Onthe contrary, it is precisely the formation of new bone material which confirms that bothmaterials are biocompatible, highlighting the biodegradation rate of the chitosan and thelevel of purification of AgNPs in the colloidal AgNP-Q chitosan dispersion.Nanomaterials 2023, 13, 321 11 of 18Nanomaterials 2023, 13, x FOR PEER REVIEW 11 of 19 3.7. Histological Analysis For a qualitative evaluation of the inflammatory response in the tissues, the levels of response observed were defined as: (1) when the grafts do not stimulate any inflammatory response; (2) when there is a presence of macrophages and plasma cells, which means that the material has been detected in the local tissue and presented to B lymphocytes; (3) a moderate immune response with the presence of giant cells; and (4) a severe inflammatory response accompanied by necrosis [20]. In our study, the histological evaluation in proto-col A (biocompatibility) showed that the repair process was normal with no changes such as pro-inflammatory cells, bone resorption, any degree of inflammation, or necrosis. How-ever, for Group 1 (colloidal NHAP-Q chitosan dispersion), the histological results re-vealed a slight bone growth on the margins toward the central part of the defect, presum-ably because the implanted material was uniformly distributed throughout the defect. Still, some particles remained present after 2 weeks (data not shown) and 4 weeks with few signs of resorption (Figure 5A). Figure 5. Histological analyses of the defect implanted in the animals. (A) The defect was implanted with colloidal NHAP-Q chitosan dispersion (protocol A, Group 1, 4 weeks). (B) The defect was im-planted with colloidal AgNP-Q chitosan dispersion (protocol A, Group 2, 4 weeks). No signs of significant inflammation were observed in both images. Magnification, 10×. Hematoxylin and eosin were used for staining. The red circle corresponds to the area of the defect. For Group 2 (colloidal AgNP-Q chitosan dispersion), the histological observation showed small amounts of residual material at 2 weeks (data not shown) with improved resorption of the implanted material at 4 weeks, as well as the formation of trabecular bone very similar to basal bone (Figure 5B). It is important to mention that none of the materials tested in this study stimulated the implanted materials’ pro-inflammatory or rejection process. On the contrary, it is precisely the formation of new bone material which confirms that both materials are biocompatible, highlighting the biodegradation rate of the chitosan and the level of purification of AgNPs in the colloidal AgNP-Q chitosan dis-persion. On the other hand, in protocol B, whose main objective was to evaluate the regener-ative capacity of the materials in a critical-size defect, histological sections of the calvarias were assessed at 4 weeks after implantation of the defects with colloidal NHAP-Q chi-tosan dispersion. Histological examination showed the formation of bone trabeculae, es-pecially in the central part of the defect and some areas near the edges, and connective tissue with fewer osteoblasts and osteocytes. The presence of connective tissue suggests the presence of residual material that prevents correct repair of the bone with some signs of degradation. After 8 weeks, a considerable change in mineralized tissue was observed in the cen-tral part of the defects and very close to the borders contiguous to the basal bone (Figure 6A). A representative clinical view of the defect area, which decreased 8 weeks after the placement of the grafts, is depicted in Figure 6B. In addition, remnants composed of chi-tosan fibers and NHAPs surrounding osteoid material were observed, reducing the A Bi . i t l i l l f t f t i l t i t i l . ( ) f t i l tith colloidal P- chitosan dispersion (protocol A, Group 1, 4 weeks). (B) The defect wasimplanted with colloidal AgNP-Q chitosan dispersion (protocol A, Group 2, 4 weeks). No signs ofsignificant inflammation were observed in both images. Magnification, 10×. Hematoxylin and eosinwere used for staining. The red circle corresponds to the area of the defect.On the other hand, in protocol B, whose main objective was to evaluate the regenerativecapacity of the materials in a critical-size defect, histological sections of the calvarias wereassessed at 4 weeks after implantation of the defects with colloidal NHAP-Q chitosandispersion. Histological examination showed the formation of bone trabeculae, especiallyin the central part of the defect and some areas near the edges, and connective tissue withfewer osteoblasts and osteocytes. The presence of connective tissue suggests the presenceof residual material that prevents correct repair of the bone with some signs of degradation.After 8 weeks, a considerable change in mineralized tissue was observed in thecentral part of the defects and very close to the borders contiguous to the basal bone(Figure 6A). A representative clinical view of the defect area, which decreased 8 weeksfter the placement of the grafts, is depicted in Figure 6B. In addition, remnants composedof chitosan fibers and NHAPs surrounding osteoid material ere observed, reducing t ere idual material, similar to the results obtained at 6 w eks in he same anim l model butwith the demineralized bone matrix [27].Nanomaterials 2023, 13, x FOR PEER REVIEW 12 of 19 residual material, similar to the results obtained at 6 weeks in the same animal model but with the demineralized bone matrix [27]. Figure 6. Histological analyses of the defect implanted in the animals. (A) The defect was implanted with colloidal NHAP-Q chitosan dispersion (protocol B, Group 1, 4 weeks). Mineralized tissue is shown in yellow, and the black arrow points to connective tissue. (B) The defect was implanted with colloidal NHAP-Q chitosan dispersion (protocol B, Group 1, 8 weeks). Black arrows point to the formation of laminar bone, and white arrows point to the bony bridge between the new and basal bone. (C) Clinical view at 8 weeks using (1) NHAP-Q and (2) colloidal AgNP-Q chitosan dispersion. Magnification, 10×. Hematoxylin and eosin. A significant amount of laminar bone is present at the edges of the defects in the central part of the calvaria after implantation of colloidal AgNP-Q chitosan dispersion at 4 weeks. On the other hand, near-complete defect closure was carried out, and the thick-ness of the new bone formed was similar to that of the original bone (Figure 6B). At 8 weeks, the mineralized material was slightly higher than at 4 weeks. However, the bony bridge between the newly formed bone and the basal bone was only present in some small areas and was not entirely uniform. Moreover, a homogeneous distribution of the bone tissue was observed, with little or no residual material being distinguished by the pres-ence of new mineralized material with osteoblasts and osteocytes within its matrix (Figure 6C). 3.8. Radiographic Evaluation Once the calvarias were dissected, a conventional periapical X-ray was taken and digitized for processing with the ImageJ imaging program. The evaluation of the 8 mm defects grafted with colloidal NHAP-Q chitosan dispersion at 4 weeks of evolution showed an average decrease in its initial area of 37.5 ± 4.0%, while a 75.1 ± 12.1% reduction was observed at 8 weeks. On the other hand, a decrease of 67.5 ± 5.7% was observed at 8 weeks, which was more significant (79.3 ± 11%) at 8 weeks of evolution for defects im-planted with colloidal AgNP-Q chitosan dispersion (Table 2). Table 2. Mean bone formation (n = 5) expressed as % ± SD. Weeks * Colloidal NHAP-Q Chitosan Dispersion Colloidal AgNP-Q Chitosan Dispersion 4 37.5 ± 4.0 67.5 ± 5.7 8 75.1 ± 12 79.3 ± 11 * Time after the procedure. A B CFigure 6. His ological analyses of the defect implanted in the animals. (A) The defect was implantedwith colloidal NHAP-Q chitosan dispersion ( rotocol B, Group 1, 4 weeks). Mineralized tissue iss own in yello , and the red arrow point to connective tissue. (B) The defect was implanted withcolloidal NHAP-Q chit san dispersi n (protocol B, Group 1, 8 weeks). Red arrows point to theformatio of lamin bone, and yellow arr ws po nt to the bony bridge between the new and basalbone. (C) Clinical view at 8 weeks using (1) NHAP-Q and (2) collo dal AgNP-Q chitosan dispersion.Magnification, 10×. Hematoxylin and eosin.A significant amount of laminar bone is present at the edges of the defects in thecentral part of the calvaria after implantation of colloidal AgNP-Q chitosan dispersion atNanomaterials 2023, 13, 321 12 of 184 weeks. On the other hand, near-complete defect closure was carried out, and the thicknessof the new bone formed was similar to that of the original bone (Figure 6B). At 8 weeks,the mineralized material was slightly higher than at 4 weeks. However, the bony bridgebetween the newly formed bone and the basal bone was only present in some small areasand was not entirely uniform. Moreover, a homogeneous distribution of the bone tissuewas observed, with little or no residual material being distinguished by the presence ofnew mineralized material with osteoblasts and osteocytes within its matrix (Figure 6C).3.8. Radiographic EvaluationOnce the calvarias were dissected, a conventional periapical X-ray was taken anddigitized for processing with the ImageJ imaging program. The evaluation of the 8 mmdefects grafted with colloidal NHAP-Q chitosan dispersion at 4 weeks of evolution showedan average decrease in its initial area of 37.5 ± 4.0%, while a 75.1 ± 12.1% reduction wasobserved at 8 weeks. On the other hand, a decrease of 67.5 ± 5.7% was observed at 8 weeks,which was more significant (79.3 ± 11%) at 8 weeks of evolution for defects implanted withcolloidal AgNP-Q chitosan dispersion (Table 2).Table 2. Mean bone formation (n = 5) expressed as % ± SD.Weeks * Colloidal NHAP-Q ChitosanDispersionColloidal AgNP-Q ChitosanDispersion4 37.5 ± 4.0 67.5 ± 5.78 75.1 ± 12 79.3 ± 11* Time after the procedure.3.9. Scanning Electron Microscopy Evaluation of the CalvariasThe interfaces between the new bone formed, the basal bone, and the center of thegraft were analyzed (Figure 7A), including the image of the de novo-formed bone with thepresence of bone trabeculae formation and gaps (Figure 7B). The interconnection pointswith the basal bone support the integration of the de novo-formed bone. In a close-upanalysis, small cell bodies were observed, corresponding to osteoblasts and osteocytesin the bone trabeculae and the lagoons, respectively (Figure 7C). These osteoblasts areessential for depositing new non-mineralized tissue (osteoid) that will later be mineralized(Figure 7D), where structures called burr-like projections connect the osteoblasts. Thesestructures are evidence of the deposition of mineralizable substances [28].3.10. Blood AnalysisAnalysis of the blood of animals treated with the colloidal chitosan dispersions versusthe untreated control showed that only AST was significantly different. The rest of theblood parameters showed no significant difference, suggesting that the use of the col-loidal chitosan dispersions is safe for different organs (liver and kidney), as no significantdifferences in ALT, urea, or creatinine were measured (Table S1).3.11. Distribution of Silver in OrgansOur study analyzed the Ag content in the heart, liver, kidneys, spleen, and skin. Theliver and kidneys were assessed due to their function as excretion systems in the body. Incontrast, the spleen was analyzed because it functions as a lymphoid organ and recyclesthe erythrocytes. In the case of the heart and skin, they were interpreted as potential placesfor Ag deposition through blood circulation or direct contact with the colloidal chitosandispersion. Histological analysis of these organs showed no changes in the ultrastructureand morphology of the tissues (Figure S4).The results showed a low accumulation of Ag in the tested tissues (Table S2). Althougha similar study showed higher accumulation in the kidneys and liver [29], this differencecould be related to the size of the nanoparticles used (36 nm versus 13 nm in our study).Nanomaterials 2023, 13, 321 13 of 18Nanomaterials 2023, 13, x FOR PEER REVIEW 13 of 19 3.9. Scanning Electron Microscopy Evaluation of the Calvarias The interfaces between the new bone formed, the basal bone, and the center of the graft were analyzed (Figure 7A), including the image of the de novo-formed bone with the presence of bone trabeculae formation and gaps (Figure 7B). The interconnection points with the basal bone support the integration of the de novo-formed bone. In a close-up analysis, small cell bodies were observed, corresponding to osteoblasts and osteocytes in the bone trabeculae and the lagoons, respectively (Figure 7C). These osteoblasts are essential for depositing new non-mineralized tissue (osteoid) that will later be mineralized (Figure 7D), where structures called burr-like projections connect the osteoblasts. These structures are evidence of the deposition of mineralizable substances [28]. Figure 7. Scanning electron microscopy images of the calvaria at 8 weeks post-treatment. (A) De novo-formed bone; (B) de novo-formed bone showing bone trabeculae formation (white arrows) and gaps. NB, de novo-formed bone; BB, basal bone. (C) Small cell bodies correspond to osteoblasts and osteocytes. (D) Osteoid deposit after mineralization (red rectangle) . Scale bars are indicated at the bottom of each image. 3.10. Blood Analysis Analysis of the blood of animals treated with the colloidal chitosan dispersions ver-sus the untreated control showed that only AST was significantly different. The rest of the blood parameters showed no significant difference, suggesting that the use of the colloidal chitosan dispersions is safe for different organs (liver and kidney), as no significant dif-ferences in ALT, urea, or creatinine were measured (Table S1). 3.11. Distribution of Silver in Organs Our study analyzed the Ag content in the heart, liver, kidneys, spleen, and skin. The liver and kidneys were assessed due to their function as excretion systems in the body. In contrast, the spleen was analyzed because it functions as a lymphoid organ and recycles the erythrocytes. In the case of the heart and skin, they were interpreted as potential places for Ag deposition through blood circulation or direct contact with the colloidal chitosan lacunae trabeculaeSmall bodiesNBBBA BCFigure 7. Scanning electron microscopy images of the calvaria at 8 weeks post-treatment. (A) Denovo-formed bone; (B) de novo-formed bone showing bone trabeculae formation (white arrows) andgaps. NB, de novo-formed bone; BB, basal bone. (C) Small cell bodies correspond to osteoblasts andosteocytes. (D) Osteoid deposit after mineralization (red rectangle). Scale bars are indicated at thebottom of each image.4. DiscussionIt is widely known that colloidal chitosan dispersions have a wide variety of character-istics that make them unique and ideal for use in biomedicine, such as oxygen permeability,the presence of metabolites, and good mechanical properties that mimic tissues [30]. Thisis especially relevant when transplants are needed for bone regeneration. However, au-tografts can have many c mplications and be hard to source for are s such as the oralcavity [31].Our study developed colloidal chitosan dispersions combined with AgNP-Q andNHAP-Q with high efficacy in bone healing. This effect is unsurprising as previous studieshave shown that combined chitosan and HA composites have promising properties in boneregeneration. These include controllable porosity, water retention, tensile strength, and theability to have pre-osteoblast cells (MC3T3-E1) attach and proliferate, creating a suitableenvironment for cell crawling and growth, without an impact on cell morphology andviability [32,33]. In addition, it has b en show that a chito an-HA membrane would bemo e effective than chitosan al ne for bone formation in the early stages [34].A cytotoxic effect was measured in both cell lines in an in vitro study using osteoblastand osteoclast cell lines exposed to 50 nm AgNPs (surface area: 11.54 m2/g). The cytotoxiceffect was based on the size- and area-dependent release of Ag+ from the NPs, with3 µm microparticles (surface area: 0.19 m2/g) displaying weak cytotoxic effects and 30 µmmicroparticles (surface area: 0.019 m2/g) having no inhibitory effects at all. Osteoblastsseemed to be more sensitive, with viability and differentiation significantly decreased atAgNP treatments of 128 µg/mL and 64 µg/mL, respectively, while osteoclasts experiencedsignificant decreases at 256 µg/mL and 128 µg/mL, respectively [35]. However, thisNanomaterials 2023, 13, 321 14 of 18study also found that the antibacterial effects on S. epidermidis were only effective at Ag+concentrations of 2–4× the cytotoxic levels. This underlines the importance of customizingNP size to optimize toxicity and antibacterial activity.Interestingly, mesenchymal stem cells (MSCs) were exposed to AgNPs in the stageof osteoblast differentiation. The results showed that AgNPs induced the expression ofthe gene CBFA1 responsible for the osteoblast differentiation [36]. Although we did nottest the sole effect of AgNPs in osteoblasts, our results showed osteoblast proliferationusing colloidal chitosan dispersions containing AgNPs, suggesting that the presence of thecolloidal chitosan dispersion component did not affect the growth of osteoblasts.Regarding the biocompatibility of our colloidal chitosan dispersions, our resultsindicated that they are biocompatible as a faster reparation of the defect in the in vivostudy (protocol B) was observed. In addition, it is well known that AgNPs have a potentantibacterial activity not present in the NHAPs. On the other hand, using AgNPs aloneraises concerns about their distribution in organs when used in an in vivo protocol. Thoseare the main reasons why we chose to perform more studies with AgNPs than NHAPs.Other studies reported similar results of biocompatibility compared to our colloidalchitosan dispersions. For example, good compatibilities were obtained when naturalrubber latex-AgNP composites were applied to regenerate bone defects [37]. Similar resultswere obtained when AgNPs were added to a gelatin/alginate scaffold used for skulldefect treatment [38]. Another composite prepared with polyurethane and tannic acidsupplemented with AgNP and NHAP reported similar results compared to our study forthe calvaria regeneration [39].One study attempted to use Ag and its innate antimicrobial abilities to combat implantinfection, creating an Ag coordination polymer network presented as a nanostructured coat-ing on metallic implant substrates. These networks showed dose-dependent antibacterialeffects on S. epidermidis and S. aureus in agar inhibition assays and prevent of S. epidermidisimplant infection in murine models. Some studies have attributed the toxicity of AgNPsto a wide variety of mechanisms, including the release of ions from NPs, oxidative stress,protein or DNA binding, and the generation of reactive oxygen species [40]. In addition,the mechanism of toxicity depends on the characteristics of NPs, such as their surface area,size, shape, capping agent, surface charge, particle purity, structural distortion, and thebioavailability of the individual particles [40].AgNPs have also been found to promote osteogenic activity, induce actin polymeriza-tion, activate RhoA (which enhances actin cytoskeletal tension and induces osteogenesis),and increase the expression of multiple genes, including RUNX2, ALP, BMP-2, COL1A1,OCN, and OPN when compared to AgNO3 exposure. Low AgNP levels that were notcytotoxic to urine-derived mesenchymal stem cells (≤4 µg/mL) still increased ALP activ-ity and matrix mineralization [41]. Another study reported the in vitro effect of AgNPson cell calcification and mineralization by bone cells. A remarkable cell mineralizationenhancement coincided with AgNP exposure, followed by HANP exposure. In addition,AgNPs were found to alter the microRNA expression of transcriptional factors associatedwith bone formation and lead to miRNA regulation of several gene-target BMPs not foundin the controls. Cytotoxicity studies performed with the NP concentrations used in theexperiments did not indicate any onset of cytotoxic effects [41].Furthermore, the addition of Ag into scaffolds has been performed in many studies.An in vitro study with human osteoblast-like cells (MG63) showed cytocompatibility withthe treatment of a membrane made from a slurry of Ag ion-substituted nano-HA/TiO2NPs and polyamide 66 (Ag-nHA/TiO2/PA66) causing no significant viability change afterup to 7 days of culture time. Another study found that adding AgNPs to a gelatin alginatescaffold increased pore size, porosity, water absorption, and degradation rates. Moreover,the 200 µM treatment was the most effective, showing the most de novo-formed bone inrabbit skull defects [38]. Contrarily, another study found that adding Ag to a chitosan nHAscaffold decreased the biodegradation rate. This could enable scaffold availability duringan extended period of bone growth. In addition, the composite was antibacterial againstNanomaterials 2023, 13, 321 15 of 18S. aureus and E. coli while remaining non-toxic to rat primary calvarial osteoblasts and ahuman osteosarcoma cell line [42]. As compounds in each scaffold differ, it is complicatedto compare results, as any discrepancies can be due to the properties of selected materials.However, it remains clear that Ag addition to composites contributes as an antibacterialcomponent and aids in refining parameters.Simultaneously, it is important to balance the unique properties with toxicity to ensurecytocompatibility. In vivo studies showed significant changes in rats’ ALP and cholesterollevels upon AgNP exposure, suggesting that exposure to more than 300 mg of AgNPs mightresult in mild liver damage [43,44]. Another study reported a toxicological evaluation ofmale guinea pigs exposed to three concentrations of AgNPs (100, 1000, and 10,000 ppm) ina sub-chronic model for 13 weeks. A close correlation between the dermal exposure andtissue levels of AgNPs was found (p < 0.05), and tissue uptake of AgNPs happened in adose-dependent manner with the following ranking: kidney > muscle > bone > skin > liver> heart > spleen. In the same study, histopathological analyses showed severe proximalconvoluted tubule and distal convoluted tubule degeneration in the kidneys of the middle-and high-dose animals. Separate lines and marrow space narrowing were determinedas two significant signs of bone toxicities observed in the three different dose levels ofAgNPs. Moreover, an increased dermal dose of AgNPs caused cardiocyte deformity,congestion, and inflammation [45]. In contrast, the concentration of Ag in our colloidalchitosan dispersions was 1 mg/mL. No toxicity or histological changes in the tibiae andcalvarias were observed at this concentration. On the other hand, when the control skin wascompared with the treated skin (Figure S2), a minor inflammatory infiltrate was observedin the treated skin, with high fibroblastic activity and collagen deposits. These activitiessuggest an accumulation of the AgNPs on the treated site. However, this is a weak responseto the colloidal chitosan dispersion but not to a foreign implanted body that would producegiant cells and it did not alter the tissue repair process [46].Our results showed that colloidal NHAP-Q chitosan dispersion regenerated the boneto 75% at 8 weeks. This result was superior to some other studies; for example, a boneformation of 67%, 57%, 50%, 19%, and 11% was measured when the macroporous biphasiccalcium phosphate, Bio-Oss (Geistlich, Princeton, NJ, USA), demineralized freeze-driedbone, allograft, Stypro, and empty control were evaluated, respectively [47]. A studyusing chitosan combined with an absorbable collagen sponge reported a defect closure of2.83± 1.06% and a new bone area of 4.84± 0.88% at 8 weeks. Interestingly, there was also asignificant decrease in bone density at 8 weeks compared with controls, which the authorsattributed to the remainder of residual materials, fatty marrow, and fibrovascular tissue [10].However, one study reported the complete healing of 5 mm defects in rat calvarias after8 weeks of implantation using an Ag-nHa/TiO2/PA66 membrane [48]. Again, becausethe tested substrates are different in experiments, it is hard to compare them with ourresults directly. Additionally, many studies reported a qualitative observation rather than apercentage. For example, one study (using a membrane formulated with HA and chitosan)reported improved local bone tissue growth in an 8 mm rat calvarial critical-size defectat 2 and 8 weeks [34]. The efficacy was significantly higher than the control membrane,which did not contain HA. Another study using colloidal chitosan dispersions made withhyaluronate and MegaGen synthetic bone reported colloidal chitosan dispersion resorptionand partial substitution to the lamellar bone after 8 weeks of implantation [49].5. ConclusionsThe biological implication of our study is that our colloidal chitosan dispersion mimicsan extracellular matrix, which is not similar to a conventional rigid graft. Moreover, ourporous chitosan colloidal dispersions allow cellular dynamics in any direction where cellu-lar integration is facilitated [30], showed biocompatibility, and promoted osteoregeneration.Moreover, we demonstrated significant antibacterial activity against clinical multidrug-resistant bacteria, and in vivo studies confirmed significant healing of CSDs. This studyshowed a potential for future use of our nanocomposites in biomedical applications. TheNanomaterials 2023, 13, 321 16 of 18advantage of the proposed use of the nanocomposites developed in this study is that infuture applications the concentrations of the AgNPs, HANP, and chitosan could be adaptedto a specific application for better efficacy. For example, the concentration of the AgNPscould be increased according to the presence of multidrug-resistant bacteria.Supplementary Materials: The following supporting information can be downloaded at:https://www.mdpi.com/article/10.3390/nano13020321/s1, Figure S1: Surgical sites in ProtocolA (Biocompatibility); Figure S2: Clinical observation of the defects after 4 weeks post-treatment;Figure S3: Characterization of the NPs used in this study by zeta potential; Figure S4: Representativeimages of organ histology; Table S1: Blood parameters were measured in animals of animals at8 weeks expressed as a mean of n = 5; Table S2: Tissue levels of Ag expressed as mg/g wet tissue,n = 5.Author Contributions: M.A.C.-S., Y.N.S. and P.Z. performed the experiments. N.N.-M. was involvedin the conceptualization, supervision, writing, and editing of the manuscript. H.B. was involved inthe supervision, data evaluation, writing, and editing of the manuscript. G.A.M.-C. was involvedin the conceptualization, data evaluation, supervision, writing, and editing of the manuscript. Allauthors shared in the data evaluation and the manuscript writing. All authors have read and agreedto the published version of the manuscript.Funding: H.B. acknowledges support from the Antibody Engineering and Proteomics, Immunityand Infection Research Centre, Vancouver, Canada.Institutional Review Board Statement: The animal study protocol was approved by the EthicsCommittee of the University Autonoma de San Luis Potosi, Mexico (protocol code CIS-FE-028-014,2014). The experiments were conducted following the Mexican Legislation Standard of NOM-063-SSA1-1993 and the General Health Law (Guide for the Care and Use of Laboratory Animals, TheNational Academies Press).Data Availability Statement: The data that support the findings of this study are available from thecorresponding author upon reasonable request.Conflicts of Interest: The authors declare no conflict of interest.References1. Egermann, M.; Lill, C.A.; Griesbeck, K.; Evans, C.H.; Robbins, P.D.; Schneider, E.; Baltzer, A.W. Effect of BMP-2 Gene Transfer onBone Healing in Sheep. Gene Ther. 2006, 13, 1290–1299. [CrossRef] [PubMed]2. Isaksson, S.; Alberius, P. Comparison of Regenerative Capacity Elicited by Demineralized Bone Matrix of Different EmbryonicOrigins. J. Cranio Maxillofac. Surg. 1992, 20, 73–80. [CrossRef] [PubMed]3. Schortinghuis, J.; Ruben, J.L.; Raghoebar, G.M.; Stegenga, B. Ultrasound to Stimulate Mandibular Bone Defect Healing: APlacebo-Controlled Single-Blind Study in Rats. 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silver nanoparticles
hydroxyapatite particles
chitosan
critical-size defect
animal model
Osteoregeneration of Critical-Size Defects Using Hydroxyapatite–Chitosan and Silver–Chitosan Nanocomposites
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