Materials
Biosilica
Marine sponge Dragmacidon reticulatum specimens were used for BS extraction. Samples were collected at the north coast of São Paulo (Praia Grande (23o49'23.76 "S, 45o25'01.79" W, São Sebastião, Brazil and in Araçá Bay area (23o81'73.78 "S, 45o40'66.39 "W, São Sebastião, Brazil). After collection, sponges were washed with MiliQ water, cut into small pieces and treated with 5% (v/v) sodium hypochlorite to degrade all organic components of the sponge. Afterwards, material was repeatedly washed with MilliQ water (10 times) to remove sodium hypochlorite solution and transferred to a beaker, with nitric acid/sulfuric acid (1:4). Following this step, the material was submitted to several washes with MilliQ water to reach a final pH ≥6.0 (WEAVER et al., 2003). BS samples were dried at room temperature and fine powder BS particles were obtained (particle size: 106 – 126µm).
Spongin-like collagen (SPG)
Aplysina fulva marine sponges were used for SPG extraction. Samples were collected in Praia Grande (23o49'23.76 "S, 45o25'01.79" W, São Sebastião, Brazil) and in Araçá Bay area (23o81'73.78 "S, 45o40'66.39 "W, São Sebastião, Brazil). SPG was extracted based on the method described by Swatschek et al (SWATSCHEK et al., 2002). In the lab, samples were washed 3 times with Milli-Q water for cell debris removal and sponges were cut into small pieces. Then, Tris-HCl buffer (100 mM, pH 9.5, 10 mM EDTA, 8 M urea, 100 mM 2-mercaptoethanol) was added to the samples and the pH was adjusted to 9 (with NaOH solution). Solution was transferred into a stirred beaker over 24 h and, centrifuged for 5 minutes at 2oC. The pellet was discarded, supernatant removed and pH was adjusted to 4 with acetic acid. A precipitate was formed and then resuspended in Milli-Q water, centrifuged again and lyophilized for preservation (SWATSCHEK et al., 2002).
Preparation of the scaffolds
For this study, scaffolds were manufactured following the protocol described in a previous study (SILVA, 2021). Table 1 demonstrates the amount of BS and SPG used for scaffold manufacturing for each group. Materials were inserted into a 2 ml plastic syringe containing 0.5 g of sodium dibasic phosphate (Na2HPO4) powder (used as the porogenic agent). Then 1 ml of 2% Na2HPO4 solution was added and the mixture was vigorously stirred for 20 seconds in an amalgamator (Silamat, Vivadent, Schaan, Liechtenstein). Finally, the composites were injected into hollow molds measuring 0.3 cm in diameter by 0.2 cm in height.
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Material characterization
Scanning Electron Microscopy (SEM)
The analysis of SEM was performed for analysing the morphology of the scaffolds. For this purpose, samples were mounted on stubs with carbon tape and sputter coated with gold and the SEM analysis was performed.
Fourier transform infrared spectroscopy (FTIR)
FTIR was used for characterizing the chemical bonds present in the samples used in this study. Analyses were done using pulverized samples in the range of 520–4000 cm−1 with a resolution of 2 cm−1. The samples were scanned 100 times for each FTIR measurement and the spectrum acquired was the average of all these scans.
Animals and experimental groups
For this experiment, 60 healthy female Wistar rats, 3 months old (average weight of 250-300 g) were used. Animals were allowed to acclimatize for 7 days and housing was provided per pair in a standard type 3 cage. In this study, the experimental model of ovariectomy (OVX) was used for inducing osteoporosis (RONG et al., 2022). After 9 weeks post OVX surgery, animals were submitted to another surgical procedure to create a bone defect in the tibia.
Animals were randomly assigned to 3 experimental groups as described below:
- Control Group (CG): osteoporotic rats submitted to the surgical procedure to induce bone defects with no treatment.
- Biosilica Group (BS): osteoporotic rats submitted to the surgical procedure to induce bone defects and treated with BS scaffolds.
- Biosilica/Spongin Group (BS/SPG): osteoporotic rats submitted to the surgical procedure to induce bone defects and treated with BS/SPG scaffolds.
All groups were divided into 2 sub-groups (n=10) with different periods until euthanasia at 15- and 30-days after bone surgery.
Ovariectomy (OVX)
All experimental animals were submitted to the OVX surgery. For surgery, animals received an intraperitoneal anesthesia with Ketamine (80 mg/kg) and xylazine (8 mg/kg) and the abdominal area was shaved using a razor blade. The shaved area was cleaned with iodopovidone and an incision was made with a scalpel blade. After the incision, the ovaries were visually located in the abdominal cavity and a bilateral ovariectomy was performed (RONG et al., 2022). At the end of the procedure, the wound was closed internally with resorbable thread and externally with nylon sutures. A period of 9 weeks was allowed for the development of osteoporosis (RONG et al., 2022).
Experimental model of tibia bone defect
Nine weeks post-surgery, a bone defect was created in both tibias of each animal. Pre-operatively pain medication was provided by an injection of Carprofen (Rimadyl®, Pfizer Animal Health, New York, USA) in dosage of 5 mg/kg, given 15 min before surgery. Anesthesia was induced and maintained by Isoflurane inhalation (Rhodia Organique Fine Limited) combined with oxygen delivered by mask. Before creating the defects, both hind limbs of the rats were shaved and disinfected with povidone iodine. The rats were immobilized in the supine position and with the knee maximally flexed a longitudinal incision through skin and muscle was made on the medial surface. Then, noncritical sized bone defects (3 mm diameter) were created using a motorized drill (Beltec®, Araraquara, SP, Brazil), in the right tibia, under copious irrigation, with saline solution at the upper third of the tibia (10 mm distal of the knee joint). To ensure adequate postoperative analgesia, before closing the wound, local drop anaesthesia was applied using Lidocaïne (10 mg/ml Lidocaïne FNA, Centrafarm B.V., Etten-Leur, the Netherlands) and Bupivacaïne (5 mg/ml Bupivacaïne Actavis, Actavis B.V., Baarn, the Netherlands) diluted with NaCl using ~1 ml per rat (containing 1 mg of Lidocaïne and 0.25 mg of Bupivacaïne). After the experimental periods, animals were euthanized by anaesthetic overdose (Ketamine: 240 mg/Kg e Xilazine: 24 mg/Kg).
Histopathological analysis
Histopathological analysis was performed with the aim of describing the morphological findings at the site of the bone defect in all experimental groups. After euthanasia, tibias were removed and immediately fixated in 10% formalin (Merck, Darmstadt, Germany) for 2 days and submitted to decalcification using 4% ethylenediaminetetraacetic acid (EDTA) (Merck, Darmstadt, Germany). Samples were then submitted to the process of dehydration using a graded series of ethanol and embedding in paraffin blocks. Histological sections (5 µm) were prepared using a microtome with a blade (Leica Microsystems SP 1600, Nussloch, Germany). Laminae were stained with hematoxylin and eosin (Merck, Darmstadt, Germany) and examined using light microscopy (Leica Microsystems AG, Wetzlar, Germany, Darmstadt-Germany). Qualitative analysis of each lamina for the presence of granulation tissue, newly formed bone, material particles and the border defect was performed by blinded 2 observers (M.A.C. and J.R.P.) (FERNANDES et al., 2017).
Histomorphometric analysis
To further characterise tissue morphology in the experimental groups, histomorphometry was performed using a semi-automatic image-analysing OsteoMeasure System (Osteometrics, Atlanta, GA, USA). Samples were quantified separately considerate of the following parameters: bone volume fraction (BV/TV, %), percentage of bone surface occupied by osteoblast (Ob.S/BS), and osteoblast number per tissue area (N.Ob/T.Ar, /mm²). Analysis was performed by 2 experienced but blinded observers (M.F.S. and J.R.P.).
Collagen analysis by picrosirius evaluation
Histological sections were stained by Sirius-Red and analyzed under polarized light to differentiate collagen fiber types I (yellow and red) and III (green). The volume fraction (Vv) of collagen type I and type III as well as the sum of each type (VvTotal) were obtained. For this purpose, 10 photomicrographs of the region of the defect per animal were obtained, through a computerized imaging device (Axio Visio 4.5 Zeiss®) attached to a microscope (Axio Observer D1, Zeiss®) with a 40x objective. The photomicrographs were blinded and submitted to a test system containing 36 crosses (MANDARIM-DE-LACERDA, 2003). To obtain the Vv, a test system was randomly positioned on the photomicrograph and when type I or type III collagen fibers touched the upper quadrant of each cross, they were counted. The total number of points (Pt) that coincided on the total area analyzed was determined, as well as the partial number of points on each collagen type fiber analyzed (Pcollagen type I, III or total) and the percentage of interest structure was calculated by applying the formula Vv = ∑ Ptype I, III or total collagen ∕ ∑ Pt x 100 (BAPTISTA et al., 2019).
Immunohistochemistry analysis
To understand whether the chosen biomaterials influence the bone immune-inflammatory response, the streptavidin–biotin-peroxidase method was used for the immunohistochemistry analysis (FERNANDES et al., 2017). Xylene was used for removal of the paraffin from the sections, samples were then dehydrated in graded ethanol and pre-treated with 0.01 M citric acid buffer (pH 6) in a steamer for 5 min. Hydrogen peroxide in phosphate-buffered saline (PBS) was used for endogenous peroxidase inactivation for 5 min and blocked with 5% normal goat serum in PBS for 10 min. Primary antibody was incubated with anti-Runt-related transcription factor 2 (Runx-2) polyclonal (code: sc-8566, Santa Cruz Biotechnology, USA) at a concentration of 1:100 and anti-Osteoprotegerin (OPG) monoclonal (code: sc-7269, Santa Cruz Biotechnology, USA), overnight at 4ºC. Immunostaining was performed using 0.05% solution of 3-3’-diaminobenzidine solution and restrained with Harris haematoxylin (Merck) for 10 secs. A qualitative analysis was performed considerate of the presence and location of the immunomarkers.
Statistical Analysis
Data were expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Shapiro-Wilk normality test was used to check distribution. Mann-Whitney or Kruskal-Wallis test and Dunn post hoc were used for nonparametric data. T test and two-way analysis of variance (ANOVA) with Tukey multiple comparisons post-tests were used for parametric data. Differences were considered significant at p ≤ 0.05.