Due to bioactive properties, introducing spongin-like collagen (SPG) into the biosilica (BS) extracted from marine sponges, would present an enhanced biological material for improving osteoporotic fracture healing by increasing bone formation rate. Our aim was to characterize the morphology of the BS/SPG scaffolds by scanning electron microscopy (SEM), the chemical bonds of the material by Fourier transform infrared spectroscopy (FTIR) and evaluate the orthotopic in vivo response of BS/SPG scaffolds in tibial defects of osteoporotic fractures in rats (histology, histomorphometry and immunohistochemistry) in 2 experimental periods (15 and 30 days). SEM showed that scaffolds were porous, showing the spicules of BS and fibrous aspect of SPG. FTIR showed characteristic peaks of BS and SPG. For the in vivo studies, after 30 days BS and BS/SPG showed a higher amount of newly formed bone compared to the first experimental period, observed both in the periphery and in the central region of the bone defect. For histomorphometry, BS/SPG presented higher %BV/TV compared to the other experimental groups. After 15 days, BS presented higher volumes of collagen type I. After 30 days, all groups demonstrated higher volumes of collagen type III compared to volumes at 15 days. After 30 days, BS/SPG presented higher immunostaining of osteoprotegerin compared to the other experimental groups at the same experimental period. The results showed that BS and BS/SPG scaffolds were able of improving bone healing. Future researches should focus on the effects of BS/SPG on longer periods in vivo studies.
Research Article
In vivo effects of biosilica and spongin-like collagen scaffolds on the healing process in osteoporotic rats
https://doi.org/10.21203/rs.3.rs-4283707/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 17 Aug, 2024
You are reading this latest preprint version
bone tissue engineering
Biosilica
Spongin-like collagen
marine sponges
in vivo studies
osteoporosis
The World Health Organization (WHO) defines osteoporosis as a ‘progressive systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture’ (KANIS, 2018; SLAIDINA et al., 2023). Osteoporosis is responsible for a significant increase in the risk of bone fractures, particularly of the hip, vertebrae, distal forearm and humerus (KOBAYASHI et al., 2022; LIU et al., 2019). The occurrence of osteoporotic fractures is related to high rates of morbidity and mortality and high economic costs (LIU et al., 2019).
Due to reduced bone mass, osteoporosis results in impaired ability for bone repair after fracture, leading to an increased risk for the development of pseudoarthrosis and fracture non-union (KANIS et al., 2018). Depending on the severity of the osteoporotic fracture, surgical interventions may be needed (ARCOS et al., 2014). In osteoporotic hip fractures, for example, metallic rigid fixation devices such as intramedullary nails, bridge plates and tension band constructs are commonly used in surgical procedures to restore the mechanical properties and capacity of supporting the loads of daily life activities (ANDERSON et al., 2020; HOLLENSTEINER et al., 2019). However, there are disadvantages to such efforts including significant bone stiffness causing stress shielding of the underlying bone, poor wound healing, infection and the occurrence of implant failure (mainly due to the compromised osteoporotic bone mass and insufficient osteogenesis, compromising the implant integration) (SCHUETZE et al., 2019).
Considering these important issues, the use of effective biomaterials (metallic implants and active cements) for fracture fixation and stimulation of bone ingrowth to improve tissue-implant osteointegration has been of significant interest (KANNO et al., 2022). Metallic implants are used as primary fixation devices and synthetic bioactive ceramics are used mainly as reinforcement of the metallic hardware. Synthetic biomaterials, especially the ceramics including calcium phosphate, bioglasses and calcium sulfate cements, have been widely used for bone tissue regeneration (BARRETO et al., 2023). More recently, natural biomaterials, especially the ones extracted from marine biodiversity, have been emerging as a promising alternative for manufacturing bone substitutes (SILVA et al., 2014). Among the marine biodiversity, marine sponges (the phylum Porifera) are one of the most promising sources of biological elements and molecules, with a vast therapeutic potential for a wide range of applications due mainly to the antitumor, antiviral, anti-inflammatory and antibiotic effects (SILVA et al., 2014). The inorganic part of the sponges, the glassy amorphous silica called biosilica (BS), and the organic part, the spongin-like collagen (SPG), have demonstrated beneficial effects on osteoblast and fibroblast cell proliferation and on the stimulation of tissue healing (DE ALMEIDA CRUZ et al., 2020; GABBAI-ARMELIN et al., 2018). Because silica ions are known as an important element to stimulate bone formation, BS (glassy amorphous silica- SiO2) containing water and small amounts of Al, Ca, Cl, Cu, Fe, K, Na, S, and Zn (EHRLICH et al., 2010) has been demonstrated to have osteogenic effects in in vitro and in vivo studies (WANG et al., 2014). Gabbai-Armelin et al.(GABBAI-ARMELIN et al., 2019) using a cytotoxicity assay, demonstrated that BS positively influenced osteoblast cell viability and increased Runx2 and BMP4 gene expression, concluding that BS has a huge potential to be used for bone tissue engineering applications. Also, SPG has been considered a natural compound for tissue bioregeneration presenting biocompatibility and capability of supporting human skin cell growth (GREEN et al., 2003; IWATSUBO et al., 2015). Parisi et al. (PARISI et al., 2018) demonstrated that the introduction of collagen from marine sponges into a scaffold of biosilicate (a biomaterial with high osteogenic properties) showed biocompatibility and it was capable of supporting bone formation in a critical bone defect in rats.
As both biocompounds have been shown to have positive biological outcomes, investigation of the combination of both biomaterials would be a natural approach to progress the field. In particular, the effects of the composite biomaterials on the process of bone healing in the presence of osteoporosis. In this context, we hypothesized that the introduction of SPG into BS would present an enhanced biological material for improving osteoporotic bone defect healing by increasing bone formation rate and volumes. Therefore, the aim of the present work was to evaluate the orthotopic in vivo response of BS/SPG samples in the process of bone healing. For this purpose, the composite biomaterials were implanted into experimentally induced tibial defects of osteoporotic fractures in rats, and bone responses (histology, histomorphometry and immunohistochemistry) were evaluated after 15 and 30 weeks after implantation.
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.
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.
SEM
Figure 1 presents the SEM morphological examination of BS and BS/SPG scaffolds. It is possible to observe in Figures 1A and C, the spicule shaped particles of the BS scaffolds, with the presence of pores. For BS/SPG scaffolds it is possible to observe the spicule shaped particles and pore formation throughout the scaffold (Figure 1B). Figure 1D demonstrates the fibrillar shaped particles which is a characteristic of SPG.
FTIR
Figure 2 shows the FTIR spectrum of BS and SPG analysis. For BS, characteristic peaks compatible with silicon oxide clusters (Si-O/Si-O-Si) were observed at 1034 cm-1 and 788 cm-1 wavelengths, respectively. The FTIR spectrum of SPG demonstrates the peaks compatible with amine (NH) at the wavelengths of 1594 and 3362 cm-1 and carbon oxides (C-O) in a wavelength ranging from 1000-1500 cm-1.
Histopathological analysis
Figure 3 presents the histological findings of the experimental periods at 15- and 30-days post-surgery.
15-days post-surgery
At 10X magnitude, it was possible to observe that for GC, the defect border was still apparent and granulation tissue was seen filling the central region of the defect, with the presence of newly formed bone tissue surrounding the periphery (Figure 3A). For BS, the border of defect could still be observed, and the bone defect was filled mainly with granulation tissue, especially in the central region, with newly formed bone and particles of the biomaterial at the periphery (Figure 3C). For BS/SPG, the defect border could still be seen, with granulation tissue and residual biomaterial present at the central defect area. Newly formed bone was also observed at the periphery (Figure 3E).
30-days post-surgery
For GC, the defect border could still be seen but it was less demarcated compared to the shorter experimental period. Granulation tissue could still be observed in the central region of the defect (but to a lesser extent) with ingrowth of newly formed bone from the periphery to the center (Figure 3B).
For BS, 30 days post-surgery, the borders could still be observed. The bone defects were consistently filled with degraded biomaterial particles and granulation tissue in the central region. A high amount of newly formed bone compared to the first 15-day experimental period was observed both in the periphery and in the central region (Figure 3D). For BS/SPG, similar findings of those found in BS were seen, with a discrete border of the defect and material particles, with signs of degradation. The central region was filled with granulation tissue and newly formed bone (growing from the periphery to the central region) (Figure 3F).
Histomorphometric analysis
Figure 4 represents the values of the histomorphometric analysis. In the first 15-day experimental period, no significant differences were observed in the intergroup analysis. At 30 days post-surgery, the intragroup analysis demonstrated a higher value of %BV/TV for all groups compared to the first 15-day period (p= 0,0085, p= 0,0184 and p= <0,0001, respectively). Moreover, 30 days post-surgery, the intergroup analysis, demonstrated that BS/SPG showed higher values for this variable compared to BS (p= 0,0002) (Figure 4A). No other difference was observed.
Figure 4B demonstrates that, for Ob.S/BS (%), a significantly higher value (p = 0,009) was found for BS/SPG compared to BS 15 days post-surgery. No differences were observed among CG and the treated groups. Also, for Ob.S/BS (%), significant higher values (p = 0,0097) were found for BS 30 days post-surgery compared to BS 15 days post-surgery. No other difference was observed.
Figure 4C demonstrates a higher value for N.Ob/T.Ar (mm2) for BS/SPG when compared to BS (p = 0,0093) in the first experimental period (15 days post-surgery). The intragroup analysis demonstrated higher BS values at 30 days post-surgery compared to the values found 15 days post-surgery (p= 0,0196). No other difference was observed.
Collagen analysis by Picrosirius staining
Figure 5 demonstrates the evaluation of the collagen fibers for all groups. For CG, in the first experimental period, the collagen fiber meshes were seen in the region of the newly formed bone, surrounding the trabeculae. Both BS and BS/SPG groups presented collagen arrangement in a networked way (Figure 5A, C and E). After 30 days, the network of collagen fibers was distributed more densely and in parallel bundle arrangement. The same findings were seen for both biomaterial-treated groups (Figures 5 B, D and F).
The values found in the quantitative analysis of collagen are demonstrated in Figure 6. For BS, the volume fraction (Vv) of Type I collagen fibers in the first 15-day experimental period was higher compared to CG (p=0,000) (Figure 6A). BS/SPG presented lower values of this variable compared to BS (p=0,000). Thirty days post-surgery, no statistical difference was observed among groups (Figure 6 A), although a lower value of collagen fibers Type I (Vv) was observed 30 days after the surgery for BS compared to the first period of 15 days (p=0.001). For collagen fiber type III, 15 days post-surgery, a higher value of volume fraction (Vv) was seen for BS/SPG compared to BS (p=0,003) and CG (p=0,000) (Figure 6B). After 30 days, BS presented lower values compared to CG (p=0,000). For BS 30 days post-surgery, a higher value of Vv was seen for collagen type III fibers compared to the period of 15 days (p=0.001) and a higher (Vv) value for BS/SPG 30 days after the surgery compared to the 15 day period (p=0.001) (Figure 6B).
When comparing the total volume fraction (Vv) of collagen fiber types I and III, it can be observed that 15 days post-surgery, BS/SPG demonstrated lower values compared to BS (p=0,028) (Figure 6C). After 30 days post-surgery, BS presented lower values for these variables compared to CG (p=0,000) and BS/SPG demonstrated higher values compared to BS (p=0,000) (Figure 6C). In addition, a lower (Vv) value was observed for BS 30 days after the surgery compared to the 15 day period (p=0.001).
Immunohistochemistry
Runx-2
Figure 7 shows the qualitative analysis of Runx-2 for CG, BS and BS/SPG. For CG, the Runx-2 immunostaining was observed mainly in the granulation tissue in the first experimental period (Figure 7A). Thirty days post-surgery, the immunomarker was seen mainly in the newly formed bone (Figure 7B). For BS, the immunostaining was observed mainly in the granulation tissue areas and around BS particles for both experimental periods. At 30 days post-surgery RUNX-2 immunostaining was also observed in the newly formed bone (Figures 7C and 7D). Runx-2 immunostaining for BS/SPG was observed mostly through the granulation tissue and at the newly formed bone for both experimental periods (Figures 7E and 7F, respectively).
Figure 7G presents the results of the quantitative analysis for immunostaining of Runx-2. No statistical differences were observed for any groups or experimental periods (p>0.5).
OPG
Figure 8 shows the qualitative evaluation of OPG immunostaining. For CG, the immunostaining was observed mostly in the granulation tissue area at 15 days post-surgery and in the newly formed bone tissue at 30 days post-surgery (Figures 8A and 8B, respectively). For BS, OPG immunostaining was observed mainly through the granulation tissue and in the newly formed bone tissue at 15 days post-surgery (Figure 8C). Also, OPG immunostaining was mainly observed at newly formed bone tissue for the 30 days post-surgery (Figure 8D). For BS/SPG after 15 days, OPG immunostaining was observed mostly at granulation tissue areas and around BS particles. Thirty days post-surgery the immunostaining of OPG was observed at the newly formed bone tissue (Figures 8E and 8F).
Figure 8G presents the semiquantitative analysis of OPG immunomarker. Fifteen days post-surgery, no statistical differences were observed among the groups. After 30 days post-surgery, CG and BS presented statistically lower values compared to BS/SPG (p= 0.481).
This study investigated the in vivo performance of BS/SPG scaffolds in the process of bone healing in osteoporotic rats. The characterization tests demonstrated the spicules of BS and the fibrous irregular structure of SPG. The FTIR showed the elements of the scaffolds with the presence of silicon and amines. Histological analysis showed that no adverse reactions were seen at the site of the implant, with a biomaterial degradation of the material along the experimental time and substitution by newly formed bone for both biomaterials treated groups. Histomorphometry demonstrated a tendency for higher values in all variables in the composite BS/SPG biomaterial, with statistical differences for BV/TV. Higher amounts of collagen type I were seen for BS; and for collagen type III higher values were observed for BS/SPG. No differences for RUNX-2 immunostaining were observed and higher OPG immunostaining was observed for BS/SPG.
Some studies have demonstrated that both BS and SPG from marine sponges are able to stimulate bone cell proliferation and healing in healthy animals (GABBAI-ARMELIN et al., 2019; PARISI et al., 2018, 2020; SANTANA et al., 2021). To further investigate the effects of scaffolds manufactured with both biomaterials in the process of bone healing, we used the model of tibial bone defect in osteoporotic rats. As previously described, animals treated with BS and BS/SPG scaffolds demonstrated ingrowth of newly bone in the region of the defect, with no adverse reaction. Based on our results, it is possible to state that BS and BS/SPG scaffolds are biocompatible as we did not see any signs of biomaterial rejection with no excessive inflammatory response, nor the presence of giant cells or any capsule involving the material particles (LEE et al., 2019). The material presented some degree of degradation over time, releasing ions from BS which may attract osteoprogenitor cells, increasing the rate of bone formation into BS/SPG scaffolds (GRANITO et al., 2009; HENCH; JONES, 2015; PARISI et al., 2019). Some authors have demonstrated that SPG implants are able to stimulate cell proliferation (SILVA, 2021) and evoking no inflammatory response in experimental bone defects, evidencing in vivo biocompatibility (SANTANA et al., 2021).
The quantitative histological analyses showed higher values for animals treated woth BS/SPG compared to BS only, indicating that the composite scaffolds presented a superior biological performance to stimulate bone healing in the osteoporotic rats. The combination of materials from different sources and classes, known as composites, has been shown to be an optimized therapeutic strategy for stimulating tissue ingrowth (TATARA; MIKOS, 2016; XIAO et al., 2019). In this context, the combination of an inorganic material, like BS with an organic material such as SPG, may be a closer alternative to mimic the structure and composition of the bone tissue constituting a biomaterial with enhanced biological properties and performance (KIDO et al., 2019; KWAK et al., 2013; VARGAS et al., 2013).
Collagens constitute a family of proteins present in the extracellular matrix of connective tissues and are responsible for contributing to maintenance of the structural integrity of bone tissue. Bone is unique in connective tissue healing because it heals entirely by cellular regeneration and the production of a mineral matrix rather than just collagen deposition known as scar (KIM et al., 2020; MARX, 2007). Moreover, type I collagen primarily provides structural and mechanical support, acting as a natural scaffold for the other extracellular matrix proteins beyond their core purpose in mineral deposition initiation (LICINI et al., 2022). For the quantitative collagen analysis, an increase of type I collagen for BS and of type III collagen for BS/SPG were found. These findings suggest that the scaffolds promoted different collagen stimuli. Although the importance of type I collagen in structural and mechanical support, it is important to highlight that bone fracture callus formation presents with considerable amounts of type III collagen which is later replaced by type I collagen (HILTUNEN; VUORIO; ARO, 1993; LANE et al., 1986). Volk et al.(VOLK et al., 2014) stated that type III collagen regulates osteoblastogenesis and the quantity of trabecular bone. Miedel et al.(MIEDEL et al., 2015) using a tibial bone defect in mouses, showed that a decreased amount of type III collagen leads to an impairment of the capacity of bone formation and alterations in remodeling during fracture healing. The results of the present work demonstrated that, in addition to increased type III collagen in BS/SPG group, BS/SPG histomorphometry results demonstrated higher values in bone volume fraction (BV/TV% evaluated 30 days post-surgery), percentage of bone surface occupied by osteoblast (OB.S/BS%, 15 days post-surgery) and osteoblast number per tissue area (N.OB/T.Ar/mm2, 15 days post-surgery), corroborating the hypothesis that the type III collagen modulates fracture callus bone formation.
Immunohistochemistry analysis showed immunostaining of Runx-2 and OPG for all samples. Runx-2 immunofactor is mainly expressed in osteoblasts and it is required for the differentiation of mesenchymal progenitors toward osteoblast cell lineage. It is well known that Runx-2 is fundamental for upregulation of other osteoblastic markers, like osteocalcin, osteopontin and alkaline phosphatase (MOON et al., 2014; NOTOYA et al., 2004), which may have influenced osteoblast cell differentiation and consequently, bone formation and deposition. OPG, also known as osteoclastogenesis inhibitory factor (OCIF) or tumour necrosis factor receptor superfamily member 11B (TNFRSF11B), is a cytokine receptor of the tumour necrosis factor (TNF) receptor superfamily encoded by the TNFRSF11B gen(CARRILLO-LÓPEZ et al., 2021). OPG is largely expressed by osteoblast lineage cells of bone, epithelial cells of the gastrointestinal tract, lung, breast and skin vascular endothelial cells, as well as B-cells and dendritic cells in the immune system (TOBEIHA et al., 2020). In the present work, BS/SPG presented higher OPG immunostaining. This outcome may indicate the increased presence of osteoclasts in an attempt to degrading the biomaterial but further research is required to confirm whether this is the case.
The results of the present work confirmed the hypotheses that BS/SPG scaffolds presented improved biological responses in the process of bone healing of osteoporotic rats. To further understand the effects of composite BS/SPG, information on the long-term performance of the material remains to be examined.
The process of bone healing in osteoporotic tissues can be compromised by low bone mass. In this context, natural material such as BS and BS/SPG scaffolds have been emerging as promising alternatives for synthetic biomaterials. The present study demonstrated that BS/SPG scaffolds were able to improve bone healing in osteoporotic rats without observable deleterious effects noted in the measures used in this study. Taken together, the results suggest that BS/SPG is biocompatible and presents non-cytotoxicity in the in vivo studies; and can be considered as an alternative source of biomaterials for bone tissue engineering proposals. Future research should focus on investigating the effects of BS/SPG on long term periods of implantation in animal bone defect models.
Competing Interests and Funding
This research was funded by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), grant number 2019/10228-5.
Institutional Review Board Statement
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. This study was approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal University of São Paulo (2019/ 2629220119).
Data Availability
The data presented in this study are available on request from the corresponding author.
Conflicts of Interest:
The authors declare no conflict of interest.
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No competing interests reported.