Biomaterials synthesis
The scaffolds containing chitosan (CH) and polyvinyl alcohol (PVA) as mixtures of both types of polymers named CH/PVA were prepared by freeze-drying method. The scaffolds were prepared as follows: CH was dissolved in 1% acetic acid solution for 2-3 h till formation of a clear solution. Then, risedronate (10 mg/g) was added to the CH solution and stirred for another 1 h at room temperature. And then, PVA was gradually added to the above solution at 60 ± 5 °C with continuous stirring for an additional hour to dissolve PVA. After complete dissolution, the prepared solution was sonicated (Digital sonicator, MTI, USA) for 30 mins to eliminate air bubbles and get homogeneous solutions. The prepared solutions were poured in molds (diameter of 15 mm) to create disks and layers and frozen overnight at −80 °C before freeze-drying process. Finally, the frozen scaffolds were freeze dried in a freeze dryer (Christ freeze dryer, ALPHA 2-4 LD plus, Germany) for 24 h to obtain dried scaffolds. For CH/PVA scaffolds, 10% w/w of CH, PVA was used in weight ratios; 30:70 blank non-medicated scaffold containing no drug were prepared using the same procedure. Each scaffolds contained 10% w/w risedronate. The solid content in the CH/PVA scaffold forming solutions was 10% w/v. Furthermore, the glass was added in percentage 10% in the CH/PVA 3:7 scaffolds (Oudadesse et al., 2013; Bui et al., 2010; Sun et al., 2013).
Narrative description of accomplishments
Selection of the most reliable anti-osteoporotic drug
For the management of osteoporosis, risedronate sodium was selected as one of the most distinguished anti-osteoporotic drugs. Risedronate sodium belongs to the bisphosphonate category that exerts its effect in the treatment of osteoporosis through binding to hydroxyapatite crystals at the bone matrix and through inhibiting of bone resorption by inhibiting osteoclast activity. Risedronate has a relative potency 2000 times higher than that for the reference bisphosphonate and etidronate. On the other hand, risedronate renal clearance is about 87% of total clearance, indicating that only a small proportion of a systematically available dose is associated with the bone.
Characterization of the prepared scaffolds has been undertaken through porosity measurements
The scaffold porosity was measured using a pycnometer (Ultrapyc 1200e Quantachrome pycnometer, USA). All the prepared scaffolds had porosity values higher than 75%. Such high percentages of porous scaffold structure with interconnected pores are required to enhance bone formation. Furthermore, such a scaffold structure would also facilitate cell migration, adhesion, and proliferation. Addition of the drug lowers the percentage porosity to some extent.
Experimental studies
In vivo animal studies were designed to evaluate the osteogenic potential of different bone scaffolds on the healing of experimentally induced critical-sized mandibular bone defect in dogs.
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Study I (effect of bio-glass)—was designed to evaluate the osteogenic potential of different concentrations (10% and 30%) of bio-glass (BG) combined with chitosan-polyvinyl acid (CH/PVA) as an osteoconductive scaffold for the healing of experimentally induced critical-sized mandibular bone defect in dogs.
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Study II (effect of risedronate drug)—these groups were designed to evaluate the osteogenic potential of risedronate (M) combined with chitosan-polyvinyl acid (CH/PVA) with or without bio-glass (BG) osteoconductive scaffold for the healing of experimentally induced critical-sized mandibular bone defect in dogs.
Scaffold drug-loaded CH/PVA-BG and physico-chemical characterization. Newly developed scaffold has been prepared from a mixture of two types of polymers without/with bioactive glass and the selected antiosteoporosis drug (risedronate).
Animals
The present study was conducted on 12 skeletally mature mongrel dogs (Canis lupus familiaris). Before enrollment in the study, each dog was given a complete clinical, physical and radiographic examination to exclude the evidence of systemic, orthopedic, and neurologic disease. Dogs included in the study were with a 15-kg body weight and aged 20 months. The animals were kept under normal laboratory conditions and given free access of food and water. All study procedures were done in accordance to and approved by the “Institutional Animal Care and Use Committee (IUCUC)” of Faculty of Veterinary Medicine- Cairo University.
All dogs were divided into four equal groups (n = 3 each):
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Group 1: CH.PVA with no BG. (NM)
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Group 2: CH.PVA with no BG. (M)
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Group 3: CH.PVA with BG. (NM)
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Group 4: CH.PVA with BG. (M)
Dogs were housed individually in separate cages at the Department of Veterinary Surgery, Anesthesiology & Radiology; Faculty of Veterinary Medicine, Cairo University. Before enrolment in the study, dogs were quarantined for 2 weeks, kennels were sprayed with 6/1000 ml Neocidal diazinone (Diaenone® 60 EC; Ciba-Geigy, Switzerland), dogs were bathed in 1/1000 Neocidal diazinone, and Ivermectine (Ivomec® ; merc, Sharp and Dome, USA) was injected in a dose of 0.1 mg/kg body weight subcutaneously to guard against ecto-parasitic and endoparasitic infestation.
Surgery
After initial preparation, surgery was performed. A straight approximately 5 cm wide incision was performed on the right side of the mandible. Dissection and division were performed by planes up to the periosteum. With the surgical field exposed, monocortical bone cavities were prepared using 5 mm trephine burs 12 mm in diameter under thorough irrigation with saline. A small chisel and mallet were used to facilitate the removal of the cortical disk with great care was taken to avoid injury of the inferior alveolar canal. The surgical procedure was performed unilaterally (Fig. 1a). The defect of the right side was obliterated by the sterile graft particles, the material was mixed with blood (Fig. 1b), and the obliterated bone defect was covered with resorbable tissue guiding membrane (Biocollagen membrane). The operated wound was closed in a routine manner (Fig. 1c).
Radiological measurements
The mandibular defect was radiographed using X-ray potentiality of 45 KV, 15 MA, and 1/10 s.
Laboratory measurements
Blood samples were collected from all animals before and after surgery, weekly intervals for 3 months, using sterile disposable syringes. Part of the blood sample will be left to clot and centrifuged at 3000 rpm for 15 min and then stored at −70 °C until further processing, finally sera were processed for estimation of the following:
Statistical analysis
Data were collected and analyzed by computer program SPSS “version 17” ((The Statistical Package for the Social Science Program), Chicago, USA). All data were expressed as mean ± SD and percentages. Unpaired t test was used to compare a quantitative variable between two independent groups in parametric data. Mann Whitney test was used to compare quantitative variables between two independent groups when data were nonparametric (SD > 25% of mean).
