In Vivo Investigation of Polymer-Ceramic PCL/HA and PCL/β-TCP 3D Composite Scaffolds and Electrical Stimulation for Bone Regeneration

Critical bone defects are a major clinical challenge in reconstructive bone surgery. Polycaprolactone (PCL) mixed with bioceramics, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), create composite scaffolds with improved biological recognition and bioactivity. Electrical stimulation (ES) aims to compensate the compromised endogenous electrical signals and to stimulate cell proliferation and differentiation. We investigated the effects of composite scaffolds (PCL with HA; and PCL with β-TCP) and the use of ES on critical bone defects in Wistar rats using eight experimental groups: untreated, ES, PCL, PCL/ES, HA, HA/ES, TCP, and TCP/ES. The investigation was based on histomorphometry, immunohistochemistry, and gene expression analysis. The vascular area was greater in the HA/ES group on days 30 and 60. Tissue mineralization was greater in the HA, HA/ES, and TCP groups at day 30, and TCP/ES at day 60. Bmp-2 gene expression was higher in the HA, TCP, and TCP/ES groups at day 30, and in the TCP/ES and PCL/ES groups at day 60. Runx-2, Osterix, and Osteopontin gene expression were also higher in the TCP/ES group at day 60. These results suggest that scaffolds printed with PCL and TCP, when paired with electrical therapy application, improve bone regeneration.

Effect of a Decellularized Omentum Scaffold with Combination of Mesenchymal Stem Cells and Platelet-Rich Plasma on Healing of Critical-Sized Bone Defect: A Rat Model

The high costs and extensive time needed for the treatment of critical-sized bone defects are still major clinical concerns in orthopedic surgery; therefore, researchers continue to look for more cost and time-effective methods. This study aims to investigate the effects of a decellularized omentum scaffold with a combination of platelet-rich plasma (PRP) and mesenchymal stem cells on the healing of critical-sized bone defects. Wistar albino rats (n = 30) were investigated in five groups. Critical-sized bone defects were formed on bilateral radius shafts. No scaffold, decellularized omentum, omentum with PRP and omentum + mesenchymal stem cells was used in group 1 (control group), 2, 3 and 4, respectively. In addition, omentum with a combination of mesenchymal stem cells +PRP was used in group 5. After 6 weeks, both radiological and histological healing were evaluated comparatively among the groups. After the use of a decellularized omentum scaffold, vitality of new cells was maintained, and new bone formation occurred. When compared to the control group, radiological healing was significantly better (p = 0.047) in the omentum and omentum + PRP-treated groups. Furthermore, histological healing was better in the omentum and omentum + PRP-treated groups than the control group (p = 0.001). The use of a decellularized omentum scaffold is suitable in the healing of critical bone defects.

Dose-Dependent Effects of a Novel Selective EP4 Prostaglandin Receptor Agonist on Treatment of Critical Size Femoral Bone Defects in a Rat Model

Difficulties in treating pseudarthrosis and critical bone defects are still evident in physicians’ clinical routines. Bone morphogenetic protein 2 (BMP-2) has shown promising osteoinductive results but also considerable side effects, not unexpected given that it is a morphogen. Thus, the bone regenerative potential of the novel selective, non-morphogenic EP4 prostaglandin receptor agonist KMN-159 was investigated in this study. Therefore, mineralized collagen type-1 matrices were loaded with different amounts of BMP-2 or KMN-159 and implanted into a 5 mm critical-sized femoral defect in rats. After 12 weeks of observation, micro-computed tomography scans were performed to analyze the newly formed bone volume (BV) and bone mineral density (BMD). Histological analysis was performed to evaluate the degree of defect healing and the number of vessels, osteoclasts, and osteoblasts. Data were evaluated using Kruskal-Wallis followed by Dunn’s post hoc test. As expected, animals treated with BMP-2, the positive control for this model, showed a high amount of newly formed BV as well as bone healing. For KMN-159, a dose-dependent effect on bone regeneration could be observed up to a dose optimum, demonstrating that this non-morphogenic mechanism of action can stimulate bone formation in this model system.

Biophysical Stimuli as the Fourth Pillar of Bone Tissue Engineering

The repair of critical bone defects remains challenging worldwide. Three canonical pillars (biomaterial scaffolds, bioactive molecules, and stem cells) of bone tissue engineering have been widely used for bone regeneration in separate or combined strategies, but the delivery of bioactive molecules has several obvious drawbacks. Biophysical stimuli have great potential to become the fourth pillar of bone tissue engineering, which can be categorized into three groups depending on their physical properties: internal structural stimuli, external mechanical stimuli, and electromagnetic stimuli. In this review, distinctive biophysical stimuli coupled with their osteoinductive windows or parameters are initially presented to induce the osteogenesis of mesenchymal stem cells (MSCs). Then, osteoinductive mechanisms of biophysical transduction (a combination of mechanotransduction and electrocoupling) are reviewed to direct the osteogenic differentiation of MSCs. These mechanisms include biophysical sensing, transmission, and regulation. Furthermore, distinctive application strategies of biophysical stimuli are presented for bone tissue engineering, including predesigned biomaterials, tissue-engineered bone grafts, and postoperative biophysical stimuli loading strategies. Finally, ongoing challenges and future perspectives are discussed.

3D-printed Poly-Lactic Co-Glycolic Acid (PLGA) scaffolds in non-critical bone defects impede bone regeneration in rabbit tibia bone

BACKGROUND: An increasing number of bone graft materials are commercially available and vary in their composition, mechanism of action, costs, and indications. OBJECTIVE: A commercially available PLGA scaffold produced using 3D printing technology has been used to promote the preservation of the alveolar socket after tooth extraction. We examined its influence on bone regeneration in long bones of New Zealand White rabbits. METHODS: 5.0-mm-diameter circular defects were created on the tibia bones of eight rabbits. Two groups were studied: (1) control group, in which the bone defects were left empty; (2) scaffold group, in which the PLGA scaffolds were implanted into the bone defect. Radiography was performed every two weeks postoperatively. After sacrifice, bone specimens were isolated and examined by micro-computed tomography and histology. RESULTS: Scaffolds were not degraded by eight weeks after surgery. Micro-computed tomography and histology showed that in the region of bone defects that was occupied by scaffolds, bone regeneration was compromised and the total bone volume/total volume ratio (BV/TV) was significantly lower. CONCLUSION: The implantation of this scaffold impedes bone regeneration in a non-critical bone defect. Implantation of bone scaffolds, if unnecessary, lead to a slower rate of fracture healing.

Calcium-Silicate-Incorporated Gellan-Chitosan Induced Osteogenic Differentiation in Mesenchymal Stromal Cells

Gellan-chitosan (GC) incorporated with CS: 0% (GC-0 CS), 10% (GC-10 CS), 20% (GC-20 CS) or 40% (GC-40 CS) w/w was prepared using freeze-drying method to investigate its physicochemical, biocompatible, and osteoinductive properties in human bone-marrow mesenchymal stromal cells (hBMSCs). The composition of different groups was reflected in physicochemical analyses performed using BET, FTIR, and XRD. The SEM micrographs revealed excellent hBMSCs attachment in GC-40 CS. The Alamar Blue assay indicated an increased proliferation and viability of seeded hBMSCs in all groups on day 21 as compared with day 0. The hBMSCs seeded in GC-40 CS indicated osteogenic differentiation based on an amplified alkaline-phosphatase release on day 7 and 14 as compared with day 0. These cells supported bone mineralization on GC-40 CS based on Alizarin-Red assay on day 21 as compared with day 7 and increased their osteogenic gene expression (RUNX2, ALP, BGLAP, BMP, and Osteonectin) on day 21. The GC-40 CS–seeded hBMSCs initiated their osteogenic differentiation on day 7 as compared with counterparts based on an increased expression of type-1 collagen and BMP2 in immunocytochemistry analysis. In conclusion, the incorporation of 40% (w/w) calcium silicate in gellan-chitosan showed osteoinduction potential in hBMSCs, making it a potential biomaterial to treat critical bone defects.

Effect of a Decellularized Omentum Scaffold With Combination of Mesenchymal Stem Cells and Platelet Rich Plasma on Healing of Critical-sized Bone Defect

Background: High-cost and the long time needed for the treatment of critical-sized bone defects is still major clinical concern in orthopedic surgery, therefore new research continues to identify more cost and time-effective methods. In this study, we aimed to investigate the effect of a decellularized omentum scaffold with a combination of platelet-rich plasma (PRP) and mesenchymal stem cells on the healing of critical- sized bone defects. Materlials and methods: Wistar albino rats (n­=30) were investigated in five groups. Critical-sized bone defects were formed on bilateral radius shafts. To increase healing, no scaffold, decellularized omentum, omentum with with PRP and omentum mesenchymal stem cells was used in group 1 (control group) , 2, 3 and 4 respectively. In addition omentum with a combination of mesenchymal stem cells and PRP was used in group 5. After 6 weeks, both radiological and histological healing were evaluated comparatively among groups. Results: After use of decellularized omentum scaffold, vitality of new cells maintained, new bone formation occurred. When compared to control group, radiological healing was statistically significantly better in omentum and omentum with PRP-treated groups (p=0.047). Furthermore histological healing was better in the omentum and omentum with PRP-treated groups than both control and omentum with mesenchymal stem cell-treated groups according to Salkeld Scoring System (p=0.001).Conclusion: The use of decellularized omentum scaffold is suitable in the healing of critical bone defects.

Analysis of the behavior of a biomaterial based on wollastonite/TCP in the implant process of an experimental model of critical bone defects

This study analyzes the clinical, macroscopic and radiographic characteristics of a biomaterial with different proportions of wolastonite (W) and tricalcium phosphate (TCP) on bone tissue regeneration during the implantation process of an experimental model of critical bone defects. Fifteen Wistar rats were used, randomly distributed in 5 groups (n = 3), with a bone defect created on an 8.0 mm diameter calvaria. 4 groups received implants with different proportions of W%/TCP%, referred to as W20/TCP80, W40/TCP60, W60/TCP40 and W80/TCP20. The fifth control group (GC) was filled with blood clot only. Clinical evaluation was performed every 24 hours, and after 7 days, the animals were euthanized. The calvaria were dissected and analyzed macroscopically and by radiography. All study groups showed a satisfactory clinical evolution. The macroscopic analysis showed filling of the bone defect with granules surrounded by newly formed tissue, and the radiographic analysis showed different patterns of displacement of the biomaterial. The study concluded that the different proportions of W%/TCP% were well tolerated by the study groups and demonstrated biocompatibility. The enhanced hydrophilic behavior of the W40/TCP60, W60/TCP40 and W80/TCP20 groups favored the application in the experimental model in vivo.

Risk factors of ankle osteoarthritis in the treatment of critical bone defects using ilizarov technique

Background Distraction osteogenesis using the Ilizarov external circular fixator has been applied in lower limb reconstructive surgery widely. The increasing ankle osteoarthritis (OA) progression and severity are often associated with the period of external fixator and the greater relative instability of the ankle joint, but few studies have quantified risk factors directly during this technique. Methods The study was conducted on 236 patients who underwent bone transport surgery for tibias using the Ilizarov external circular fixator from 2008 to 2018. The cumulative incidence of ankle OA diagnoses in patients after the Ilizarov technique treatment was calculated and stratified by risk factors from preoperative and postoperative management. After the data were significant through the Mann-Whitney U test analyzed, odds ratios were calculated using logistic regression to describe factors associated with the OA diagnosis including gender, age, BMI, location of bone defect, diabetes, hypertension, osteoporosis, the history of metal allergy and glucocorticoid intake, the American Orthopaedic Foot & Ankle Society (AOFAS) ankle-HF scale scores, defect size (DS), the type of bone transport, the bone union time, external fixator time (EFT), and external fixator index (EFI). Results There were 199 males and 37 females with a mean age of 47 years (range 28–59 years). Out of 236 patients, 49 had an additional treatment for ankle OA after the Ilizarov technique treatment of bone defects (average follow-up time 2.1 years, range 1.6–4.2 years). The incidence of postoperative ankle OA was 20.8 %, with 19 patients classified as K&L grade 3 and seven patients as grade 4. The top five risk factors included double-level bone transport (OR3.79, P = 0.005), EFI > 50days/cm (OR3.17, P = 0.015), age > 45years (OR2.29, P = 0.032), osteoporosis (OR1.58, P < 0.001), BMI > 25 (OR1.34, P < 0.001). Male, BMI > 25, diabetes, osteoporosis, and AOFAS ankle-HF scale scores are the independent risk factors. Conclusions Ilizarov external circular fixator is a safe and effective method of treatment for critical bone defects. The double level bone transport, EFI > 50days/cm, age > 45years, osteoporosis, BMI > 25 are the top five relevant risk factors of ankle OA. The probability of developing ankle OA among patients having three or more risk factors is 50–70 %.

Evaluation of Guided Bone Regeneration in Critical Defects Using Bovine and Porcine Collagen Membranes: Histomorphometric and Immunohistochemical Analyses

Guided bone regeneration (GBR) is a technique used to facilitate bone regeneration, which uses a biocompatible membrane acting as a physical barrier to prevent the adjacent connective tissue from invading the bone defect. The aim of this study was to evaluate and compare the effectiveness of bovine and porcine collagenous membranes as barriers to connective tissue invasion during the repair of critical bone defects in rat calvaria, using histological, histometric, and immunohistochemical analyses. For this study, 72 rats were divided into three groups: clot group (CG), bovine collagen group (BCG), and porcine collagen group (PCG). Analyses were performed on days 7, 15, 30, and 60. The histological results showed that the PCG exhibited bone neoformation starting from day 7, and after 30 days of repair, the surgical defect was completely filled in some animals. For the BCG, there was little bone neoformation activity in the initial periods, and from day 30 onwards, there was an increase in bone neoformation, with a greater increase on day 60. The data obtained in the histometric analysis reveal that, on day 30, the neoformed bone area did not vary greatly between the PCG and the BCG, though both varied from the CG. By day 60, the PCG presented a greater area of neoformation than the BCG. These results were corroborated by the immunohistochemistry results. In view of the results obtained, it can be concluded that all membranes studied in this research promoted GBR.