Effect of Local Sustainable Release of BMP2-VEGF from Nano-Cellulose Loaded in Sponge Biphasic Calcium Phosphate on Bone Regeneration.

Bone regeneration is a coordinated process mainly regulated by multiple growth factors. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and bone morphogenetic proteins (BMPs) induce osteogenesis during bone healing process. The aim of this study was to investigate how these growth factors released locally and sustainably from nano-cellulose (NC) simultaneously effect bone formation. A biphasic calcium phosphate (BCP)-NC-BMP2-VEGF (BNBV) scaffold was fabricated for this purpose. The sponge BCP scaffold was prepared by replica method and then loaded with 0.5% NC containing BMP2-VEGF. Growth factors were released from NC in a sustainable manner from 1 to 30 days. BNBV scaffolds showed higher cell attachment and proliferation behavior than the other scaffolds loaded with single growth factors. Bare BCP scaffolds and BNBV scaffolds seeded with rat bone marrow mesenchymal stem cells were implanted ectopically and orthotopically in nude mice for 4 weeks. No typical bone formation was exhibited in BNBV scaffolds in ectopic sites. BMP2 and VEGF showed positive effects on new bone formation in BNBV scaffolds, with and without seeded stem cells, in the orthotopic defects. This study demonstrated that the BNBV scaffold could be beneficial for improved bone regeneration. Stem cell incorporation into this scaffold could further enhance the bone healing process.

Nanoparticle biphasic calcium phosphate loading on gelatin-pectin scaffold for improved bone regeneration.

Biphasic calcium phosphate (BCP) nanoparticles were loaded with porous gelatin-pectin (GE-P) scaffolds. The biodegradable gelatin-pectin-BCP scaffolds were produced as miscible mixtures with well-defined interconnected pores to facilitate osteoconductivity and enhance bone formation. It was observed that the compressive strength increased with the loading of BCP nanoparticles. From in vitro results, cell adhesion, viability, and proliferation were found in the GE-P-10 scaffolds in comparison with those without BCP, resulting in high alkaline phosphate (ALP), and osteopontin (OPN) expression at 21 days. Micro-computed tomography data, hematoxylin and eosin staining, and immunohistochemistry (OPN, OCN, COL I, and COL II) confirmed rapid new bone formation in rabbit models. Our results provide a novel and simple method to provide an adequate scaffold, and thus GE-P-BCP porous scaffolds may be appropriate candidates for bone tissue engineering.

Platelet-rich plasma encapsulation in hyaluronic acid/gelatin-BCP hydrogel for growth factor delivery in BCP sponge scaffold for bone regeneration.

Microporous calcium phosphate based synthetic bone substitutes are used for bone defect healing. Different growth factor loading has been investigated for enhanced bone regeneration. The platelet is a cellular component of blood which naturally contains a pool of necessary growth factors that mediate initiation, continuation, and completion of cellular mechanism of healing. In this work, we have investigated the encapsulation and immobilization of platelet-rich plasma (PRP) with natural polymers like hyaluronic acid (HA) and gelatin (Gel) and loading them in a biphasic calcium phosphate (BCP) scaffold, for a synthetic-allologous hybrid scaffold. Effect of PRP addition in small doses was evaluated for osteogenic potential in vitro and in vivo. BCP (10%) mixed HA-Gel hydrogel with or without PRP, was loaded into a BCP sponge scaffold. We investigated the hydrogel-induced improvement in mechanical property and PRP-mediated enhancement in biocompatibility. In vitro studies for cytotoxicity, cell attachment, and proliferation were carried out using MC3T3-E1 pre-osteoblast cells. In in vitro studies, the cell count, cell proliferation, and cell survival were higher in the scaffold with PRP loading than without PRP. However, in the in vivo studies using a rat model, the PRP scaffold was not superior to the scaffold without PRP. This discrepancy was investigated in terms of the interaction of PRP in the in vivo environment.

In vitro and in vivo studies of BMP-2-loaded PCL-gelatin-BCP electrospun scaffolds.

To confirm the effect of recombinant human bone morphogenetic protein-2 (BMP-2) for bone regeneration, BMP-2-loaded polycaprolactone (PCL)-gelatin (Gel)-biphasic calcium phosphate (BCP) fibrous scaffolds were fabricated using the electrospinning method. The electrospinning process to incorporate BCP nanoparticles into the PCL-Gel scaffolds yielded an extracellular matrix-like microstructure that was a hybrid system composed of nano- and micro-sized fibers. BMP-2 was homogeneously loaded on the PCL-Gel-BCP scaffolds for enhanced induction of bone growth. BMP-2 was initially released at high levels, and then showed sustained release behavior for 31 days. Compared with the PCL-Gel-BCP scaffold, the BMP-2-loaded PCL-Gel-BCP scaffold showed improved cell proliferation and cell adhesion behavior. Both scaffold types were implanted in rat skull defects for 4 and 8 weeks to evaluate the biological response under physiological conditions. Remarkable bone regeneration was observed in the BMP-2/PCL-Gel-BCP group. These results suggest that BMP-2-loaded PCL-Gel-BCP scaffolds should be considered for potential bone tissue engineering applications.

A combination of biphasic calcium phosphate scaffold with hyaluronic acid-gelatin hydrogel as a new tool for bone regeneration.

A novel bone substitute was fabricated to enhance bone healing by combining ceramic and polymer materials. In this study, Hyaluronic acid (HyA)-Gelatin (Gel) hydrogel was loaded into a biphasic calcium phosphate (BCP) ceramic, and the resulting scaffold, with unique micro- and macroporous orientation, was evaluated for bone regeneration applications. The fabricated scaffold showed high interconnected porosity, with an average compressive strength of 2.8±0.15 MPa, which is usually recommended for cancellous bone substitution. In vitro cytocompatibility studies were conducted using bone marrow mesenchymal stem cells. The HyA-Gel-loaded BCP scaffold resulted in a significant increase in cell proliferation at 3 (p<0.05) and 7 days (p<0.001) and high alkaline phosphatase activities at 14 and 21 days. Furthermore, the in vivo studies showed that the implanted HyA-Gel-loaded BCP scaffold begins to degrade within 3 months after implantation. Histological sections also confirmed a rapid new bone formation and a high rate of collagen mineralization. A bone matrix formation was confirmed by positive immunohistochemistry staining of osteopontin, osteocalcin, and collagen type I. In vivo expression of extracellular matrix proteins demonstrated that this novel bone substitute holds great promise for use in stimulating new bone regeneration.