Andrew R. Padalhin

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.

A hybrid electrospun PU/PCL scaffold satisfied the requirements of blood vessel prosthesis in terms of mechanical properties, pore size, and biocompatibility

In this study, a novel hybrid polyurethane/polycaprolactone (PU/PCL) tubular scaffold was fabricated using the electrospinning process for blood vessel prosthesis applications. The detailed microstructure and material properties such as porosity, tensile and bust strength, contact angle, and biocompatibility were investigated and compared with those of monolithic PU and PCL scaffolds. The mechanical properties of the hybrid PU/PCL scaffold (tensile strength: 18 MPa, pressure strength: 590 mmHg) were found to be within the range needed for artificial blood vessel applications. The pore sizes of the PU/PCL scaffold ranged from 5–150 um in diameter, are sufficient enough to allow nutrient diffusion across the membrane. The reduced hydrophobic property of the PU/PCL scaffold was the result of the addition of relatively less hydrophobic PU compared with monolithic PCL scaffold. The biocompatibility of the PU/PCL scaffold was evaluated through cytotoxicity testing, and morphological observation by scanning electron microscopy and confocal microscopy using cow pulmonary artery endothelial cells and fibroblast like cells (L929).

Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends

In this study, the optimized formulations of polycaprolactone (PCL) combined with poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL), and biphasic calcium phosphate (BCP) were analyzed in terms of cytocompatibility with bone-related cells, hemocompatibility, and in vivo bone-regenerating capacity to determine their potentials for bone tissue regeneration. Fiber morphology of PCL/GEL and PCL/BCP electrospun mats considerably differs from that of the PCL membrane. Based on the contact angle analyses, the addition of GEL and PLGA was shown to reduce the hydrophobicity of these membranes. The assessment of in vitro cytocompatibility using MC3T3-E1 cells indicated that all of the membranes were suitable for pre-osteoblast proliferation and adhesion, with PCL/BCP having a significantly higher reading after seven days of incubation. The results of the in vitro hemocompatibility of the different fibrous scaffolds suggest that coagulation and platelet adhesion were higher for hydrophobic membranes (PCL and PCL/PLGA), while hemolysis can be associated with fiber morphology. The potential of the membranes for bone regeneration was determined by analyzing the microCT data and tissue sections of samples implanted in 5 mm sized defects (one and two months). Although all of the membranes were suitable for pre-osteoblast proliferation, in vivo bone regeneration after two months was found to be significantly higher in PCL/BCP (p < 0.001).

A Study of BMP-2-Loaded Bipotential Electrolytic Complex around a Biphasic Calcium Phosphate-Derived (BCP) Scaffold for Repair of Large Segmental Bone Defect.

A bipotential polyelectrolyte complex with biphasic calcium phosphate (BCP) powder dispersion provides an excellent option for protein adsorption and cell attachment and can facilitate enhanced bone regeneration. Application of the bipotential polyelectrolyte complex embedded in a spongy scaffold for faster healing of large segmental bone defects (LSBD) can be a promising endeavor in tissue engineering application. In the present study, a hollow scaffold suitable for segmental long bone replacement was fabricated by the sponge replica method applying the microwave sintering process. The fabricated scaffold was coated with calcium alginate at the shell surface, and genipin-crosslinked chitosan with biphasic calcium phosphate (BCP) dispersion was loaded at the central hollow core. The chitosan core was subsequently loaded with BMP-2. The electrolytic complex was characterized using SEM, porosity measurement, FTIR spectroscopy and BMP-2 release for 30 days. In vitro studies such as MTT, live/dead, cell proliferation and cell differentiation were performed. The scaffold was implanted into a 12 mm critical size defect of a rabbit radius. The efficacy of this complex is evaluated through an in vivo study, one and two month post implantation. BV/TV ratio for BMP-2 loaded sample was (42±1.76) higher compared with hollow BCP scaffold (32±0.225).

Preformed chitosan cryogel-biphasic calcium phosphate: a potential injectable biocomposite for pathologic fracture

The increasing interest in chitosan-based biomaterials stems from its desirable physicochemical properties. Although calcium phosphates have been mixed with chitosan to form injectable scaffolds, its application for bone tissue engineering has been limited and is still being explored to improve its clinical translatability. We report a biocomposite comprised of preformed chitosan cryogel with dispersed biphasic calcium phosphate that can flow under moderate pressure allowing passage through a small gauge needle, while maintaining sufficient integrity and strength during injection for gel recovery. The formed samples were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction analysis and protein adsorption measurements. Composite with 1% w/v biphasic calcium phosphate (CSG1) resulted in a homogeneous and rigid final structure. Injectable composite cryogel CSG1 (2.5 ± 0.2 N, 23G needle) exhibited good protein adsorption and biocompatibility. Results of subcutaneous implantation in rats reveal relatively high presence of polymorphonuclear cells but with no fibrous encapsulation with the composites, allowing further infiltration of cells within the sample implants. The biocomposite system presents a less-invasive delivery of bone filling material for stabilizing pathologic fractures.

Phosphonate-chitosan functionalization of a multi-channel hydroxyapatite scaffold for interfacial implant-bone tissue integration

Nonunion associated with long bone defects continues to be highly researched both experimentally and clinically. A porous hydroxyapatite (HAp) scaffold has been recognized as a bone repair and substitute material clinically, but its use in segmental bone defects has been limited by poor integration and stability, as a consequence of scaffold strength unmatched with the native bone. Herein, we designed a multi-channel HAp-based scaffold for application in segmental bone defects, with a specific geometry and design. It possesses the required porosity for bone tissue regeneration with sufficient mechanical properties. We also developed a surface functionalization/modification method with the goal of early scaffold integration and stability. Initial functionalization with poly(vinyl phosphonic acid), PVPA, allowed simple attachment of a chitosan polymeric layer. The modification improved the biocompatibility of the scaffold and attachment of rat bone marrow-derived mesenchymal stem cells (rBMSC) in vitro. The modification also served as a buffer between the implant scaffold and bone tissue. Significant improvement in the integration behavior with better interlocking of the scaffold to bone tissue was observed for the modified scaffolds implanted in rabbit tibiae. The modified HAp scaffolds exhibited early interfacial implant-bone tissue integration with enhanced new bone formation and high potential for use in segmental bone defects.

A hybrid composite system of biphasic calcium phosphate granules loaded with hyaluronic acid–gelatin hydrogel for bone regeneration

An ideal bone substitute should be made of biocompatible materials that mimic the structure, characteristics, and functions of natural bone. Many researchers have worked on the fabrication of different bone scaffold systems including ceramic–polymer hybrid system. In the present study, we incorporated hyaluronic acid–gelatin hydrogel to micro-channeled biphasic calcium phosphate granules as a carrier to improve cell attachment and proliferation through highly interconnected porous structure. This hybrid system is composed of ceramic biphasic calcium phosphate granules measuring 1 mm in diameter with seven holes and hyaluronic acid–gelatin hydrogel. This combination of biphasic calcium phosphate and hyaluronic acid–gelatin retained suitable characteristics for bone regeneration. The resulting scaffold had a porosity of 56% with a suitable pore sizes. The mechanical strength of biphasic calcium phosphate granule increased after loading hyaluronic acid–gelatin from 4.26 ± 0.43 to 6.57 ± 0.25 MPa, which is highly recommended for cancellous bone substitution. Swelling and degradation rates decreased in the hybrid scaffold compared to hydrogel due to the presence of granules in hybrid scaffold. In vitro cytocompatibility studies were observed by preosteoblasts (MC3T3-E1) cell line and the result revealed that biphasic calcium phosphate/hyaluronic acid–gelatin significantly increased cell growth and proliferation compared to biphasic calcium phosphate granules. Analysis of micro-computed tomography data and stained tissue sections from the implanted samples showed that the hybrid scaffold had good osseointegration and better bone formation in the scaffold one and two months postimplantation. Histological section confirmed the formation of dense collagenous tissue and new bone in biphasic calcium phosphate/hyaluronic acid–gelatin scaffolds at two months. Our study demonstrated that such hybrid biphasic calcium phosphate/hyaluronic acid–gelatin scaffold is a promising system for bone regeneration.

Bone Regeneration Using Hydroxyapatite Sponge Scaffolds with In Vivo Deposited Extracellular Matrix.

There is currently an increased interest in studying the extracellular matrix (ECM) and its potential applications for tissue engineering and regenerative medicine. The ECM plays an important role by providing adhesive substrates to cells during migration, morphogenesis, differentiation, and homeostasis by signaling biochemical and biomechanical cues to cells. In this study, the ECM was incorporated into hydroxyapatite by implanting sponge replica scaffolds in subcutaneous pockets in rats, and the implants were tested for bone regeneration potential. The resulting scaffolds were characterized using scanning electron microscopy, confocal microscopy, DNA and RNA quantification, tissue staining, energy dispersive X-ray spectroscopy analysis, compressive strength testing, porosity, and pore size distribution analysis using bare scaffolds as a control reference. Biocompatibility was assessed using MC3T3-E1 preosteoblast cells and in vivo studies were carried out by implanting decellularized scaffolds in 11 mm radial defects in New Zealand rabbits for 4 and 8 weeks to determine the effect of the in vivo deposited ECM. Material characterization indicated that a 2-week decellularized scaffold was the best among the samples, with an evenly distributed ECM visible on hematoxylin and eosin-stained tissue sections, a compressive strength of 2.53 ± 0.68 MPa, a porosity of 58.08 ± 3.32% and a pore size distribution range of 10-150 μm. In vivo results showed no severe inflammation, with increased cell infiltration followed by dense matrix deposition after 4 weeks and new bone formation at 8 weeks. The results indicate that incorporation of an in vivo deposited ECM into ceramic scaffolds can potentially improve bone regeneration.

Evaluation of egg white ovomucin-based porous scaffold as an implantable biomaterial for tissue engineering

Studies have shown the technological and functional properties of ovomucin (OVN) in the food-agricultural industry. But research has yet to explore its potential as an implantable biomaterial for tissue engineering and regenerative medicine. In this study we isolated OVN from egg white by isoelectric precipitation and fabricated scaffolds with tunable porosity by utilizing its foaming property. Gelatin a known biocompatible material was introduced to stabilize the foams, wherein different ratios of OVN and gelatin had a significant effect on the degree of porosity, pore size and stability of the formed hydrogels. The porous scaffolds were crosslinked with EDC resulting in stable scaffolds with prolonged degradation. Improved cell proliferation and adhesion of rat bone marrow-derived mesenchymal stem cells were observed for OVN containing scaffolds. Although, scaffolds with 75% OVN showed decrease in cell proliferation for L929 fibroblast type of cells. Further biocompatibility assessment as implant material was determined by subcutaneous implantation in rats of selected scaffold. H&E staining showed reasonable vascularization over time and little evidence of severe fibrosis at the implant site. Persistent polarization of classically activated macrophage was not observed, potentially reducing inflammatory response, and showed increased expression of alternatively activated macrophage cells that is favorable for tissue repair. Analysis of IgE levels in rat serum after implantation indicated minimal and resolvable allergic response to the OVN implants. The results demonstrate OVN as an acceptable implant scaffold that could provide new opportunities as an alternative natural biocompatible and functional biomaterial in various biomedical applications.

Bilayer electrospun poly(vinyl alcohol)–gelatin mat and biphasic calcium phosphate–pectin–gelatin hydrogel for application in bone hemorrhage:

A bilayer composite scaffold consisting of a biphasic calcium phosphate–pectin–gelatin hydrogel and an electrospun poly(vinyl alcohol)–gelatin matrix was developed and investigated for application in bone hemorrhage. The aim of this research is to develop a new biomaterial system that provides a hemostatic effect on bone hemorrhage and does not interfere with native bone regeneration. The role of electrospun poly(vinyl alcohol)–gelatin in scaffolds is to provide the covering of the wound, while encapsulation and absorption of red blood cells are attributed to the biphasic calcium phosphate–pectin–gelatin hydrogel. Cell viability and cell proliferation were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Live/Dead assay, and immunofluorescence assay using pre-osteoblast MC3T3-E1 cells. The effects with and without hemostatic bilayer scaffolds on rat calvaria were compared at 1 and 3 weeks. The hemostatic bilayer agent showed good blood absorption behavior and efficient bone healing properties after 3 weeks, as observed by micro-computed tomography and hematoxylin and eosin staining.