Microwave sintering and in vitro study of defect-free stable porous multilayered HAp-ZrO2 artificial bone scaffold.

Continuously porous hydroxyapatite (HAp)/t-ZrO2 composites containing concentric laminated frames and microchanneled bodies were fabricated by an extrusion process. To investigate the mechanical properties of HAp/t-ZrO2 composites, the porous composites were sintered at different temperatures using a microwave furnace. The microstructure was designed to imitate that of natural bone, particularly small bone, with both cortical and spongy bone sections. Each microchannel was separated by alternating lamina of HAp, HAp-(t-ZrO2) and t-ZrO2. HAp and ZrO2 phases existed on the surface of the microchannel and the core zone to increase the biocompatibility and mechanical properties of the HAp-ZrO2 artificial bone. The sintering behavior was evaluated and the optimum sintering temperature was found to be 1400 °C, which produced a stable scaffold. The material characteristics, such as the microstructure, crystal structure and compressive strength, were evaluated in detail for different sintering temperatures. A detailed in vitro study was carried out using MTT assay, western blot analysis, gene expression by polymerase chain reaction and laser confocal image analysis of cell proliferation. The results confirmed that HAp-ZrO2 performs as an artificial bone, showing excellent cell growth, attachment and proliferation behavior using osteoblast-like MG63 cells.

Bone Regeneration by Multichannel Cylindrical Granular Bone Substitute for Regeneration of Bone in Cases of Tumor, Fracture, and Arthroplasty.

In orthopedics, a number of synthetic bone substitutes are being used for the repair and regeneration of damaged or diseased bone. The nature of the bone substitutes determines the clinical outcome and its application for a range of orthopedic clinical conditions. In this study, we aimed to demonstrate the possible applications of multichannel granular bone substitutes in different types of orthopedic clinical conditions, including bone tumor, fracture, and bone defect with arthroplasty. A clinical investigation on a single patient for every specific type of disease was performed, and patient outcome was evaluated by physical and radiographic observation. Brief physical characterization of the granular bone substitute and in vivo animal model investigation were presented for a comprehensive understanding of the physical characteristics of the granules and of the performance of the bone substitute in a physiological environment, respectively. In all cases, the bone substitute stabilized the bone defect without any complications, and the defect regenerated slowly during the postoperative period. Gradual filling of the defect with the newly regenerated bone was confirmed by radiographic findings, and no adverse effects, such as osteolysis, graft dispersion, and non-union, were observed. Homogeneous bone formation was observed throughout the defect area, showing a three-dimensional bone regeneration. High-strength multichannel granules could be employed as versatile bone substitutes for the treatment of a wide range of orthopedic conditions.

Initial biocompatibility and enhanced osteoblast response of Si doping in a porous BCP bone graft substitute.

Granular shape biphasic calcium phosphate (BCP) bone grafts with and without doping of silicon cations were evaluated in regards to biocompatibility and MG-63 cellular response. To do this we studied Cellular cytotoxicity, cellular adhesion and spreading behavior and cellular differentiation with alizarin red S staining. Gene expression in MG-63 cells on the implanted bone substitutes was also examined at different time points using RT-PCR. In comparison, the Si-doped BCP granule showed more cellular viability than the BCP granule without doping in MTT assay. Moreover, cell proliferation was much higher when Si doping was employed. The cells grown on the silicon-doped BCP substitutes had more active filopodial growth with cytoplasmic webbing that proceeded to the flattening stage, which was indicative of well cellular adhesion. When these cells were exposed to Si-doped BCP granules for 14 days, well differentiated MG-63 cells were observed. Osteonectin and osteopontin genes were highly expressed in the late stage of differentiation (14 days), whereas collagen type I mRNA were found to be highly expressed during the early stage (day 3). These combined results of this study demonstrate that silicon-doped BCP enhanced osteoblast attachment/spreading, proliferation, differentiation and gene expression.

In vitro and in vivo assessment of biomedical Mg-Ca alloys for bone implant applications.

BACKGROUND: Magnesium (Mg)-based alloys are considered to be promising materials for implant application due to their excellent biocompatibility, biodegradability, and mechanical properties close to bone. However, low corrosion resistance and fast degradation are limiting their application. Mg-Ca alloys have huge potential owing to a similar density to bone, good corrosion resistance, and as Mg is essential for Ca incorporation into bone. The objective of the present work is to determine the in vitro degradation and in vivo performance of binary Mg- xCa alloy ( x = 0.5 or 5.0 wt%) to assess its usability for degradable implant applications. METHODS: Microstructural evolutions for Mg- xCa alloys were characterized by optical, SEM, EDX, and XRD. In vitro degradation tests were conducted via immersion test in phosphate buffer saline solution. In vivo performance in terms of interface, biocompatibility, and biodegradability of Mg- xCa alloys was examined by implanting samples into rabbit femoral condyle for 2 and 4 weeks. RESULTS: Microstructural results showed the enhancement in intermetallic Mg2Ca phase with increase in Ca content. Immersion tests revealed that the dissolution rate varies linearly, with Ca content exhibiting more hydrogen gas evolution, increased pH, and higher degradation for Mg-5.0Ca alloy. In vivo studies showed good biocompatibility with enhanced bone formation for Mg-0.5Ca after 4 weeks of implantation compared with Mg-5.0Ca alloy. Higher initial corrosion rate with prolonged inflammation and rapid degradation was noticed in Mg-5.0Ca compared with Mg-0.5Ca alloy. CONCLUSIONS: The results suggest that Mg-0.5Ca alloy could be used as a temporary biodegradable implant material for clinical applications owing to its controlled in vivo degradation, reduced inflammation, and high bone-formation capability.

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).

Brushite-based calcium phosphate cement with multichannel hydroxyapatite granule loading for improved bone regeneration.

In this work, we report brushite-based calcium phosphate cement (CPC) system to enhance the in vivo biodegradation and tissue in-growth by incorporation of micro-channeled hydroxyapatite (HAp) granule and silicon and sodium addition in calcium phosphate precursor powder. Sodium- and silicon-rich calcium phosphate powder with predominantly tri calcium phosphate (TCP) phase was synthesized by an inexpensive wet chemical route to react with mono calcium phosphate monohydrate (MCPM) for making the CPC. TCP nanopowder also served as a packing filler and moderator of the reaction kinetics of the setting mechanism. Strong sintered cylindrical HAp granules were prepared by fibrous monolithic (FM) process, which is 800 µm in diameter and have seven micro-channels. Acid sodium pyrophosphate and sodium citrate solution was used as the liquid component which acted as a homogenizer and setting time retarder. The granules accelerated the degradation of the brushite cement matrix as well as improved the bone tissue in-growth by permitting an easy access to the interior of the CPC through the micro-channels. The addition of micro-channeled granule in the CPC introduced porosity without sacrificing much of its compressive strength. In vivo investigation by creating a critical size defect in the femur head of a rabbit model for 1 and 2 months showed excellent bone in-growth through the micro-channels. The granules enhanced the implant degradation behavior and bone regeneration in the implanted area was significantly improved after two months of implantation.

Hard tissue regeneration using bone substitutes: an update on innovations in materials.

Bone is a unique organ composed of mineralized hard tissue, unlike any other body part. The unique manner in which bone can constantly undergo self-remodeling has created interesting clinical approaches to the healing of damaged bone. Healing of large bone defects is achieved using implant materials that gradually integrate with the body after healing is completed. Such strategies require a multidisciplinary approach by material scientists, biological scientists, and clinicians. Development of materials for bone healing and exploration of the interactions thereof with the body are active research areas. In this review, we explore ongoing developments in the creation of materials for regenerating hard tissues.

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.

Bioactive glass incorporation in calcium phosphate cement-based injectable bone substitute for improved in vitro biocompatibility and in vivo bone regeneration.

In this work, we fabricated injectable bone substitutes modified with the addition of bioactive glass powders synthesized via ultrasonic energy-assisted hydrothermal method to the calcium phosphate-based bone cement to improve its biocompatibility. The injectable bone substitutes was initially composed of a powder component (tetracalcium phosphate, dicalcium phosphate dihydrate and calcium sulfate dehydrate) and a liquid component (citric acid, chitosan and hydroxyl-propyl-methyl-cellulose) upon which various concentrations of bioactive glass were added: 0%, 10%, 20% and 30%. Setting time and compressive strength of the injectable bone substitutes were evaluated and observed to improve with the increase of bioactive glass content. Surface morphologies were observed via scanning electron microscope before and after submersion of the samples to simulated body fluid and increase in apatite formation was detected using x-ray diffraction machine. In vitro biocompatibility of the injectable bone substitutes was observed to improve with the addition of bioactive glass as the proliferation/adhesion behavior of cells on the material increased. Human gene markers were successfully expressed using real time-polymerase chain reaction and the samples were found to promote cell viability and be more biocompatible as the concentration of bioactive glass increases. In vivo biocompatibility of the samples containing 0% and 30% bioactive glass were evaluated using Micro-CT and histological staining after 3 months of implantation in male rabbits' femurs. No inflammatory reaction was observed and significant bone formation was promoted by the addition of bioactive glass to the injectable bone substitute system.

The effects of dimethyl 3,3'-dithiobispropionimidate di-hydrochloride cross-linking of collagen and gelatin coating on porous spherical biphasic calcium phosphate granules.

Collagen- and gelatin-coated porous spherical granule was prepared by slurry dripping process using biphasic calcium phosphate powder. The coating was stabilized by cross-linking with dimethyl 3,3'-dithiobispropionimidate di-hydrogenchloride (DTBP). Afer DTBP cross-linking, the nanostructure of collagen- and gelatin-coated surfaces was changed from smooth to fibrous and net-like structure. Excellent cross-linking of the coating was seen as indicated by the differential scanning calorimetry thermogram and the Fourier transform infrared spectroscopy spectra. After cross-linking the relative intensities of the Fourier transform infrared spectroscopy peaks were decreased and amide bands were shifted to the left. The interaction of gelatin with DTBP cross-linking agent was stronger than that with collagen according to differential scanning calorimetry and Fourier transform infrared spectroscopy results. The compressive strength of the granular bone substitutes increased significantly after the coating process and gelatin coated biphasic calcium phosphate granules showed highest value at 3.68 MPa after cross-linking. Porosity was greater than 63% and did not change significantly with coating. Biocompatibility investigation by in vitro and in vivo showed that the coating improved the cell proliferation marginally. However, the cross-linking process did not jeopardize the excellent biocompatibility of collagen and gelatin. The in vivo study confirms better bone formation behavior of the cross-linked gelatin and collagen coated samples investigated for 8 weeks in vivo.

Fabrication of porous hydroxyapatite scaffolds as artificial bone preform and its biocompatibility evaluation.

In this study, a novel porous hydroxyapatite scaffold was designed and fabricated to imitate natural bone through a multipass extrusion process. The conceptual design manifested unidirectional microchannels at the exterior part of the scaffold to facilitate rapid biomineralization and a central canal that houses the bone marrow. External and internal fissures were minimized during microwave sintering at 1,100 °C. No deformation was noted, and a mechanically stable scaffold was fabricated. Detailed microstructure of the fabricated artificial bone was examined by scanning electron microscope and X-ray diffractometer, and material properties like compressive strength were evaluated. The initial biocompatibility was examined by the cell proliferation of MG-63 osteoblast-like cells using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Preliminary in vivo investigation in a rabbit model after 4 weeks and 8 weeks of implantation showed full osteointegration of the scaffold with the native tissue, and formation of bone tissue within the pore network, as examined by microcomputed tomography analyses and histological staining. Osteon-like bone microarchitecture was observed along the unidirectional channel with microblood vessels. These confirm a biomimetic regeneration model in the implanted bone scaffold, which can be used as an artificial alternative for damaged bone.

Fabrication of multilayer ZrO2-biphasic calcium phosphate-poly-caprolactone unidirectional channeled scaffold for bone tissue formation.

We developed a continuously porous scaffold with laminated matrix and bone-like microstructure by a multi-pass extrusion process. In this scaffold, tetragonal ZrO2, biphasic calcium phosphate and poly-caprolactone layers were arranged in a co-axially laminated unit cell with a channel in the center. The entire matrix phase had a laminated microstructure of alternate lamina of tetragonal ZrO2, biphasic calcium phosphate and poly-caprolactone--biphasic calcium phosphate with optimized designed thickness and channeled porosity. Each of the continuous pores was coaxially encircled by the poly-caprolactone--biphasic calcium phosphate layer, biphasic calcium phosphate layer and finally tetragonal ZrO2 layer, one after the other. Before extrusion, 5 vol% graphite powder was mixed with tetragonal ZrO2 to ensure pores in the outer layer and connectivity among the lamellas. The design strategy is aimed to incorporate a lamellar microstructure like the natural bone in the macro-scaled ceramic body to investigate the strengthening phenomenon and pave the way for fabricating complex microstructure of natural bone could be applied for whole bone replacement. The final fabricated scaffold had a compressive strength of 12.7 MPa and porosity of 78 vol% with excellent cell viability, cell attachment and osteocalcin and collagen expression from cultured MG63 cells on scaffold.

Fabrication of bioglass infiltrated Al(2)O (3)-(m-ZrO (2)) composites.

Using 80 vol.% of poly methyl methacrylate (PMMA) as a pore-forming agent to obtain interconnected porous bodies, porous Al(2)O(3)-(m-ZrO(2)) bodies were successfully fabricated. The pores were about 200 microm in diameter and were homogeneously dispersed in the Al(2)O(3)-25 vol.% (m-ZrO(2)) matrix. To obtain Al(2)O(3)-(m-ZrO(2))/bioglass composites, the molten bioglass was infiltrated into porous Al(2)O(3)-(m-ZrO(2)) bodies at 1400 degrees C. The material properties of the Al(2)O(3)-(m-ZrO(2))/bioglass composites, such as relative density, hardness, compressive strength, fracture toughness and elastic modulus were investigated.