Preparation and characterisation of tri-calcium phosphate scaffolds with tunnel-like macro-pores for bone tissue engineering

Calcium Phosphate ceramics have been widely used in tissue engineering due to their excellent biocompatibility and biodegradability. In the physiological environment, they are able to gradually degrade, absorbed and promote bone growth. Ultimately, they are capable of replacing damaged bone with new tissue. However, their low mechanical properties limit calcium phosphate ceramics in load-bearing applications. To obtain sufficient mechanical properties as well as high biocompatibility is one of the main focuses in biomaterials research. Therefore, the current project focuses on the preparation and characterization of porous tri-calcium phosphate (TCP) ceramic scaffolds. Hydroxapatite (HA) was used as the raw material, and normal calcium phosphate bioglass was added to adjust the ratio between calcium and phosphate. It was found that when 20% bioglass was added to HA and sintered at 1400oC for 3 hours, the TCP scaffold was obtained and this was confirmed by X-ray diffraction (XRD) analysis. Test results have shown that by applying this method, TCP scaffolds have significantly higher compressive strength (9.98MPa) than those made via TCP powder (<3MPa). Moreover, in order to further increase the compressive strength of TCP scaffolds, the samples were then coated with bioglass. For normal bioglass coated TCP scaffold, compressive strength was 16.69±0.5MPa; the compressive strength for single layer mesoporous bioglass coated scaffolds was 15.03±0.63MPa. In addition, this project has also concentrated on sizes and shapes effects; it was found that the cylinder scaffolds have more mechanical property than the club ones. In addition, this project performed cell culture within scaffold to assess biocompatibility. The cells were well distributed in the scaffold, and the cytotoxicity test was performed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay. The Alkaline Phosphatase (Alp) activity of human bone marrow mesenchymal stem cell system (hBMSCs) seeded on scaffold expressed higher in vitro than that in the positive control groups in osteogenic medium, which indicated that the scaffolds were both osteoconductive and osteoinductive, showing potential value in bone tissue engineering.

Strong and bioactive tri-calcium phosphate scaffolds with tube-like macropores

Calcium phosphate ceramic scaffolds have been widely investigated for bone tissue engineering due to their excellent biocompatibility and biodegradation. Unfortunately, they have the shortcoming of low mechanical properties. In order to provide strong, bioactive, and biodegradable scaffolds, a new approach of infiltrating the macro-tube ABS (acrylontrile butadiene styrene) templates with a hydroxyapatite/bioactive glass mixed slurry was developed to fabricate porous Si-doped TCP (tri-calcium phosphate) scaffolds. The porous Si-doped TCP ceramics with a high porosity (~65%) and with interconnected macrotubes (~0.8mm in diameter) and micropores (5-100 m) had a high compressive strength (up to 14.68+0.2MPa), which was comparable to that of a trabecular bone and was much higher than those of pure TCP scaffolds. Additional cell attachment study and MTT cytotoxicity assay proved the bioactivity and biocompatibility of the new scaffolds. Thus a potential bioceramic material and a new approach to make the potential scaffolds were developed for bone tissue engineering.

Preparation of porous calcium phosphate ceramic scaffold in bone tissue engineering

Calcium Phosphate ceramic has been widely used in bone tissue engineering due to its excellent biocompatibility and biodegradability. However, low mechanical properties and biodegradability limit their potential applications. In this project, hydroxyapatite (HA) and calcium phosphate bioglass were used to produce porous tri-calcium phosphate (TCP) bio-ceramic scaffolds. It was found that porous TCP bioceramic could be obtained when 20wt percent bioglass addition and sintered in 1400 degrees celsius for 3 h. Significantly higher compressive strength (9.98 MPa) was achieved in the scaffolds as compared to those produced from tCP power (<3 MPa). The biocompatibility of the scaffold was also estimated.

Some aspects of calcium phosphate chemistry in sugarcane clarification

This paper reviews some aspects of calcium phosphate chemistry since phosphate in juice is an important parameter in all sugar juice clarification systems. It uses basic concepts to try and explain the observed differences in clarification performance obtained with various liming techniques. The paper also examines the current colorimetric method used for the determination of phosphate in sugar juice. In this method, a phosphomolybdate blue complex formed due to the addition of a dye is measured at 660 nm. Unfortunately, at this wavelength there is interference of the colour arising from within the juice and results in the underestimation of the amount of soluble inorganic phosphate content of juice. It is suggested that phosphate analysis be conducted at the higher wavelength of 875 nm where the interference of the juice colour is minimised.

The influence of cellular source on periodontal regeneration using calcium phosphate coated polycaprolactone scaffold supported cell sheets

Cell-based therapy is considered a promising approach to achieving predictable periodontal regeneration. In this study, the regenerative potential of cell sheets derived from different parts of the periodontium (gingival connective tissue, alveolar bone and periodontal ligament) were investigated in an athymic rat periodontal defect model. Periodontal ligament (PDLC), alveolar bone (ABC) and gingival margin-derived cells (GMC) were obtained from human donors. The osteogenic potential of the primary cultures was demonstrated in vitro. Cell sheets supported by a calcium phosphate coated melt electrospun polycaprolactone (CaP-PCL) scaffold were transplanted to denuded root surfaces in surgically created periodontal defects, and allowed to heal for 1 and 4 weeks. The CaP-PCL scaffold alone was able to promote alveolar bone formation within the defect after 4 weeks. The addition of ABC and PDLC sheets resulted in significant periodontal attachment formation. The GMC sheets did not promote periodontal regeneration on the root surface and inhibited bone formation within the CaP-PCL scaffold. In conclusion, the combination of either PDLC or ABC sheets with a CaP-PCL scaffold could promote periodontal regeneration, but ABC sheets were not as effective as PDLC sheets in promoting new attachment formation.

Effect of culture conditions and calcium phosphate coating on ectopic bone formation

This study investigated the effect of a calcium phosphate (CaP) coating onto a polycaprolactone melt electrospun scaffold and in vitro culture conditions on ectopic bone formation in a subcutaneous rat model. The CaP coating resulted in an increased alkaline phosphatase activity (ALP) in ovine osteoblasts regardless of the culture conditions and this was also translated into higher levels of mineralisation. A subcutaneous implantation was performed and increasing ectopic bone formation was observed over time for the CaPcoated samples previously cultured in osteogenic media whereas the corresponding non-coated samples displayed a lag phase before bone formation occurred from 4 to 8 weeks post-implantation. Histology and immunohistochemistry revealed bone fill through the scaffolds 8 weeks post-implantation for coated and non-coated specimens and that ALP, osteocalcin and collagen 1 were present at the ossification front and in the bone tissues. Vascularisation in the vicinity of the bone tissues was also observed indicating that the newly formed bone was not deprived of oxygen and nutrients.We found that in vitro osteogenic induction was essential for achieving bone formation and CaP coating accelerated the osteogenic process. We conclude that high cell density and preservation of the collagenous and mineralised extracellular matrix secreted in vitro are factors of importance for ectopic bone formation.

Stoichiometry, crystallinity, and nano-scale surface morphology of the graded calcium phosphate-based bio-ceramic interlayer on Ti-Al-V

A plasma-assisted concurrent Rf sputtering technique for fabrication of biocompatible, functionally graded CaP-based interlayer on Ti-6Al-4V orthopedic alloy is reported. Each layer in the coating is designed to meet a specific functionality. The adherent to the metal layer features elevated content of Ti and supports excellent ceramic-metal interfacial stability. The middle layer features nanocrystalline structure and mimics natural bone apatites. The technique allows one to reproduce Ca/P ratios intrinsic to major natural calcium phosphates. Surface morphology of the outer, a few to few tens of nanometers thick, layer, has been tailored to fit the requirements for the bio-molecule/protein attachment factors. Various material and surface characterization techniques confirm that the optimal surface morphology of the outer layer is achieved for the process conditions yielding nanocrystalline structure of the middle layer. Preliminary cell culturing tests confirm the link between the tailored nano-scale surface morphology, parameters of the middle nanostructured layer, and overall biocompatibility of the coating.

Reactive species in RF plasma sputtering deposition of nanocrystalline calcium phosphate bio-active films

Optical emission of reactive plasma species during the synthesis of functionally graded calcium phosphate-based bioactive films has been investigated. The coatings have been deposited on Ti-6Al-4V orthopedic alloy by co-sputtering of hydroxyapatite (HA) and titanium targets in reactive plasmas of Ar + H2O gas mixtures. The species, responsible for the Ca-P-Ti film growth have been non-intrusively monitored in situ by a high-resolution optical emission spectroscopy (OES). It is revealed that the optical emission originating from CaO species dominates throughout the deposition process. The intensities of CaO, PO and CaPO species are strongly affected by variations of the operating pressure, applied RF power, and DC substrate bias. The optical emission intensity (OEI) of reaction species can efficiently be controlled by addition of H2O reactant.

Comparison of silicate-substituted calcium phosphate (Actifuse®) with rhBMP-2 (Infuse®) in posterolateral instrumented lumbar fusion

Study Design This was a randomised controlled trial in patients with degenerative disc disease (DDD) who underwent instrumented posterolateral lumbar fusion (PLF) surgery. Objective The aim of this study was to assess the efficacy of the bone grafting substitute, silicate-substituted calcium phosphate (SiCaP) compared with bone morphogenetic protein (rhBMP-2) and to evaluate clinical outcomes over a period of two years. Methods Patients undergoing PLF surgery for DDD at a single centre were recruited and randomised to one of two groups; SiCaP (n=9) or rhBMP-2 (n=10). One patient withdrew prior to randomisation and another from the rhBMP-2 group after randomisation. The radiological and clinical outcomes were examined and compared. Fusion was assessed at 12 months with computed tomography (CT) and plain radiographs. Clinical outcomes were evaluated by recording measures of pain, quality of life, disability and neurological status from six weeks to two years postoperatively. Results In the SiCaP and rhBMP-2 groups, fusion was observed in 9/9 and 8/9 patients respectively. Pain and disability scores were reduced and quality of life increased in both groups. Leg pain, disability and satisfaction scores were similar between the groups at each postoperative time point, however, back pain was less at six weeks and quality of life was higher at six months in the SiCaP group than the rhBMP-2 group. Conclusions SiCaP and rhBMP-2 were comparable in terms of achieving successful bone growth and fusion. Both groups similarly alleviated pain and improved quality of life, neurological, satisfaction and return to work outcomes following PLF surgery.

Calcium phosphate flocs and the clarification of sugar cane juice from whole of crop harvesting

Sugar cane biomass is one of the most viable feedstocks for the production of renewable fuels and chemicals. Therefore, processing the whole of crop (WC) (i.e., stalk and trash, instead of stalk only) will increase the amount of available biomass for this purpose. However, effective clarification of juice expressed from WC for raw sugar manufacture is a major challenge because of the amounts and types of non-sucrose impurities (e.g., polysaccharides, inorganics, proteins, etc.) present. Calcium phosphate flocs are important during sugar cane juice clarification because they are responsible for the removal of impurities. Therefore, to gain a better understanding of the role of calcium phosphate flocs during the juice clarification process,the effects of impurities on the physicochemical properties of calcium phosphate flocs were examined using small-angle laser light scattering technique, attenuated total reflectance Fourier transformed infrared spectroscopy, and X-ray powder diffraction. Results on synthetic sugar juice solutions showed that the presence of SiO2 and Na+ ions affected floc size and floc structure. Starch and phosphate ions did not affect the floc structure; however, the former reduced the floc size, whereas the latter increased the floc size. The study revealed that high levels of Na+ ions would negatively affect the clarification process the most, as they would reduce the amount of suspended particles trapped by the flocs. A complementary study on prepared WC juice using cold and cold/intermediate liming techniques was conducted. The study demonstrated that, in comparison to the one-stage (i.e., conventional) clarification process, a two-stage clarification process using cold liming removed more polysaccharides (≤19%),proteins (≤82%), phosphorus (≤53%), and SiO2 (≤23%) in WC juice but increased Ca2+ (≤136%) and sulfur (≤200%)

The influence of impurities on calcium phosphate floc structure and size in sugar solutions

Settling, dewatering and filtration of flocs are important steps in industry to remove solids and improve subsequent processing. The influence of non-sucrose impurities (Ca2+, Mg2+, phosphate and aconitic acid) on calcium phosphate floc structure (scattering exponent, Sf), size and shape were examined in synthetic and authentic sugar juices using X-ray diffraction techniques. In synthetic juices, Sf decreases with increasing phosphate concentration to values where loosely bound and branched flocs are formed for effective trapping and removal of impurities. Although, Sf did not change with increasing aconitic acid concentration, the floc size significantly decreased reducing the ability of the flocs to remove impurities. In authentic juices, the flocs structures were marginally affected by increasing proportions of non-sucrose impurities. However, optical microscopy indicated the formation of well-formed macro-floc network structures in sugar cane juices containing lower proportions of non-sucrose impurities. These structures are better placed to remove suspended colloidal solids.

Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo

The use of polycaprolactone (PCL) as a biomaterial, especially in the fields of drug delivery and tissue engineering, has enjoyed significant growth. Understanding how such a device or scaffold eventually degrades in vivo is paramount as the defect site regenerates and remodels. Degradation studies of three-dimensional PCL and PCL-based composite scaffolds were conducted in vitro (in phosphate buffered saline) and in vivo (rabbit model). Results up to 6 months are reported. All samples recorded virtually no molecular weight changes after 6 months, with a maximum mass loss of only about 7% from the PCL-composite scaffolds degraded in vivo, and a minimum of 1% from PCL scaffolds. Overall, crystallinity increased slightly because of the effects of polymer recrystallization. This was also a contributory factor for the observed stiffness increment in some of the samples, while only the PCL-composite scaffold registered a decrease. Histological examination of the in vivo samples revealed good biocompatibility, with no adverse host tissue reactions up to 6 months. Preliminary results of medical-grade PCL scaffolds, which were implanted for 2 years in a critical-sized rabbit calvarial defect site, are also reported here and support our scaffold design goal for gradual and late molecular weight decreases combined with excellent long-term biocompatibility and bone regeneration. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 90A: 906-919, 2009

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features.