Strontium ion reinforced bioceramic scaffold for load bearing bone regeneration.

Bone defects in load bearing areas require bone reconstruction with strong biomaterial having mechanical characteristics like cortical bone. Bioceramics are biomaterials that support bone formation as well as provide adequate mechanical properties. A strontium substitution of the bioceramic is expected to further increase its bioactivity by enhancing osteogenesis and protect the bone from osteoclastic resorption. The study involves development, characterization and in vivo testing of a newly developed strontium substituted hydroxyapatite based bioceramic scaffold (SrHAB) with sufficient biomechanical properties. Optimal concentration of strontium ion required for enhanced osteogenic differentiation was identified by comparing three compositions of SrHAB scaffold; namely Sr10HAB, Sr30HAB and Sr50 HAB for their Alkaline phosphatase activity in vitro. The selected Sr10HAB scaffold demonstrated in vivo bone formation with osteogenic differentiation of stromal derived mesenchymal stem cells (MSC) from human and ovine sources in ectopic and ovine models. Thus, Sr10HAB scaffold has a potential for application in load bearing bone requirements of orthopaedics and dentistry.

Biomimetic approaches with smart interfaces for bone regeneration.

A 'smart tissue interface' is a host tissue-biomaterial interface capable of triggering favourable biochemical events inspired by stimuli responsive mechanisms. In other words, biomaterial surface is instrumental in dictating the interface functionality. This review aims to investigate the fundamental and favourable requirements of a 'smart tissue interface' that can positively influence the degree of healing and promote bone tissue regeneration. A biomaterial surface when interacts synergistically with the dynamic extracellular matrix, the healing process become accelerated through development of a smart interface. The interface functionality relies equally on bound functional groups and conjugated molecules belonging to the biomaterial and the biological milieu it interacts with. The essential conditions for such a special biomimetic environment are discussed. We highlight the impending prospects of smart interfaces and trying to relate the design approaches as well as critical factors that determine species-specific functionality with special reference to bone tissue regeneration.

Osteogenic efficacy of strontium hydroxyapatite micro-granules in osteoporotic rat model.

Excessive demineralization in osteoporotic bones impairs its self-regeneration potential following a defect/fracture and is of great concern among the aged population. In this context, implants with inherent osteogenic ability loaded with therapeutic ions like Strontium (Sr2+) may bring forth promising outcomes. Micro-granular Strontium incorporated Hydroxyapatite scaffolds have been synthesized and in vivo osteogenic efficacy was evaluated in a long-term osteoporosis-induced aged (LOA) rat model. Micro-granules with improved surface area are anticipated to resorb faster and together with the inherent bioactive properties of Hydroxyapatite with the leaching of Strontium ions from the scaffold, osteoporotic bone healing may be promoted. Long-term osteoporosis-induced aged rat model was chosen to extrapolate the results to clinical osteoporotic condition in the aged. Micro-granular 10% Strontium incorporated Hydroxyapatite synthesized by wet precipitation method exhibited increased in vitro dissolution rate and inductively coupled plasma studies confirmed Strontium ion release of 0.01 mM, proving its therapeutic potential for osteoporotic applications. Wistar rats were induced to long-term osteoporosis-induced aged model by ovariectomy along with a prolonged induction period of 10 months. Thereafter, osteogenic efficacy of Strontium incorporated Hydroxyapatite micro-granules was evaluated in femoral bone defects in the long-term osteoporosis-induced aged model. Post eight weeks of implantation in vivo regeneration efficacy ratio was highest in the Strontium incorporated Hydroxyapatite implanted group (0.92 ± 0.04) compared to sham and Hydroxyapatite implanted group. Micro CT evaluation further substantiated the improved osteointegration of Strontium incorporated Hydroxyapatite implants from the density histograms. Thus, the therapeutical potential of micro-granular Strontium incorporated Hydroxyapatite scaffolds becomes relevant, especially as bone void fillers in osteoporotic cases of tumor resection or trauma.

Zinc oxide nanoparticles induced oxidative stress in mouse bone marrow mesenchymal stem cells.

Engineered nanoparticles are developed for various applications in industrial, electrical, agricultural, pharmaceutical and medical fields due to their unique properties. Nanoparticles such as TiO(2) and ZnO are widely used in cosmetics for UV protection. The toxicological investigations of ZnO NPs are highly recommended because of the increasing use in various industrial and consumer products. The toxic potential of ZnO NPs was assumed to be caused by the release of free Zn+ ions in the medium. Many of the in vivo studies suggest the toxic nature of ZnO NPs, the in vitro studies are certainly important to elucidate the mechanism of toxicity. This study examined the toxicity of ZnO NPs with the average size of 6-8 nm on the isolated mice bone marrow mesenchymal stem cells. The study focuses on the cytotoxicity and oxidative stress-mediated cellular responses upon exposure to ZnO NPs. The results indicated that the exposure to ZnO NPs significantly affects cellular viability in a dose-dependent manner. Formation of reactive oxygen species (ROS) was found to be the mechanism of cellular toxicity. The release of Zn(+) ions from the nanoparticles, due to the instability of ZnO NPs in the acidic compartment of lysosomes, also increases the ROS generation. In addition to increased ROS production, damage of lysosomal membrane and the activation of executioner caspase-3 and caspase-7 were observed, which eventually ends in apoptosis.

An in vitro study on the interaction of hydroxyapatite nanoparticles and bone marrow mesenchymal stem cells for assessing the toxicological behaviour.

Mesenchymal stem cells or multipotent progenitor cells isolated from bone marrow presents close resemblance to the natural in vivo milieu and hence preferred more than the conventional cell culture systems to predict the toxicological behavior of bio-nano interaction. The objective of the present study is to evaluate the molecular toxicity of hydroxyapatite nanoparticles (HANPs) using mouse bone marrow mesenchymal stem cells (BMSCs). In-house synthesized HANPs (50 nm) were used to study the cytotoxicity, nano particle uptake, effect on cyto skeletal arrangement, oxidative stress response and apoptotic behavior with the confluent BMSCs as per standard protocols. The results of the MTT assay indicated that HANPs does not induce cytotoxicity up to 800 µg/mL. It was also observed that oxidative stress related apoptosis and reactive oxygen species (ROS) production following nanoparticle treatment was similar to that of control (cells without treatment). Hence it can be concluded that the in-house synthesized HANPs are non-toxic/safe at the molecular level suggesting that the HANPs are compatible to BMSCs. Further, the in vitro BMSCs cell culture can be used as a model for evaluating the preliminary toxicity of nanomaterials.

Interfacing of dextran coated ferrite nanomaterials with cellular system and delayed hypersensitivity on Guinea pigs.

The study focused on the interfacing of dextran coated ferrite nanomaterials (DFNM) with the cellular system and delayed hypersensitivity on Guinea pigs. In vitro study investigated the cytotoxic potential of DFNM on L929 cells, effect on antioxidant enzymes and Lipid peroxides (LPO) production on rat brain homogenates. DFNM was also repeatedly exposed topically to Guinea pigs for the evidence of skin sensitization and toxicity at the molecular level. Biochemical and hematological parameters were estimated. Liver and brain of Guinea pigs were homogenized and evaluated for the induction of LPO, glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD) and 8-hydroxyl-2-deoxyguanosine (8-OHdG). The results of the study demonstrated that there was no significant alternation in the level of antioxidant defense enzymes, LPO, hematological, biochemical or oxidative stress related DNA damage. Hence, it can be concluded that the synthesized DFNM was non-skin irritant or non-toxic at the molecular level under the laboratory conditions.

Calcium phosphate cement as an alternative for formocresol in primary teeth pulpotomies.

BACKGROUND: Formocresol remains to be the preferred medicament in pulpotomy, despite the concerns regarding tissue devitalization and systemic toxicity. Several materials were used as alternatives, but none proved significantly advantageous. Of recent, calcium phosphate cement (CPC) has been projected as an ideal pulpotomy material considering its tissue compatibility and dentinogenic properties. This study explores the suitability of a CPC formulation for pulpotomy, in comparison with formocresol. MATERIALS AND METHODS: This comparative case study included 10 children (8-12 age group) having a pair of non-carious primary canines (both maxillary and mandibular) posted for extraction. Pulpotomy was performed with CPC in the right canines and formocresol in the left and sealed with IRM ® (Dentsply). The teeth were extracted at 70 ± 5 days and sectioned and stained for the histopathological evaluation. Parameters such as pulpal inflammation, tissue reaction to material, dentine bridge formation, location of dentine bridge, quality of dentine formation in bridge, and connective tissue in bridge etc. were evaluated. RESULTS: The histological assessment after 70 days showed no statistically significant difference between the two groups in any of the parameters. However, CPC gave more favorable results in pulpal inflammation, with a lower score of 1.6 against 2.6 for formocresol. CPC samples showed better formation of dentine bridge in quantity and quality. The mean scores for CPC for the extent of dentine bridge formation, quality of dentine bridge and connective tissue in the bridge, were 2.0, 1.4, and 1.2 respectively, whereas the corresponding values for formocresol were 0.8, 0.2, and 1.0. CONCLUSION: CPC is more compatible to pulp tissues than formocresol and it shows good healing potential. CPC is capable of inducing dentine formation without an area of necrosis.

Cells-nano interactions and molecular toxicity after delayed hypersensitivity, in guinea pigs on exposure to hydroxyapatite nanoparticles.

The aim of the study was to evaluate the cells-nanoparticle interactions and molecular toxicity after delayed hypersensitivity in Guinea pigs, exposed to hydroxyapatite nanoparticles (HANP). The study focuses on synthesizing and characterizing HANPs and gaining an insight into the cytotoxicity, molecular toxicity, hypersensitivity and oxidative stress caused by them in vitro and in vivo. HANP was synthesized by chemical method and characterized by standard methods. Cytotoxicity was assessed on L929 cells by MTT assay and in vitro studies were carried out on rat liver homogenate. In vivo study was carried out by topical exposure of Guinea pigs with HANP, repeatedly, and evaluating the skin sensitization potential, blood parameters, oxidative stress in liver and brain and DNA damage (8-hydroxyl-2-deoxyguanosine: 8-OHdG) in liver. The results of the study indicated that there was no cytotoxicity (up to 600µg/mL) and oxidative damage (up to 100µg/mL), when exposed to HANPs. It was also evident that, there was no skin sensitization and oxidative damage when HANP were exposed to Guinea pigs.

Nanohydroxyapatite incorporated electrospun polycaprolactone/polycaprolactone-polyethyleneglycol-polycaprolactone blend scaffold for bone tissue engineering applications.

The present work is a comparative evaluation of physical and biological properties of electrospun biodegradable fibrous scaffolds based on polycaprolactone (PCL) and its blend with polycaprolactone-polyethyleneglycol-polycaprolactone (CEC) with and without nanohydroxyapatite (nHAP) particles. The fiber morphology, porosity, surface wettability, and mechanical properties of electrospun PCL were distinctly influenced by the presence of both copolymer CEC and nHAP. The degradation in hydrolytic media affected both morphological and mechanical properties of the scaffolds and the tensile strength decreased by 58% for PCL, 83% for PCL/CEC, 36% for PCL/nHAP and 75% for PCL/CEC/nHAP in 90 days of PBS ageing. MTT assay using mouse fibroblast L929 cells proved all the scaffolds to be non-cytotoxic. An overall enhanced performance was shown by PCL/CEC/nHAP scaffold in cell viability (LPH) and proliferation (Picogreen). Simultaneously, ELF assay of ALP activity (bone marker) confirmed the presence of osteogenic-induced Rabbit adipose-derived mesenchymal stem cells (ADMSCs) on all the scaffolds. In comparison, the results reveal the potential of the cytocompatible PCL/CEC/nHAP scaffold for the fabrication of living bony constructs for tissue engineering applications.

Strontium calcium phosphate for the repair of leporine (Oryctolagus cuniculus) ulna segmental defect.

Scaffolds to aid in repair, replacement, or regeneration of bony tissues have been developed using a wide spectra of materials. Under clinical conditions, assessment of healing and implant placement is guided radiographically. In this context, strontium's role in osteostimulation and its relevance in radio-opacity are known. Therefore to aid in assessment and to ensure tissue regeneration, a bone mimetic porous strontium calcium phosphate (SrCaPO(4) ) was synthesized in-house, which was non-cytotoxic (ISO 10993 (Part V) and subsequently characterized for its crystallinity, functional groups, and 3D porous topography. Furthermore, to assess the feasibility of the bioactive ceramic scaffolds in bone repair, SrCaPO(4) and hydroxyapatite (HA-Control) scaffolds were implanted in the segmental ulna bone critical-sized defect (1.5 cm) of New Zealand White Rabbits (leporine model-Oryctolagus cuniculus) for a period of 4 and 12 weeks, respectively. Healing of the defects was uneventful without any inflammation or infection. Radio-opacity of SrCaPO(4) within the defect site enabled easy assessment of implant placement and osteointegration. Again, histological evaluation coupled with micro-CT and histomorphometrical analysis indicated that SrCaPO(4) favored significant de novo bone formation in par with material degradation at 4 and 12 weeks post-implantation compared to HA at 4 and 12 weeks. Investigations on this radio-opaque SrCaPO(4) established its role in the repair of critical-sized segmental defects, proposing it as a suitable bone substitute for clinical reconstructive surgery with easy radiographic evaluation.

Short-term studies using ceramic scaffolds in lapine model for osteochondral defect amelioration.

This study was undertaken to glean preliminary information on the role of triphasic ceramic coated hydroxyapatite (HASi) and biphasic (alpha-tricalcium phosphate and hydroxyapatite based) calcium phosphate (BCP) for the development of osteochondral constructs. The proposed constructs were tested for performance in vitro with rabbit adipose-derived mesenchymal stem cells (RADMSCs) and further analysed in vivo in a lapine model for osteochondral defect amelioration. Desirable scaffolding architecture ensuring favourable conditions for cell attachment, nutrient exchange and neo-tissue organization was achieved by the synthesis of porous ceramic blocks and characterizations were carried out using x-ray diffraction and Fourier transform infrared spectroscopy. The cytocompatibility of the scaffold-cell combination product was evaluated using microscopy techniques that proved the scaffold to be non-cytotoxic and favourable for cell growth and proliferation. Short-term implantation studies were conducted with bare cylindrical HASi and BCP scaffolds, press fit deep into the bony bed of the median femoral condyles of the rabbit, which resulted in favourable specific in vivo response of de novo cartilage-like cells on the surface and sub-surface bony trabeculae. The generated pilot data will help to assess the severity of proposed procedures before embarking on scaled-up efforts.

Development and evaluation of two PVD-coated ß-titanium orthodontic archwires for fluoride-induced corrosion protection.

The present research was aimed at developing surface coatings on ß titanium orthodontic archwires capable of protection against fluoride-induced corrosion. Cathodic arc physical vapor deposition PVD (CA-PVD) and magnetron sputtering were utilized to deposit thin films of titanium aluminium nitride (TiAlN) and tungsten carbide/carbon (WC/C) coatings on ß titanium orthodontic archwires. Uncoated and coated specimens were immersed in a high fluoride ion concentration mouth rinse, following a specially designed cycle simulating daily use. All specimens thus obtained were subjected to critical evaluation of parameters such as electrochemical corrosion behaviour, surface analysis, mechanical testing, microstructure, element release, and toxicology. The results confirm previous research that ß titanium archwires undergo a degradation process when in contact with fluoride mouth rinses. The study confirmed the superior nature of the TiAlN coating, evident as many fewer changes in properties after fluoride treatment when compared with the WC/C coating. Thus, coating with TiAlN is recommended in order to reduce the corrosive effects of fluorides on ß titanium orthodontic archwires.

Ultrastructural evaluation of in vitro mineralized calcium phosphate phase on surface phosphorylated poly(hydroxy ethyl methacrylate-co-methyl methacrylate).

The in vitro functionality of surface phosphorylated poly(hydroxy ethyl methacrylate-co-methyl methacrylate), poly(HEMA-co-MMA) to induce bioinspired mineralization of calcium phosphate phase is evaluated. The primary nucleation of calcium phosphate on the surface phosphorylated copolymer occurs within 3 days of immersion when immersed in 1.5x simulated body fluid and the degree of mineralization is proportional to the hydroxy ethyl methacrylate content in the copolymer. The calcium phosphate phase is identified as hydroxyapatite by X-Ray diffraction analysis. The transmission electron microscopic evaluation combined with selected area diffraction pattern and energy dispersive analysis exemplified that the primary nuclei of amorphous calcium phosphate transforms to crystalline needle like calcium rich apatite, within a period of 3 days immersion in simulated body fluid. The atomic force microscopic results corroborate the c-axis growth of the crystals within 3 days immersion in SBF.

Reconstruction of goat femur segmental defects using triphasic ceramic-coated hydroxyapatite in combination with autologous cells and platelet-rich plasma.

Segmental bone defects resulting from trauma or pathology represent a common and significant clinical problem. In this study, a triphasic ceramic (calcium silicate, hydroxyapatite and tricalcium phosphate)-coated hydroxyapatite (HASi) having the benefits of both HA (osteointegration, osteoconduction) and silica (degradation) was used as a bone substitute for the repair of segmental defect (2 cm) created in a goat femur model. Three experimental goat femur implant groups--(a) bare HASi, (b) osteogenic-induced goat bone marrow-derived mesenchymal stem cells cultured HASi (HASi+C) and (c) osteogenic-induced goat bone marrow-derived mesenchymal stem cells cultured HASi+platelet-rich plasma (HASi+CP)--were designed and efficacy performance in the healing of the defect was evaluated. In all the groups, the material united with host bone without any inflammation and an osseous callus formed around the implant. This reflects the osteoconductivity of HASi where the cells have migrated from the cut ends of host bone. The most observable difference between the groups appeared in the mid region of the defect. In bare HASi groups, numerous osteoblast-like cells could be seen together with a portion of material. However, in HASi+C and HASi+CP, about 60-70% of that area was occupied by woven bone, in line with material degradation. The interconnected porous nature (50-500 microm), together with the chemical composition of the HASi, facilitated the degradation of HASi, thereby opening up void spaces for cellular ingrowth and bone regeneration. The combination of HASi with cells and PRP was an added advantage that could promote the expression of many osteoinductive proteins, leading to faster bone regeneration and material degradation. Based on these results, we conclude that bare HASi can aid in bone regeneration but, with the combination of cells and PRP, the sequence of healing events are much faster in large segmental bone defects in weight-bearing areas in goats.

Bioinspired mineralization and cell adhesion on surface functionalized poly(vinyl alcohol) films.

Poly(vinyl alcohol) (PVA) films, when surface functionalized by phosphorylation, induced biomimetic nucleation and growth of calcium phosphate in a simulated physiological environment. The surface phosphorylation on PVA was ensured by attenuated total reflectance infrared spectroscopy. The morphology of the calcium phosphate phase grown on surface-phosphorylated PVA (PPVA) was analysed using scanning electron microscopy coupled with an energy-dispersive X-ray detector. The primary nucleation of calcium phosphate occurs in 3 days and secondary nucleation occurs after 10 days. The energy-dispersive X-ray analysis shows that the Ca/P ratio of the coating increases with time of exposure to the simulated physiological fluid and reaches 1.67 at 10 days. The PPVA supports in vitro cell adhesion and promotes in vitro biomineralization in the presence of cells, evaluated using human osteosarcoma cells.

In vitro investigations of bone remodeling on a transparent hydroxyapatite ceramic.

The light microscopic examination of cells directly on bioceramic materials in the transmission mode is impossible because many of these materials are opaque. In order to enable direct viewing of living cells and to perform time-lapse studies, nearly transparent bioceramic materials were developed. A dense and fine-grained transparent hydroxyapatite (tHA) was processed by a gel-casting route followed by low-temperature sintering (1000 degrees C). By virtue of its transparency, direct visualization of cellular events on this material is possible in transmitted light. In this study, the interaction of different bone cell types with the tHA ceramic was envisaged. Investigation of rat calvaria osteoblasts (RCO) cultured on tHA by means of transmission light microscopy indicated good cytocompatibility of tHA. Microscopic analysis of osteogenic-induced human bone marrow stromal cells (hBMSC) on tHA and quantitative analysis of their lactate dehydrogenase (LDH) activity at different time points of culture revealed favorable proliferation as well. An increase of the alkaline phosphatase (ALP) activity indicated the differentiation of osteogenic-induced hBMSC towards the osteoblastic lineage. In addition, the differentiation of human monocytes to osteoclast-like cells could also be demonstrated on tHA and was confirmed by fluorescent microscopy imaging of multinucleated cells on the transparent material.

Tissue regeneration and repair of goat segmental femur defect with bioactive triphasic ceramic-coated hydroxyapatite scaffold.

Bone tissue engineering which is a developing and challenging field of science, is expected to enhance the regeneration and repair of bone lost from injury or disease and ultimately to gain its aesthetic contour. The objective of this study was to fabricate a tissue-engineered construct in vitro using a triphasic ceramic-coated hydroxypatite (HASi) in combination with stem cells and to investigate its potential in healing segmental defect in goat model. To accomplish this attempt, mesenchymal stem cells isolated from goat bone marrow were seeded onto HASi scaffolds and induced to differentiate into the osteogenic lineage in vitro. Scanning electron microscopy and light microscopy revealed adhesion and spread-out cells, which eventually formed a cell-sheet like canopy over the scaffold. Cells migrated and distributed themselves within the internal voids of the porous ceramic. Concurrently, the neo-osteogenesis of the tissue-engineered construct was validated in vivo in comparison with bare HASi (without cells) in goat femoral diaphyseal segmental defect (2 cm) at 4 months postimplantation through radiography, computed tomography, histology, histomorphometry, scanning electron microscopy and inductively coupled plasma spectrometry. Good osteointegration and osteoconduction was observed in bare and tissue-engineered HASi. The performance of tissue-engineered HASi was better and faster which was evident by the lamellar bone organization of newly formed bone throughout the defect together with the degradation of the material. On the contrary with bare HASi, immature woven bony bridges still intermingled with scattered small remnants of the material was observed in the mid region of the defect at 4 months. Encouraging results from this preclinical study has proved the capability of the tissue-engineered HASi as a promising candidate for the reconstruction of similar bony defects in humans.

Platelet-rich plasma and fibrin glue-coated bioactive ceramics enhance growth and differentiation of goat bone marrow-derived stem cells.

New biotechnologies such as tissue engineering require functionally active cells within supportive matrices where the physical and chemical stimulus provided by the matrix is indispensable to determine the cellular behavior. This study has investigated the influence of platelet-rich plasma (PRP) and fibrin glue (FG) on the functional activity of goat bone marrow-derived mesenchymal stem cells (gBMSCs) that differentiated into the osteogenic lineage. To achieve this goal, PRP and FG were separately coated on bioactive ceramics like hydroxyapatite (HA) and silica-coated HA (HASi), on which gBMSCs were seeded and induced to differentiate into the osteogenic lineage for 28 days. The cells were then analyzed for viability (lactate dehydrogenase assay: acridine orange and ethidium bromide staining), morphology (scanning electron microscopy), proliferation (picogreen assay), cell cycle assay (propidium iodide staining), and differentiation (alkaline phosphatase [ALP] activity and real-time PCR analysis of ALP, osteocalcin, and osteopontin gene). It has been observed that PRP and FG have appreciably favored the viability, spreading, and proliferation of osteogenic-induced gBMSCs. The osteopontin and osteocalcin expression was significantly enhanced on PRP- and FG-coated HA and HASi, but PRP had effect on neither ALP expression nor ALP activity. The results of this study have depicted that FG-coated ceramics were better than PRP-coated and bare matrices. Among all, the excellent performance was shown by FG coated HASi, which may be attributed to the communal action of the stimulus emanated by Si in HASi and the temporary extracellular matrix provided by FG over HASi. Thus, we can conclude that PRP or FG in combination with bioactive ceramics could possibly enhance the functional activity of cells to a greater extent, promoting the hybrid composite as a promising candidate for bone tissue engineering applications.

Triphasic ceramic coated hydroxyapatite as a niche for goat stem cell-derived osteoblasts for bone regeneration and repair.

Current treatment strategies for the repair or replacement of bone use synthetic implants with stem cells and their progeny--a new approach to address unmet medical needs. This study has evaluated the effect of a silica-coated bioactive ceramic, namely HASi in comparison to hydroxyapatite (HA) on the adhesion, proliferation and osteogenic differentiation of goat bone marrow-derived mesenchymal stem cells in vitro in a prolonged culture of 28 days. The cellular activities were significantly enhanced on HASi signifying the role of silica to stimulate osteoblast cells. The fabrication of such a 'cell-ceramic construct using autologous MSCs' is aimed for the transplantation to a large bone defect site in the goat femur model which still remains a formidable challenge in Orthopedic surgery.