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.

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.

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.

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.

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.

Self-assembled right handed helical ribbons of the bone mineral hydroxyapatite.

A self-assembled right handed helical ribbon of bone mineral, hydroxyapatite (HAp) was crystallized in sodium meta silicate gel matrix at 27 degrees C and the physiological pH (7.4). At temperatures 37 and 47 degrees C, helical structures were followed by many Liesegang rings. The samples were characterized by FT-IR, XRD, SEM, ICP-OES and TG-DTA techniques. The helical ribbon consisted of platy Ca-deficient apatite crystals of size 2.8 microm. Liesegang ring had a continuous network of fibers with interconnected pores. The samples exhibited bioactivity when soaked in SBF.

A triphasic ceramic-coated porous hydroxyapatite for tissue engineering application.

Scaffolds which encourage the incorporation of a cell source for tissue engineering applications are critical determinants for clinical defects. Over the years, a number of biomaterials have emerged for cell support and growth, but only a few have demonstrated clinical efficacy. We therefore investigated an in-house-developed silica-based bioactive ceramic for its ability to support and sustain the growth of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. For this, MSCs aspirated from goat bone marrow were isolated and culture expanded on a novel triphasic ceramic composite coated hydroxyapatite (HASi) scaffold comprising hydroxyapatite, tricalcium phosphate and calcium silicate. The viability of cells that harbored on and within the material was ensured through fluorescence-activated cell sorting and confocal laser scanning microscope and for their anchorage sites by scanning electron microscopy. Interestingly, over the days in culture, cell-cell interactions gradually morphed into woven cell-sheets that spanned across the surface of the HASi, forming a canopy. To conclude, we have attempted to carry out the preliminary cytocompatibility studies of this novel ceramic to establish its appropriateness for bone tissue engineering application which is an important criterion in orthopaedic transplantation and regenerative surgery.

Hydroxyapatite moldable formulation using natural rubber latex as binder.

A simple but efficient processing method for shaping intricate bioceramic green bodies has been developed by using natural rubber latex as binder. Different shapes of hydroxyapatite Ca10(PO4)6(OH)2 (HAP) were molded from a composite formulation containing wet precipitated HAP, natural rubber latex (NRL), and a stabilizer. On controlled heat treatment followed by sintering, dense shapes of HAP contours were obtained. The thermal degradation profile of HAP-NRL composites shows that NRL degrades slowly without any abrupt exotherm. The results of energy dispersive X-ray analysis together with inductively coupled plasma (ICP) analysis indicate that the inorganic residue of NRL does not contain any heavy element. The sintered density of the samples increased with increased HAP content in the formulation and percentage shrinkage reduced accordingly. On varying the HAP content in the formulation from 35 to 95 wt %, the compositions with 85, 90, 92, and 95 wt % HAP showed better flexural strength in the range 40-54 MPa and a flexural modulus value in the range 36-50 GPa. The fracture morphology, as observed by the scanning electron microscope confirms that with increased HAP content in the formulation the sample microstructure attains higher uniformity. The Vickers microhardness for the samples sintered at two different temperatures (1150 and 1250 degrees C) showed that hardness increases with increase in the sintering temperature with a maximum for the highest HAP loaded formulation.

Human osteosarcoma cell adhesion behaviour on hydroxyapatite integrated chitosan-poly(acrylic acid) polyelectrolyte complex.

A novel degradable composite system has been prepared by integrating hydroxyapatite, Ca(10)(PO(4))(6)(OH)(2), (HAP) in a polyelectrolyte complex matrix of chitosan (CHI) and poly(acrylic acid) (PAA). The composite was formulated by integrating 80 wt.% HAP in the polyelectrolyte complex matrix of CHI and PAA in the ratio 40/60 (designated as CPH). The composite could be easily fabricated into clinically significant shapes by a simple moulding procedure intended for bone graft applications. The adhesion behaviour of human osteosarcoma (HOS) cells on this degradable composite system was studied by selecting the polyelectrolyte complex, CHI/PAA 40/60 (designated as CP) as control sample. Light microscopic observations show that cells around CPH retained the typical morphology of HOS cells while cells around the polyelectrolyte complex showed a cytotoxic effect. The adhesion behaviour as well as morphological responses of the seeded cells was further investigated by scanning electron microscopy. The scanning electron micrographs of the polyelectrolyte complex, CP, showed the presence of rounded cells with raised nuclear regions, indicating delayed spreading; cells adhered on CPH were flattened with filopodia and showed good attachment and spreading, indicating better adhesion onto the HAP integrated composite. Comparing the MTT assay for quantitative evaluation of cell viability, CPH showed a higher percentage of metabolically active cells compared to CP.

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.

Rapid and complete cellularization of hydroxyapatite for bone tissue engineering.

Using a tissue construct generated by cells in a scaffold in reconstructive surgery, as a substitute for autografts, is still challenging. Routine methods of incorporating cells into scaffolds are either passive, i.e. by gravity, or forced, as in a bioreactor. Extensive use of these methods is obstructed by tissue formation around the scaffold, hindrance in cell penetration and time required for cell coverage within the scaffold. In this study, human osteoblast cells as cell sheet structures were seeded to porous and dense hydroxyapatite with the hypothesis that preservation of native extracellular structures and cell-cell contacts would facilitate the cellularization process. Cellularization was assessed by fluorescence, confocal and scanning electron microscopy at intervals of 1 h, 2 days and 7 days. Cell patches with intact cell-cell and cell-extra cellular matrix contact attached and adhered on a scaffold within 1 h. The patches formed a monolayer within 2 days and complete cellularization of the scaffold was attained in 7 days. Cell viability, proliferation and function were assessed to understand the application of cell patch transfer to bone substitute. This novel approach for application in bone tissue engineering was successful in uniform distribution of intact osteoblast cell sheet structures on to bone substitute materials for rapid and complete cellularization without altering material characteristics.

Biological evaluation of pliable hydroxyapatite-ethylene vinyl acetate co-polymer composites intended for cranioplasty.

Hydroxyapatite (HAP) is undoubtedly a material suitable for repairing the defective bone tissue. However, the brittleness and non-malleability of HAP limit its clinical application as a cranioplastic analogue. To improve these properties, pliable, osteoconductive composites composed of HAP and ethylene vinyl acetate co-polymer (EVA) have been developed. This study reports the biocompatibility evaluation of the newly developed composite material. Composites of two compositions, containing 40 and 50 volume percentage of HAP, were evaluated. In vitro cell culture cytotoxity studies were carried out using L929 cell line. Intracutaneous irritation studies, and intramuscular implantation studies were carried out on rabbits. Cell culture studies showed that the composite was non-cytotoxic to mouse fibroblast cell line. Intracutaneous irritation studies did not show any gross signs of tissue reaction. Histological analysis after six months of implantation in the paravertebral muscles of rabbit showed that all the implants under study were covered with a thin soft tissue capsule. On the basis of these observations, we conclude that the composite materials are biocompatible and hence are a candidate material for implantation in the cranium.

In vitro calcium phosphate growth over surface modified PMMA film.

In vitro nucleation of calcium phosphate phase was studied over functionalized polymethyl methacrylate (PMMA) films using Fourier transform infrared spectroscopy, electron spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. PMMA films were prepared by dissolving commercial grade pellets in chloroform and cast into thin sheets. The films were immersed in a methanol solution of sodium hydroxide before treating with 1.5% solution of adenosine triphosphate (ATP) at a pH of 5.2 for 24 h. ATP treated films were then soaked in saturated lime solution for 4 days to initiate formation of calcium phosphate precursor phase over their surface. The above films immersed in simulated body fluid solution (1.5 x SBF) for more than 5 days led to the nucleation of apatitic calcium phosphate phase all over the film surface. The ATP coupled film not subjected to lime treatment did not show calcium phosphate nucleation behaviour upon immersion in SBF solution. The Ca/P ratio of the calcium phosphate phase increase with increase in soaking time in SBF solution.

Fibrin glue as an osteoinductive protein in a mouse model.

Fibrin sealant or fibrin glue (FG) has been found to be effective as a wound-healing substance in surgery. However, its role in bone fracture healing and osseous tissue response is not fully understood. This ambiguity questions the potential of FG as an inductive protein. The present study was undertaken to evaluate the osteoinductive property of FG when coated with calcium phosphate and glass ceramics and implanted in the extraskeletal site of male Swiss albino mice. Implant materials used for this study were hydroxyapatite (HA) porous granules (300-350 microm), bioactive glass system (BGS)-AW type and calcium phosphate calcium silicate system (HABGS) non-porous granules (300-350 microm). Uncoated granules (control) and coated granules with 2.5 mg FG and 5 mg FG were implanted in the quadriceps muscle of mice and sacrificed after 28 days. Histologically, HA, BGS and HABGS implanted animal groups showed good healing response. However, neo-osteogenesis was observed only in the BGS and HABGS granules impregnated with FG. Furthermore, bone formation was observed to be more conspicuous in 5 mg FG coated BGS and HABGS granules when compared with 2.5 mg FG coated BGS and HABGS granules. Fluorochrome labeling proved that mineralization had already started by day 15 with FG preadsorbed BGS and HABGS granules. On the contrary, the uncoated granules did not show any de novo bone formation. This experimental study provides an evidence of the positive role of FG as a potential osteoinductive biologic tissue adhesive.

Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method.

A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energy-dispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H3PO4 with the P content being 0.1-0.2 wt%. Phosphorylated films soaked in saturated Ca(OH)2 solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5 x SBF for more than 20 days. Phosphorylated films not treated with Ca(OH)2 did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time.

Surface reactivity of calcium phosphate based ceramics in a cell culture system.

Surface reactivity of Calcium Phosphate materials--Hydroxyapatite (HA), Tricalcium Phosphate (beta-TCP), Hydroxyapatite-Tricalcium Phosphate (HA-TCP) were elucidated in a cell culture system. MG-63 osteoblast-like cells were seeded onto the ceramic discs to evaluate changes in the cell morphology and functionality with respect to the different substrates. The dissolution and re-precipitation of calcium phosphate phases on the surface of the discs in the culture medium was found to be prominent on beta-TCP when compared with HA. Low calcium (Ca), magnesium (Mg) and alkaline phosphatase (ALP) levels and high phosphorous (P) levels in the medium of beta-TCP were observed. This indicated that P must have leached out into the medium from beta-TCP and Ca in turn deposited from the medium onto beta-TCP resulting in the apatite phase transformation. The low ALP activity in beta-TCP medium is however an indication of low osteoblastic activity. Under the phase contrast microscope, the osteoblast cells around HA material were found to be confluent and viable, while in the vicinity of beta-TCP only cellular debris was observed. In the case of HA-TCP, only a few viable cells surrounded the material amidst the debris. Scanning electron microscopy revealed numerous cells on the surface of HA showing different cell behaviour like anchorage, attachment, adhesion and spreading in the early time period as the surface was only slightly disturbed with re-crystallisation. But with time the entire surface of HA had changed due to precipitation and re-crystallization which did not support cell behaviour while the cells surrounding the material showed normal growth. On the contrary, cells were scarcely observed on the entirely changed surface of beta-TCP and HA-TCP even from the earlier days of the culture and the morphology of cells surrounding the material too started changing. These results establish that HA promoted the activity of osteoblast cells. HA surface remained unaltered for some time, while the surface of beta-TCP underwent dissolution of surface ions and resulted in the re-crystallization of apatite over the surface. The resulting changes in the surrounding milieu of beta-TCP with high phosphate and low Ca levels probably was responsible for the death of the cells.

Fully injectable calcium phosphate cement--a promise to dentistry.

Calcium phosphate cements (CPC) are self setting and biocompatible bone substitute materials with potential applications in dentistry. However, its clinical use has been challenged by poor rheological properties. A novel formulation of CPC has been developed, which gives a fully injectable and cohesive paste. This work investigates the suitability of the new "fully injectable calcium phosphate cement" (FI-CPC) for dental applications. The cementing properties, material characteristics, and the rheological properties were tested using a battery of material characteristics methods. The biocompatibility was also evaluated as per ISO 7405. The setting time (20 min) and compressive strength (>11 Mpa) of FI-CPC satisfy the clinical requirements. It underwent setting without any exothermic reaction, keeping good dimensional stability. The cement paste could be extruded through a 18-gauge needle, easily and fully. It showed excellent cohesion when immersed in water. FI-CPC was seen to set into a micro-porous mass of hydroxyapatite, the mineral part of human dentin. It showed good attachment to dentin walls, when filled in tooth perforations. FI-CPC was found non-toxic, non-allergic, non-pyrogenic, and soft-tissue compatible. The study shows that FI-CPC provides a self setting bio-compatible paste with excellent rheological properties for surgical applications. The set cement provides good and stable sealing. The osteoconductive property is an added advantage. FI-CPC proves to be an ideal material for endodontic sealing/filling and periodontic repair.

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.

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.