Sachin J. Shenoy

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.

Synthesis and characterization of dextran stabilized superparamagnetic iron oxide nanoparticles for in vivo MR imaging of liver fibrosis.

The field of medical imaging has recently seen rapid advances in the development of novel agents for enhancing image contrast. In particular, superparamagnetic iron oxide nanoparticles (SPIONs) with a variety of surface properties have been tried as effective contrast agents for magnetic resonance imaging, but with major side effects. In this study, the surface chemistry of SPIONs of size 12 nm was modified with high molecular weight dextran to yield particles of size 50 nm, without compromising the magnetic properties. A systematic characterization of the material for its size, coating efficiency, magnetic properties and biocompatibility has been carried out. The magnetic relaxivity as evaluated on a 1.5 T clinical magnet showed r2/r1 ratio of 56.28 which is higher than that reported for any other dextran stabilized ironoxide nanoparticles. Liver uptake and magnetic resonance imaging potential of dextran stabilized SPIONs (D-SPIONs) has been evaluated on liver fibrosis induced animal model, which is further supported by histopathology results.

Early osseointegration of a strontium containing glass ceramic in a rabbit model

The most important property of a bone cement or a bone substitute in load bearing orthopaedic implants is good integration with host bone with reduced bone resorption and increased bone regeneration at the implant interface. Long term implantation of metal-based joint replacements often results in corrosion and particle release, initiating chronic inflammation leading onto osteoporosis of host bone. An alternative solution is the coating of metal implants with hydroxyapatite (HA) or bioglass or the use of bulk bioglass or HA-based composites. In the above perspective, the present study reports the in vivo biocompatibility and bone healing of the strontium (Sr)-stabilized bulk glass ceramics with the nominal composition of 4.5SiO2-3Al2O3-1.5P2O5-3SrO-2SrF2 during short term implantation of up to 12 weeks in rabbit animal model. The progression of healing and bone regeneration was qualitatively and quantitatively assessed using fluorescence microscopy, histological analysis and micro-computed tomography. The overall assessment of the present study establishes that the investigated glass ceramic is biocompatible in vivo with regards to local effects after short term implantation in rabbit animal model. Excellent healing was observed, which is comparable to that seen in response to a commercially available implant of HA-based bioglass alone.

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.

Synthesis and physicochemical, in vitro and in vivo evaluation of an anisotropic, nanocrystalline hydroxyapatite bisque scaffold with parallel‐aligned pores mimicking the microstructure of cortical bone

Scaffolds for bone regeneration are mostly prepared with an isotropic, sponge‐like structure mimicking the architecture of trabecular bone. We have developed an anisotropic bioceramic with parallel aligned pores resembling the honeycomb arrangement of Haversian canals of cortical bone and investigated its potential as a scaffold for tissue engineering. Parallel channel‐like pores were generated by ionotropic gelation of an alginate–hydroxyapatite (HA) slurry, followed by ceramic processing. Organic components were thermally removed at 650 °C, whereas the pore system was preserved in the obtained HA bioceramic in the processing stage of a bisque. Even without further sintering at higher temperatures, the anisotropic HA bisque (AHAB) became mechanically stable with a compressive strength (4.3 MPa) comparable to that of native trabecular bone. Owing to the low‐temperature treatment, a nanocrystalline microstructure with high porosity (82%) and surface area (24.9 m2/g) was achieved that kept the material dissolvable in acidic conditions, similar to osteoclastic degradation of bone. Human mesenchymal stem cells (hMSCs) adhered, proliferated and differentiated into osteoblasts when osteogenically induced, indicating the cytocompatibility of the bisque scaffold. Furthermore, we demonstrated fusion of human monocytes to osteoclast‐like cells in vitro on this substrate, similar to the natural pathway. Biocompatibility was demonstrated in vivo by implantation of the bisque ceramic into cortical rabbit femur defects, followed by histological analysis, where new bone formation inside the channel‐like pores and generation of an osteon‐like tissue morphology was observed. Copyright

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, strontiums 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.

Biocompatibility property of 100% strontium-substituted SiO2–Al2O3–P2O5–CaO–CaF2 glass ceramics over 26 weeks implantation in rabbit model: Histology and micro-Computed Tomography analysis

One of the desired properties for any new biomaterial composition is its long-term stability in a suitable animal model and such property cannot be appropriately assessed by performing short-term implantation studies. While hydroxyapatite (HA) or bioglass coated metallic biomaterials are being investigated for in vivo biocompatibility properties, such study is not extensively being pursued for bulk glass ceramics. In view of their inherent brittle nature, the implant stability as well as impact of long-term release of metallic ions on bone regeneration have been a major concern. In this perspective, the present article reports the results of the in vivo implantation experiments carried out using 100% strontium (Sr)-substituted glass ceramics with the nominal composition of 4.5 SiO2 -3Al2 O3 -1.5P2 O5 -3SrO-2SrF2 for 26 weeks in cylindrical bone defects in rabbit model. The combination of histological and micro-computed tomography analysis provided a qualitative and quantitative understanding of the bone regeneration around the glass ceramic implants in comparison to the highly bioactive HA bioglass implants (control). The sequential polychrome labeling of bone during in vivo osseointegration using three fluorochromes followed by fluorescence microscopy observation confirmed homogeneous bone formation around the test implants. The results of the present study unequivocally confirm the long-term implant stability as well as osteoconductive property of 100% Sr-substituted glass ceramics, which is comparable to that of a known bioactive implant, that is, HA-based bioglass.

Transfection of Endothelial Nitric Oxide Synthase Gene Improves Angiogenic Efficacy of Endothelial Progenitor Cells in Rabbits with Hindlimb Ischemia

Background: The present study explored the effect of endothelial nitric oxide synthase (eNOS) gene transfer on the angiogenic potential of ex vivo expanded endothelial progenitor cells (EPCs) in a rabbit model of hindlimb ischemia. Methods: Rabbit peripheral blood EPCs were cultured and transfected with mammalian expression vector pcDNA3.1-eNOS containing full-length human eNOS gene. Ischemia was induced in the right hind limb of three groups of rabbits by ligation of the distal external iliac artery and excision of the common and superficial femoral arteries. In one group of animals, ten days after the surgery, autologous eNOS-EPCs were transplanted intramuscularly in the ischemic limb. Two other groups received an equivalent number of unmodified EPCs or phosphate buffered saline (PBS) respectively. Results: Two weeks after cell transplantation, the in vivo expression of eNOS was detected in limb tissue sections of eNOS-EPCs treated animals. Animals treated with eNOS-EPCs had a significant reduction in ischemic muscle necrosis and inflammation, augmentation in the capillary density (P< 0.05) and angiographic scores demonstrating distal arterial reconstitution and enhanced angiogenesis in comparison to animals transplanted with EPCs or PBS (P< 0.05). Conclusion: We conclude that modification of EPCs by eNOS constitutes an effective strategy to improve the efficacy of EPCs for therapeutic angiogenesis.

Long-term healing of mildly cross-linked decellularized bovine pericardial aortic patch

Glutaraldehyde treated bovine pericardium is extensively used in cardiovascular surgery. However, frequent occurrence of failure modes, such as calcification and structural failure, has hard pressed the need for finding an alternate technology. Decellularized bovine pericardium is an emerging technology. Mildly cross-linked decellularized bovine pericardium promotes positive remodeling with insignificant calcification and acute inflammation. In the present study, mildly cross-linked decellularized bovine pericardium was evaluated as a cardiovascular patch by studying mechanical strength as well as graft remodeling, resistance to calcific degeneration and inflammatory response using long duration porcine aortic implantation. It was observed that decellularized bovine pericardium, although thinner and less elastic had equivalent tensile properties such as tensile strength and stiffness when compared to commercially available glutaraldehyde-treated bovine pericardium. It showed the potential for site appropriate remodeling evidenced by host cell incorporation, thinner neointima, graft degradation, and neocollagenisation making it suitable for vascular patch application, whereas glutaraldehyde-treated pericardium failed to integrate with host tissue through timely degradation and host cell incorporation or neocollagenization. Conversely, it elicited persistent acute inflammation and produced calcification.