Vivek P. Soni

Enhanced mechanical strength and biocompatibility of electrospun polycaprolactone-gelatin scaffold with surface deposited nano-hydroxyapatite

In this study for the first time, we compared physico-chemical and biological properties of polycaprolactone-gelatin-hydroxyapatite scaffolds of two types: one in which the nano-hydroxyapatite (n-HA) was deposited on the surface of electrospun polycaprolactone-gelatin (PCG) fibers via alternate soaking process (PCG-HAAS) and other in which hydroxyapatite (HA) powders were blended in electrospinning solution of PCG (PCG-HAB). The microstructure of fibers was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) which showed n-HA particles on the surface of the PCG-HAAS scaffold and embedded HA particles in the interior of the PCG-HAB fibers. PCG-HAAS fibers exhibited the better Youngs moduli and tensile strength as compared to PCG-HAB fibers. Biological properties such as cell proliferation, cell attachment and alkaline phosphatase activity (ALP) were determined by growing human osteosarcoma cells (MG-63) over the scaffolds. Cell proliferation and confocal results clearly indicated that the presence of hydroxyapatite on the surface of the PCG-HAAS scaffold promoted better cellular adhesion and proliferation as compared to PCG-HAB scaffold. ALP activity was also observed better in alternate soaked PCG scaffold as compared to PCG-HAB scaffold. Mechanical strength and biological properties clearly demonstrate that surface deposited HA scaffold prepared by alternate soaking method may find application in bone tissue engineering.

Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nanofibers over hydroxyapatite: a comparative study.

The aim of this study was to compare physico-chemical and biological properties of hydroxyapatite (HA) and hardystonite (HS) based composite scaffolds. Hardystonite (Ca2ZnSi2O7) powders were synthesized by a sol-gel method while polycaprolactone-hardystonite (PCL-HS) and polycaprolactone-hydroxyapatite (PCL-HA) were fabricated in nanofibrous form by electrospinning. The physico-chemical and biological properties such as tensile strength, cell proliferation, cell infiltration and alkaline phosphatase activity were determined on both kinds of scaffolds. We found that PCL-HS scaffolds had better mechanical strength compared to PCL-HA scaffolds. Addition of HA and HS particles to PCL did not show any inhibitory effect on blood biocompatibility of scaffolds when assessed by hemolysis assay. The in vitro cellular behavior was evaluated by growing murine adipose-tissue-derived stem cells (mE-ASCs) over the scaffolds. Enhanced cell proliferation and improved cellular infiltrations on PCL-HS scaffolds were observed when compared to HA containing scaffolds. PCL-HS scaffolds exhibited a significant increase in alkaline phosphatase (ALP) activity and better mineralization of the matrix in comparison to PCL-HA scaffolds. These results clearly demonstrate the stimulatory role of Zn and Si present in HS based composite scaffolds, suggesting their potential application for bone tissue engineering.

Influence of carboxymethyl chitin on stability and biocompatibility of 3D nanohydroxyapatite/gelatin/carboxymethyl chitin composite for bone tissue engineering†

A novel three-dimensional (3D) scaffold has been developed from the unique combination of nanohydroxyapatite/gelatin/carboxymethyl chitin (n-HA/gel/CMC) for bone tissue engineering by using the solvent-casting method combined with vapor-phase crosslinking and freeze-drying. The surface morphology and physiochemical properties of the scaffold were investigated by dissolvability test, infrared absorption spectra (IR), X-ray diffraction (XRD), transmission electron microscope (TEM), scanning electron microscope (SEM), mechanical testing, and soaking in simulated body fluid (SBF). An optimized (composition and processing parameters) ratio of n-HA:gel:CMC (1:2:1), exhibited ideal porous structure with regular interconnected pores (75-250 μm) and higher mechanical strength. Result suggested that the divalent (Ca(++)), carboxyl (COO(-)), amino (NH4(+)), and phosphate (PO4(3-)) groups created favorable ionic interactions which facilitated structural stability and integrity of the composite scaffold. The SBF soaking experiment confirmed the apatite nucleation ability, induced by CMC incorporation. Furthermore, hemocompatibility (hemolysis, platelet adhesion, and protein adsorption) and biocompatibility with MG63 osteoblast cells (MTT assay, cell morphology, and confocal studies from within the 3D scaffold) indicated that the structural and dimensional stability of composite scaffold provided an optimal mechanosensory environment for enhancement of cell adhesion, proliferation, and network formation. The n-HA/gel/CMC composite, therefore, may serve as a promising composite scaffold for guided bone regeneration.

In-Vivo Efficacy of Compliant 3D Nano-Composite in Critical-Size Bone Defect Repair: a Six Month Preclinical Study in Rabbit

Bone defects above critical size do not heal completely by itself and thus represent major clinical challenge to reconstructive surgery. Numerous bone substitutes have already been used to promote bone regeneration, however their use, particularly for critical-sized bone defects along with their long term in vivo safety and efficacy remains a concern. The present study was designed to obtain a complete healing of critical-size defect made in the proximal tibia of New Zealand White rabbit, using nano-hydroxyapatite/gelatin and chemically carboxymethylated chitin (n-HA/gel/CMC) scaffold construct. The bone-implant interfaces and defect site healing was evaluated for a period up to 25 weeks using radiography, micro-computed tomography, fluorescence labeling, and histology and compared with respective SHAM (empty contra lateral control). The viscoelastic porous scaffold construct allows easy surgical insertion and post-operatively facilitate oxygenation and angiogenesis. Radiography of defect treated with scaffold construct suggested expedited healing at defect edges and within the defect site, unlike confined healing at edges of the SHAM sites. The architecture indices analyzed by micro-computed tomography showed a significant increase in percentage of bone volume fraction, resulted in reconciled cortico-trabecular bone formation at n-HA/gel/CMC constructs treated site (15.2% to 52.7%) when compared with respective SHAM (10.2% to 31.8%). Histological examination and fluorescence labeling revealed that the uniformly interconnected porous surface of scaffold construct enhanced osteoblasts’ activity and mineralization. These preclinical data suggest that, n-HA/gel/CMC construct exhibit stimulation of bones innate regenerative capacity, thus underscoring their use in guided bone regeneration.

Bone healing performance of electrophoretically deposited apatite–wollastonite/chitosan coating on titanium implants in rabbit tibiae

Bone healing of tibial defect in rabbit model was used to evaluate a composite coating of apatite–wollastonite/chitosan on titanium implant. This coating has been developed to overcome the shortcomings, such as implant loosening and lack of adherence, of uncoated titanium implant. An electrophoretic deposition technique was used to coat apatite–wollastonite/chitosan on titanium implants. The present study was designed to evaluate the bone response of coated as compared to uncoated titanium implants in an animal model. After an implantation period of 14 (group A), 21 (group B), 35 (group C) and 42 days (group D), the bone–implant interfaces and defect site healing was evaluated using radiography, scintigraphy, histopathology, fluorescence labeling and haematology. Radiography of defect sites treated with coated implants suggested expedited healing. Scintigraphy of coated implant sites indicated faster bone metabolism than uncoated implant sites. Histopathological examination and fluorescence labeling of bone from coated implant sites revealed higher osteoblastic activity and faster mineralization. Faster bone healing in the case of coated implant sites is attributed to higher cell adhesion on electrostatically charged chitosan surfaces and apatite–wollastonite‐assisted mineralization at bone–implant interfaces. Haematological studies showed no significant differences in haemoglobin, total erythrocyte and leukocyte counts, done using one way‐ANOVA, during the entire study period. Our results show that AW/chitosan‐coated implants have the advantages of faster bone healing, increased mechanical strength and good bone–implant bonding. Copyright

Mineralization of nanohydroxyapatite on electrospun poly(l-lactic acid)/gelatin by an alternate soaking process: A biomimetic scaffold for bone regeneration:

Biomimetic nanocomposite scaffolds were fabricated by electrospinning poly(l-lactic acid) and a blend of poly(L-lactic acid)/gelatin to eliminate the use of collagen. The scaffolds were mineralized via alternate soaking in calcium and phosphate solutions, whereby 66.8% nanohydroxyapatite formation was successfully induced which is similar to that of native human bone (60%). The poly(L-lactic acid)/gelatin scaffolds had uniform nanohydroxyapatite formation throughout the scaffold. The mineralization enhanced the tensile modulus and tensile strength without increasing the brittleness. The in vitro biocompatibility of scaffolds was evaluated with murine adipose tissue–derived stem cells. The scaffolds with nanohydroxyapatite aided cell attachment and promoted cell–cell interaction. The mineralization and osteocalcin expression of the murine adipose tissue–derived stem cells were maximum in the poly(L-lactic acid)/gelatin/nanohydroxyapatite scaffold. Therefore, the gelatin and nanohydroxyapatite in poly(L-lactic acid)/gelatin/nanohydroxyapatite scaffolds provided cues for the differentiation of murine adipose tissue–derived stem cells. The biochemical nature of poly(L-lactic acid)/gelatin/nanohydroxyapatite scaffold accelerated osteogenic differentiation and could be a potential candidate for bone regeneration.

Electrophoretic deposition of nanobiocomposites for orthopedic applications: influence of current density and coating duration.

Frequently metal implants undergo detachment from the host tissue due to inadequate biocompatibility and poor osteointegration. In view of this, bioactive porous apatite-wollastonite/chitosan nanocomposite coating was prepared using electrophoretic deposition (EPD) technique in the present work. The effect of coating duration and current density on surface characteristics of the nanocomposite coating was assessed using optical microscope and scanning electron microscope. EPD led to the formation of thick and homogeneous coating. Adhesion of the composite coating on titanium substrate was evaluated using tape test and bioactivity of the coatings was studied by immersing in simulated body fluid (SBF). The coating with higher current density and longer coating duration was found to be suitable with improved adhesion and bioactivity for intended metal implants.

Bioconductive 3D nano-composite constructs with tunable elasticity to initiate stem cell growth and induce bone mineralization

Bioactive 3D composites play an important role in advanced biomaterial design to provide molecular coupling and improve integrity with the cellular environment of the native bone. In the present study, a hybrid lyophilized polymer composite blend of anionic charged sodium salt of carboxymethyl chitin and gelatin (CMChNa-GEL) reinforced with nano-rod agglomerated hydroxyapatite (nHA) has been developed with enhanced biocompatibility and tunable elasticity. The scaffolds have an open, uniform and interconnected porous structure with an average pore diameter of 157±30μm and 89.47+0.03% with four dimensional X-ray. The aspect ratio of ellipsoidal pores decrease from 4.4 to 1.2 with increase in gelatin concentration; and from 2.14 to 1.93 with decrease in gelling temperature. The samples were resilient with elastic stain at 1.2MPa of stress also decreased from 0.33 to 0.23 with increase in gelatin concentration. The crosslinker HMDI (hexamethylene diisocyanate) yielded more resilient samples at 1.2MPa in comparison to glutaraldehyde. Increased crosslinking time from 2 to 4h in continuous compression cycle show no improvement in maximum elastic stain of 1.2MPa stress. This surface elasticity of the scaffold enables the capacity of these materials for adherent self renewal and cultivation of the NTERA-2 cL.D1 (NT2/D1), pluripotent embryonal carcinoma cell with biomechanical surface, as is shown here. Proliferation with MG-63, ALP activity and Alizarin red mineralization assay on optimized scaffold demonstrated ***p<0.001 between different time points thus showing its potential for bone healing. In pre-clinical study histological bone response of the scaffold construct displayed improved activity of bone regeneration in comparison to self healing of control groups (sham) up to week 07 after implantation in rabbit tibia critical-size defect. Therefore, this nHA-CMChNa-GEL scaffold composite exhibits inherent and efficient physicochemical, mechanical and biological characteristics based on gel concentrations, gelatin mixing and gelling temperature thus points to creating bioactive 3D scaffolds with tunable elasticity for orthopedic applications.

Osteogenic Nanofibrous Coated Titanium Implant Results in Enhanced Osseointegration: In Vivo Preliminary Study in a Rabbit Model

A titanium implant surface when coated with biodegradable, highly porous, osteogenic nanofibrous coating has shown enhanced intrinsic osteoinductive and osteoconductive properties. This coating mimics extracellular matrix resulting in differentiation of stem cells present in the peri-implant niche to osteoblast and hence results in enhanced osseointegration of the implant. The osteogenic nanofibrous coating (ONFC) consists of poly-caprolactone, gelatin, nano-sized hydroxyapatite, dexamethasone, ascorbic acid and beta-glycerophosphate. ONFC exhibits optimum mechanical properties to support mesenchymal stem cells and steer their osteogenic differentiation. ONFC was subjected to various characterization tests like scanning electron microscopy, Fourier-transform infrared spectroscopy, x-ray diffractometry, thermal degradation, biomineralization, mechanical properties, wettability and proliferation assay. In pre-clinical animal trials, the coated implant showed enhanced new bone formation when placed in the tibia of rabbit. This novel approach toward implant bone integration holds significant promise for its easy and economical coating thus marking the beginning of new era of electrospun osteogenic nanofibrous coated bone implants.

Comparative bone regeneration study of hardystonite and hydroxyapatite as filler in critical-sized defect of rat calvaria

There is a very significant and well-known clinical need for the development of new osteoinductive materials and the establishment of alternative therapies for the treatment of bone tissue loss or failure resulting from injury or disease as the transplantation of tissues in patients with these injury or disease is severely limited by donor scarcity and is highly associated to the risk of immune rejection and disease transfer. Herein, we studied in vivo bone response by quantifying efficacy and safety of three scaffold variations: (1) nanofibrous polycaprolactone (PCL), (2) PCL–hydroxyapatite (HA), and (3) PCL–hardystonite (HS) against SHAM as the control. Diffraction pattern from TEM showed that native HA and HS were polycrystalline and they leached higher ppm of calcium, phosphorus and zinc as compared to PCL–HA and PCL–HS in which HA, HS were incorporated in PCL nanofibers. The study was performed on 8 mm critical-sized rat calvarial defects analyzed at two timepoints, 6 and 12 weeks. The bone regenerated by PCL–HS promoted higher growth than that by SHAM and PCL alone at 12 weeks with comparable bone mineral density in all groups at both time points. PCL–HS showed potential for bone growth similar to that for PCL–HA. Histology data showed dense bone interface being formed at the site in both the PCL–HS and PCL–HA groups. Therefore, HS was found to have comparable functionality with commercial HA. No significant differences were noted in any of the blood parameters but there were differences in serum biochemistry parameters of triglyceride and creatine levels among groups, which are indirectly related to bone forming potential and directly to safety of kidney function, while the other parameters were unchanged and within the normal range. Thus, we conclude that the HS material can be a suitable substitute for bone tissue engineering.