Syam P. Nukavarapu

Electrospun nanofiber scaffolds: engineering soft tissues

Electrospinning has emerged to be a simple, elegant and scalable technique to fabricate polymeric nanofibers. Pure polymers as well as blends and composites of both natural and synthetics have been successfully electrospun into nanofiber matrices. Physiochemical properties of nanofiber matrices can be controlled by manipulating electrospinning parameters to meet the requirements of a specific application. Such efforts include the fabrication of fiber matrices containing nanofibers, microfibers, combination of nano-microfibers and also different fiber orientation/alignments. Polymeric nanofiber matrices have been extensively investigated for diversified uses such as filtration, barrier fabrics, wipes, personal care, biomedical and pharmaceutical applications. Recently electrospun nanofiber matrices have gained a lot of attention, and are being explored as scaffolds in tissue engineering due to their properties that can modulate cellular behavior. Electrospun nanofiber matrices show morphological similarities to the natural extra-cellular matrix (ECM), characterized by ultrafine continuous fibers, high surface-to-volume ratio, high porosity and variable pore-size distribution. Efforts have been made to modify nanofiber surfaces with several bioactive molecules to provide cells with the necessary chemical cues and a more in vivo like environment. The current paper provides an overlook on such efforts in designing nanofiber matrices as scaffolds in the regeneration of various soft tissues including skin, blood vessel, tendon/ligament, cardiac patch, nerve and skeletal muscle.

Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering.

Electrospun fiber matrices composed of scaffolds of varying fiber diameters were investigated for potential application of severe skin loss. Few systematic studies have been performed to examine the effect of varying fiber diameter electrospun fiber matrices for skin regeneration. The present study reports the fabrication of poly[lactic acid-co-glycolic acid] (PLAGA) matrices with fiber diameters of 150-225, 200-300, 250-467, 500-900, 600-1,200, 2,500-3,000 and 3,250-6,000 nm via electrospinning. All fiber matrices found to have a tensile modulus from 39.23+/-8.15 to 79.21+/-13.71 MPa which falls in the range for normal human skin. Further, the porous fiber matrices have porosity between 38 to 60% and average pore diameters between 10 to 14 microm. We evaluated the efficacy of these biodegradable fiber matrices as skin substitutes by seeding them with human skin fibroblasts (hSF). Human skin fibroblasts acquired a well spread morphology and showed significant progressive growth on fiber matrices in the 350-1,100 nm diameter range. Collagen type III gene expression was significantly up-regulated in hSF seeded on matrices with fiber diameters in the range of 350-1,100 nm. Based on the need, the proposed fiber skin substitutes can be successfully fabricated and optimized for skin fibroblast attachment and growth.

Osteochondral tissue engineering: current strategies and challenges.

Osteochondral defect management and repair remain a significant challenge in orthopedic surgery. Osteochondral defects contain damage to both the articular cartilage as well as the underlying subchondral bone. In order to repair an osteochondral defect the needs of the bone, cartilage and the bone-cartilage interface must be taken into account. Current clinical treatments for the repair of osteochondral defects have only been palliative, not curative. Tissue engineering has emerged as a potential alternative as it can be effectively used to regenerate bone, cartilage and the bone-cartilage interface. Several scaffold strategies, such as single phase, layered, and recently graded structures have been developed and evaluated for osteochondral defect repair. Also, as a potential cell source, tissue specific cells and progenitor cells are widely studied in cell culture models, as well with the osteochondral scaffolds in vitro and in vivo. Novel factor strategies being developed, including single factor, multi-factor, or controlled factor release in a graded fashion, not only assist bone and cartilage regeneration, but also establish osteochondral interface formation. The field of tissue engineering has made great strides, however further research needs to be carried out to make this strategy a clinical reality. In this review, we summarize current tissue engineering strategies, including scaffold design, bioreactor use, as well as cell and factor based approaches and recent developments for osteochondral defect repair. In addition, we discuss various challenges that need to be addressed in years to come.

Polyphosphazene/Nano-Hydroxyapatite Composite Microsphere Scaffolds for Bone Tissue Engineering

The nontoxic, neutral degradation products of amino acid ester polyphosphazenes make them ideal candidates for in vivo orthopedic applications. The quest for new osteocompatible materials for load bearing tissue engineering applications has led us to investigate mechanically competent amino acid ester substituted polyphosphazenes. In this study, we have synthesized three biodegradable polyphosphazenes substituted with side groups, namely, leucine, valine, and phenylalanine ethyl esters. Of these polymers, the phenylalanine ethyl ester substituted polyphosphazene showed the highest glass transition temperature (41.6 degrees C) and, hence, was chosen as a candidate material for forming composite microspheres with 100 nm sized hydroxyapatite (nHAp). The fabricated composite microspheres were sintered into a three-dimensional (3-D) porous scaffold by adopting a dynamic solvent sintering approach. The composite microsphere scaffolds showed compressive moduli of 46-81 MPa with mean pore diameters in the range of 86-145 microm. The 3-D polyphosphazene-nHAp composite microsphere scaffolds showed good osteoblast cell adhesion, proliferation, and alkaline phosphatase expression and are potential suitors for bone tissue engineering applications.

Chitosan–poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: In vitro degradation and in vivo bone regeneration studies

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation.

Differential analysis of peripheral blood‐ and bone marrow‐derived endothelial progenitor cells for enhanced vascularization in bone tissue engineering

For tissue engineering applications, effective bone regeneration requires rapid neo‐vascularization of implanted grafts to ensure the survival of cells in the early post‐implantation phase. Incorporation of autologous endothelial progenitor cells (EPCs) for the promotion of primitive vascular network formation ex vivo has offered great promise for improved graft survival, enhanced rate of vascularization and bone regeneration in vivo. For clinical usage, identification of an optimal EPC isolation source from the patient is critical. We have, for the first time, characterized and directly compared EPCs from rabbit peripheral blood and bone marrow (PB‐EPCs and BM‐EPCs, respectively). PB‐EPCs outperformed BM‐EPCs on all measures. PB‐EPCs displayed typical endothelial cell markers, such as CD31, as well as high angiogenic potential in three‐dimensional extracellular matrix in vitro. Furthermore, PB‐EPCs cultured simultaneously with mesenchymal stem cells, displayed significantly enhanced expression levels of key osteogenic and vascular markers, including alkaline phosphatase, bone morphogenetic protein 2, and vascular endothelial growth factor. On the contrary, putative BM‐EPCs did not express CD31, and instead, expressed key smooth muscle markers. BM‐EPCs further failed to display vasculogenic activity. Hence, the highly angiogenic PB‐derived EPCs may serve as an ideal cell population for enhanced vascularization and success of engineered bone tissue.

Recent Patents on Electrospun Biomedical Nanostructures: An Overview

Nanostructures in the form of tubes, wires, crystals, rods, spheres, and fibers have been fabricated and assembled into various macrostructures for a variety of high technology applications. Nanofeatures impart several amazing properties to these macrostructures including high surface area, surface functionality, and superior mechanical, optical, electrical, and magnetic properties over the parent bulk material. Polymeric nanofibers in the form of nonwoven cloth, membrane, braids and tubes are extensively used for daily needs, and in addition used as filters, protective clothing, and for a variety of industrial and biomedical applications. Electrospinning or electrostatic spinning has emerged as a very popular technique to fabricate polymeric nanofiber matrices. More than 100 different polymers of natural, synthetic origin, their blends and composites have been electrospun into different three dimensional (3-D) macrostructures. Electrospinning provides opportunities to manipulate and control surface area, fiber diameter, porosity and pore size of nanofiber matrices. These nanofiber matrices closely mimic the structure of extracellular matrix (ECM) and influence cellular activities both in vitro and in vivo. Nanofiber macrostructures have been used as a vehicle to deliver therapeutic agents, as scaffolds for engineering various tissues and also serve as an integrated part of biomedical implants. Present review will cover some of the recent important patents that use electrospun nanofiber matrices for various biomedical applications.

Dipeptide-based polyphosphazene and polyester blends for bone tissue engineering.

Polyphosphazene-polyester blends are attractive materials for bone tissue engineering applications due to their controllable degradation pattern with non-toxic and neutral pH degradation products. In our ongoing quest for an ideal completely miscible polyphosphazene-polyester blend system, we report synthesis and characterization of a mixed-substituent biodegradable polyphosphazene poly[(glycine ethyl glycinato)(1)(phenyl phenoxy)(1)phosphazene] (PNGEG/PhPh) and its blends with a polyester. Two dipeptide-based blends namely 25:75 (Matrix1) and 50:50 (Matrix2) were produced at two different weight ratios of PNGEG/PhPh to poly(lactic acid-glycolic acid) (PLAGA). Blend miscibility was confirmed by differential scanning calorimetry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Both blends resulted in higher tensile modulus and strength than the polyester. The blends showed a degradation rate in the order of Matrix2<Matrix1<PLAGA in phosphate buffered saline at 37 degrees C over 12 weeks. Significantly higher pH values of degradation media were observed for blends compared to PLAGA confirming the neutralization of PLAGA acidic degradation by polyphosphazene hydrolysis products. The blend components PLAGA and polyphosphazene exhibited a similar degradation pattern as characterized by the molecular weight loss. Furthermore, blends demonstrated significantly higher osteoblast growth rates compared to PLAGA while maintaining osteoblast phenotype over a 21-day culture. Both blends demonstrated improved biocompatibility in a rat subcutaneous implantation model compared to PLAGA over 12 weeks.

Nanotechnology and orthopedics: a personal perspective.

Bone is a nanocomposite material comprised of hierarchically arranged collagen fibrils, hydroxyapatite and proteoglycans in the nanometer scale. Cells are accustomed to interact with nanostructures, thus providing the cells with a natural bone-like environment that potentially enhance bone tissue regeneration/repair. In this direction, nanotechnology provides opportunities to fabricate as well as explore novel properties and phenomena of functional materials, devices, and systems at the nanometer-length scale. Recent studies have provided significant insights into the influence of topographical features in regulating cell behavior. Topographical features provide essential chemical and physical cues that cells can recognize and elicit desired cellular functions including preferential adhesion, migration, proliferation, and expression of specific cell phenotype to bring desired effects. The current article will address some of the nanotechnology implications in addressing issues related to orthopedic implants performance and tissue engineering approach to bone repair/regeneration.

Functionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: fabrication, in vitro and in vivo evaluation

Designing biodegradable scaffolds with bone-compatible mechanical properties has been a significant challenge in the field of bone tissue engineering and regenerative engineering. The objective of this work is to improve the polymeric scaffolds mechanical strength by compositing it with mechanically superior carbon nanotubes. Poly(lactide-co-glycolide) (PLGA) microsphere scaffolds exhibit mechanical properties in the range of human cancellous bone. On the other hand, carbon nanotubes have outstanding mechanical properties. The aim of this study is to improve further the mechanical strength of PLGA scaffolds such that they may be applicable for a wide range of load-bearing repair and regeneration applications. We have formed composite microspheres of PLGA containing pristine and modified (with hydroxyl (OH), carboxylic acid (COOH)) multi-walled carbon nanotubes (MWCNTs), and fabricated them into three-dimensional porous scaffolds. Results show that by adding only 3% MWCNTs, the compressive strength and modulus was significantly increased (35 MPa, 510.99 MPa) compared to pure PLGA scaffolds (19 MPa and 166.38 MPa). Scanning electron microscopy images showed excellent cell adhesion and proliferation. In vitro studies exhibited good cell viability, proliferation and mineralization. The in vivo study, however, indicated differences in inflammatory response throughout the 12 weeks of implantation, with OH-modified MWCNTs having the least response, followed by unmodified and COOH-modified exhibiting a more pronounced response. Overall, our results show that PLGA scaffolds containing water-dispersible MWCNTs are mechanically stronger and display good cellular and tissue compatibility, and hence are potential candidates for load-bearing bone tissue engineering.