Parthasarathy Madurantakam

Science of nanofibrous scaffold fabrication: strategies for next generation tissue-engineering scaffolds

Native extracellular matrix (ECM) provides structural support to the multicellular organism on a macroscopic scale and establishes a unique microenvironment (niche) to tissue- and organ-specific cell types. Both these functions are critical for optimal function of the organism. These natural ECMs comprise predominantly fibrillar proteins, collagen and elastin and are synthesized as monomers but undergo hierarchical organization into well-defined nanoscaled structural units. The interaction between the cells and ECM is dynamic, reciprocal and essential for tissue development, maintenance of function, repair and regeneration processes. Tissue-engineering scaffolds are synthetic, biomimetic ECM analogues that have great promise in regenerative medicine. Ongoing efforts in mimicking the native ECM in terms of composition and dimension have resulted in three strategies that permit the generation of scaffolds in nanometer dimensions. Although excellent reviews regarding the applications of these strategies in tissue engineering are available, a comprehensive review of the science behind these fabrication techniques does not exist. This review intends to fill this critical gap in the existing knowledge in the fast-expanding field of nanofibrous scaffolds. A thorough understanding of the fabrication processes would enable us to better exploit available technologies to produce superior tissue-engineering scaffolds.

Multiple factor interactions in biomimetic mineralization of electrospun scaffolds

One of the major limitations in scaffold-based bone tissue engineering has been the inability to increase the loading of biologically active inorganic mineral. The present study introduces a novel two step strategy to increase overall mineral content of electrospun scaffolds and employs multiple factor interaction as a statistic to identify the combination of factors that yields maximal scaffold mineralization. Different amounts of nHA (0, 10, 25 and 50% by wt. of polymer) were electrospun in combination with polydioxanone (PDO) or poly(glycolide: lactide) to generate composite scaffolds. Successful incorporation of nHA within, on and in between nanofibers was confirmed by transmission and scanning electron microscopy. These scaffolds were immersed in different types (conventional, revised, ionic and modified) of simulated body fluid (SBF), prepared at 1x and 4x concentrations and the incubation was carried out either in static or dynamic setting at biomimetic conditions. At 2 weeks, the total amount of mineral within the scaffold was quantified using a modified Alizarin Red-based assay. Each of the five independent factors was analyzed independently and tested for interaction using random effects ANOVA. Statistics revealed significant higher order interactions among factors and the combination of PDO containing 50% nHA incubated in 1x revised SBF resulted in maximum mineralization.

Angiogenic potential of human macrophages on electrospun bioresorbable vascular grafts

The aim of this study was to investigate macrophage interactions with electrospun scaffolds and quantify the expression of key angiogenic growth factors in vitro. This study will further help in evaluating the potential of these electrospun constructs as vascular grafts for tissue repair and regeneration in situ. Human peripheral blood macrophages were seeded in serum free media on electrospun (10 mm) discs of polydioxanone (PDO), elastin and PDO:elastin blends (50:50, 70:30 and 90:10). The growth factor secretion was analyzed by ELISA. Macrophages produced high levels of vascular endothelial growth factor and acidic fibroblast growth factor. Transforming growth factor beta-1 (TGF-beta1) secretion was relatively low and there was negligible production of basic fibroblast growth factor. Therefore, it can be anticipated that these scaffolds will support tissue regeneration and angiogenesis.

Electrospinning adipose tissue‐derived extracellular matrix for adipose stem cell culture

Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.

Evaluation of thrombogenic potential of electrospun bioresorbable vascular graft materials: acute monocyte tissue factor expression.

The purpose of this study was to quantify the acute expression of tissue factor (TF) by monocytes on interaction with electrospun bioresorbable constructs. A minimal expression of TF will demonstrate the potential for scaffolds to be used as a vascular graft without enhanced risk of failure from acute thrombotic occlusion. Polydioxanone (PDO) (60, 80, 120, and 160 mg/mL) and polycaprolactone (PCL) (80, 10, and 160 mg/mL) dissolved in 1,1,1,3,3,3 hexafluoro-2-propanol (HFP) were electrospun to form fibrous scaffolds. Circular discs (10 mm diameter) of each scaffold were disinfected and seeded with human monocytes (50,000 cells/well). The discs were statically cultured under standard conditions (37 degrees C and 5% CO2), and removed after 24 h for TF analysis with an In-Cell Western assay. Fiber diameter was calculated through ImageTool analysis of scanning electron micrographs. Acute monocyte interaction with scaffolds of PCL (120 mg/mL) resulted in the lowest amount of TF expressed (4 ng/disc), whereas scaffolds of 160 mg/mL PDO elicited the highest amount of TF expressed (51 ng/disc). TF levels expressed on all scaffolds were comparable with the amount expressed on e-PTFE (20 ng/disc). Preliminary data for TF expression on scaffolds of silk (70 mg/mL and 150 mg/mL) and silk:PCL (100 mg/mL, v/v) blends (50:50 and 70:30) resulted in values of TF expression ranging from 0 to 24 ng. Results from this study reveal electrospun grafts composed of PDO and PCL provide no greater risk of failure from an acute thrombotic occlusion due to TF expression when compared with that of the standard e-PTFE graft.

Mineralization Potential of Electrospun PDO-Hydroxyapatite-Fibrinogen Blended Scaffolds

The current bone autograft procedure for cleft palate repair presents several disadvantages such as limited availability, additional invasive surgery, and donor site morbidity. The present preliminary study evaluates the mineralization potential of electrospun polydioxanone:nano-hydroxyapatite : fibrinogen (PDO : nHA : Fg) blended scaffolds in different simulated body fluids (SBF). Scaffolds were fabricated by blending PDO : nHA : Fg in the following percent by weight ratios: 100 : 0 : 0, 50 : 25 : 25, 50 : 50 : 0, 50 : 0 : 50, 0 : 0 : 100, and 0 : 50 : 50. Samples were immersed in (conventional (c), revised (r), ionic (i), and modified (m)) SBF for 5 and 14 days to induce mineralization. Scaffolds were characterized before and after mineralization via scanning electron microscopy, Alizarin Red-based assay, and modified burnout test. The addition of Fg resulted in scaffolds with smaller fiber diameters. Fg containing scaffolds also induced sheet-like mineralization while individual fiber mineralization was noticed in its absence. Mineralized electrospun Fg scaffolds without PDO were not mechanically stable after 5 days in SBF, but had superior mineralization capabilities which produced a thick bone-like mineral (BLM) layer throughout the scaffolds. 50 : 50 : 0 scaffolds incubated in either r-SBF for 5 days or c-SBF for 14 days produced scaffolds with high mineral content and individual-mineralized fibers. These mineralized scaffolds were still porous and will be further optimized as an effective bone substitute in future studies.

Compression of Multilayered Composite Electrospun Scaffolds: A Novel Strategy to Rapidly Enhance Mechanical Properties and Three Dimensionality of Bone Scaffolds

One major limitation of electrospun scaffolds intended for bone tissue engineering is their inferior mechanical properties. The present study introduces a novel strategy to engineer stiffer scaffolds by stacking multiple layers and cold welding them under high pressure. Electrospun polydioxanone (PDO) and PDO:nanohydroxyapatite (PDO:nHA) scaffolds (1, 2, or 4 layered stacks) were compressed either before or after mineralizing treatment with simulated body fluid (SBF). After two weeks in SBF, scaffolds were analyzed for total mineral content and stiffness by Alizarin red S and uniaxial tensile testing, respectively. Scaffolds were also analyzed for permeability, pore size, and fiber diameter. Results indicated that compression of multiple layers significantly increased the stiffness of scaffolds while reducing mineralization and permeability. This phenomenon was attributed to increased density of fibers and loss of surface area due to fiber welding. Statistics revealed, the 4-layered PDO:nHA scaffold compressed first followed by mineralization in revised SBF had maximal stiffness, low permeability and pore size, and mineralization second only to noncompressed scaffolds. Within the limitations of permeability and pore size, this scaffold configuration represents an optimal midway for desired stiffness and mineral content for bone tissue engineering.

Evaluation of biological activity of bone morphogenetic proteins on exposure to commonly used electrospinning solvents

Bone tissue engineering is one of the emerging strategies for developing functionally viable bone substitutes. The recent trend in bone tissue engineering is to combine the benefits of a three-dimensional nanofibrous scaffold with biologically active molecules and responsive stem cells. Electrospinning is the most versatile of the scaffold fabrication strategies and may involve the use of an organic solvent at one stage or another. In spite of all distinct advantages of electrospinning, valid concerns about potentially denaturing interactions between the organic solvent and the biomolecules exist. Efforts are ongoing to incorporate osteoinductive molecules, such as bone morphogenetic proteins (BMPs), during the electrospinning process. The challenge lies in ensuring that the biological activity of these incorporated molecules survives the process. This study was specifically designed to investigate the effects of exposure to commonly used organic solvents on heterodimeric BMP-2/7 using slot-blot assay quantified by infrared imaging and on embryonic myoblasts stably transfected with BMP-specific response element linked to a luciferase reporter – C2C12BRA. Overall, the biological activity of these molecules significantly decreased when exposed to organic solvents but can be restored to their original values by increasing the polarity of the solvent. It was found that an aqueous buffer can effectively overcome the deleterious effects of organic solvents on BMPs, thus generating osteoinductive bone scaffolds.

Mesenchymal Stem Cells Derived from Healthy and Diseased Human Gingiva Support Osteogenesis on Electrospun Polycaprolactone Scaffolds

Periodontitis is a chronic inflammatory disease affecting almost half of the adult US population. Gingiva is an integral part of the periodontium and has recently been identified as a source of adult gingiva-derived mesenchymal stem cells (GMSCs). Given the prevalence of periodontitis, the purpose of this study is to evaluate differences between GMSCs derived from healthy and diseased gingival tissues and explore their potential in bone engineering. Primary clonal cell lines were established from harvested healthy and diseased gingival and characterized for expression of known stem-cell markers and multi-lineage differentiation potential. Finally, they were cultured on electrospun polycaprolactone (PCL) scaffolds and evaluated for attachment, proliferation, and differentiation. Flow cytometry demonstrated cells isolated from healthy and diseased gingiva met the criteria defining mesenchymal stem cells (MSCs). However, GMSCs from diseased tissue showed decreased colony-forming unit efficiency, decreased alkaline phosphatase activity, weaker osteoblast mineralization, and greater propensity to differentiate into adipocytes than their healthy counterparts. When cultured on electrospun PCL scaffolds, GMSCs from both sources showed robust attachment and proliferation over a 7-day period; they exhibited high mineralization as well as strong expression of alkaline phosphatase. Our results show preservation of ‘stemness’ and osteogenic potential of GMSC even in the presence of disease, opening up the possibility of using routinely discarded, diseased gingival tissue as an alternate source of adult MSCs.

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.