Production,blood compatibility and cytotoxicity evaluation of a single stage non-woven multicomponent electrospun scaffold mixed with sesame oil,honey and propolis for skin tissue engineering

Scaffolds used in skin tissue engineering must mimic the native function of the extracellular matrix (ECM) and facilitate the fibroblast cell response for new tissue growth. In this study, a novel dressing scaffold based on polyurethane (PU) with sesame oil, honey, and propolis was fabricated by electrospinning. Scanning electron microscopy (SEM) images showed that the diameter of the electrospun scaffolds decreased by blending sesame oil (784?±?125.46?nm) and sesame oil/honey/propolis (576?±?133.72?nm) into the PU matrix (890?±?116.911?nm). Fourier infrared (FT-IR) and thermogravimetric (TGA) analysis demonstrated the formation of hydrogen bonds and interaction between PU and sesame oil, honey, and propolis. Contact-angle measurement indicated reduced wettability of PU/sesame oil scaffold (114?±?1.732) and improved wettability (54.33?±?1.528) in the PU/sesame oil/honey/propolis scaffold. Further, tensile tests and atomic force microscopy (AFM) analysis indicated that the fabricated composite membrane exhibited enhanced mechanical strength and reduced surface roughness compared to the pristine PU. The developed composite displayed less toxicity to the red blood cells (RBC’s) compared to the pristine PU. Cytotoxicity assay showed enhanced cell viability of HDF in electrospun scaffolds than pristine PU after 72?h culture. These enhanced properties of the developed scaffolds suggest the potential of utilizing them in skin tissue engineering.     

Green synthesis of nickel oxide particles and its integration into polyurethane scaffold matrix ornamented with groundnut oil for bone tissue engineering

Physiochemical properties of the fabricated scaffolds play a crucial role in influencing the cellular response for the new tissue growth. In this study, electrospun polyurethane (PU) scaffolds incorporated with green synthesized nickel oxide nanoparticles and groundnut oil (GO) were fabricated using electrospinning technique. First, synthesis of nickel oxide (NiO) was done using leaf extract of Plectranthus amboinicus (PA) via microwave-assisted technique. Synthesized nanoparticles were confirmed through Energy-dispersive X-ray spectroscopy (EDX) analysis and size of the particles were in the range of 800–950?nm. Fiber morphology of the fabricated scaffolds was analyzed using scanning electron microscope (SEM) which showed decrease in fiber diameter for the fabricated composites compared to the pristine PU. The wettability studies showed an increase in contact angle for developed composites than the pure PU. Thermal analysis depicted an increase in thermal behavior for the PU/GO/NiO compared to the pristine PU. Surface roughness values were obtained through atomic force microscopy (AFM) which showed a decrease in roughness while adding GO and NiO to the PU. Finally, the fabricated composites showed enhanced deposition of calcium content than the pristine PU. These results corroborated that the developed composites have a significant effect on the fiber morphology, wettability, thermal behavior, surface roughness, and mineral deposition depicting its versatility for bone regeneration.     

In vitro blood compatibility and bone mineralization aspects of polymeric scaffold laden with essential oil and metallic particles for bone tissue engineering

Bone tissue engineering scaffolds necessities appropriate physicochemical and mechanical properties to support its renewal. Electrospun scaffolds have been used unequivocally in bone tissue restoration. The main intention of this research is to develop electrospun polyurethane (PU) scaffold decorated with metallic particles and essential oil with advanced properties to make them as a putative candidate. The nanocomposite scaffold exhibited appropriate wettability and suitable fiber diameter compared to the polyurethane scaffold. Interaction of the added constituents with the polyurethane was corroborated through hydrogen bonding formation. Tensile strength of the composites was enhanced compared to the polyurethane scaffold. Thermal analysis depicted the lower weight loss of the composite scaffold than the pristine PU. Blood coagulation was significantly delayed and also the composite surface rendered safe interaction with red blood cells. In vitro toxicity testing using fibroblast cells portrayed the nontoxic behavior of the fabricated material. The above-said advanced properties of the composite scaffold can be warranted for bone tissue engineering application.     

Fabrication and characterization of polyurethane patch loaded with palmarosa and cobalt nitrate for cardiac tissue engineering

Cardiac patches are attractive option in overcoming the morbidities associated with cardiac disorders. Nanofibrous scaffolds were fabricated using polyurethane (PU) added with palmarosa (PR) and cobalt nitrate (CoNO₃) using an electrospinning technique. Several characterizations were employed namely field emission scanning electron microscopy, wettability measurement, attenuated total reflectance infrared spectroscopy, thermal analysis, surface roughness measurements, and tensile testing. Further, biological response of the electrospun nanofibers were tested through coagulation study and MTS assay. As-spun composite mats showed smaller fibers than pure PU as depicted in morphology analysis. The interaction of PU with PR and CoNO₃ was confirmed in infrared spectrum and thermal analysis. The incorporation of the PR decreased the wettability and while CoNO₃ addition resulted in the hydrophilic nature as depicted in the contact angle measurements. Mechanical properties testing showed that elongation at break for the pristine PU was increased with the addition of PR and CoNO_3. The surface measurements depicted that the incorporation of PR resulted in the improvement of the surface roughness while the addition of CoNO₃ reduced the surface roughness of the pristine PU. The electrospun nanocomposites showed delayed blood clotting time compared to the pristine PU as shown in coagulation study. Both composites supported the better proliferation of fibroblast cells than pure PU. Therefore, novel composites with smaller fiber diameter, hydrophilicity, better mechanical properties, improved blood compatibility parameters, and good cell viability rates would be a promising candidate for cardiac tissue engineering.     

Nanofiber Configuration of Electrospun Scaffolds Dictating Cell Behaviors and Cell-scaffold Interactions

Electrospun nanofibers are of the same length scale as the native extracellular matrix and have been extensively reported to facilitate adhesion and proliferation of cells and to promote tissue repair and regeneration. With a primary focus on tissue repair and regeneration using electrospun scaffolds, only a few studies involved electrospun nanofiber scaffolds directing cell behaviors have been reported. In this study, we prepared electrospun nanofiber scaffolds with distinct fiber configurations, namely, random and aligned orientations of nanofibers, as well as oriented yarns, and investigated their effects on cell behaviors. Our results showed that these scaffolds supported good proliferation and viability of murine fibroblasts. Fiber configuration profoundly influenced cell morpho-logy and orientation but showed no effects on cell proliferation rate. The yarn scaffold had comparable total protein accumulation with the random and aligned scaffolds, but it supported a greater pro-liferation rate of fibroblasts with significantly elevated collagen de-position due to its porous fibrous configuration. Cell-seeded yarn scaffolds showed a greater Young's modulus compared with cell-free controls as early as 1 week. Together with its unique fiber configuration similar to the native extracellular matrix of the myocardium, the yarn scaffold might be a suitable matrix material for modeling cardiac fibrotic disorders.     

Engineered polymer matrix novel biocompatible materials decorated with eucalyptus oil and zinc nitrate with superior mechanical and bone forming abilities

Electrospun scaffolds based on polymer-matrix composites have gained wide attention recently. A novel engineered biocompatible scaffold is manufactured using polyurethane (PU) loaded with eucalyptus oil (EL) and Zinc nitrate (ZnNO₃) using the electrospinning technique. Morphological observations revealed the reduced fibre diameter for the PU/EL and PU/EL/ZnNO₃ compared to PU. Contact angle studies indicated the increase in hydrophobic behaviour of the PU/EL whereas an increase in wettability for PU/EL/ZnNO₃ compared to PU. EL and ZnNO₃ presence in the PU matrix enhanced the mechanical strength. Surface topology analysis showed a decrease in the roughness for the PU/EL and PU/EL/ZnNO₃ compared to the pristine PU. Both PU/EL and PU/EL/ZnNO₃ showed prolonged clotting time and decreased haemolytic percentage compared to the polyurethane as indicated in their anticoagulation studies. In vitro bone mineralisation testing depicted the increase in calcium deposition for the modified PU samples compared to pure polyurethane sample. Hence, PU/EL and PU/EL/ZnNO₃ scaffold with superior properties render full avenues for new bone generation.     

Fabrication of graphene‐silver/polyurethane nanofibrous scaffolds for cardiac tissue engineering

The graphene‐based nanocomposites are considered as great candidates for enhancing electrical and mechanical properties of nonconductive scaffolds in cardiac tissue engineering. In this study, reduced graphene oxide‐silver (rGO‐Ag) nanocomposites (1 and 2 wt%) were synthesized and incorporated into polyurethane (PU) nanofibers via electrospinning technique. Next, the human cardiac progenitor cells (hCPCs) were seed on these scaffolds for in vitro studies. The rGO‐Ag nanocomposites were studied by X‐ray diffraction (XRD), Raman spectroscopy, and transmission electron microscope (TEM). After incorporation of rGO‐Ag into PU nanofibers, the related characterizations were carried out including scanning electron microscope (SEM), TEM, water contact angle, and mechanical properties. Furthermore, PU and PU/nanocomposites scaffolds were used for in vitro studies, wherein hCPCs showed good cytocompatibility via 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay and considerable attachment on the scaffold using SEM studies. Real‐time polymerase chain reaction (PCR) and immunostaining studies confirmed the upregulation of cardiac specific genes including GATA‐4, T‐box 18 (TBX 18), cardiac troponin T (cTnT), and alpha‐myosin heavy chain (α‐MHC) in the PU/rGO‐Ag scaffolds in comparison with neat PU ones. Therefore, these nanofibrous rGO‐Ag–reinforced PU scaffolds can be considered as suitable candidates in cardiac tissue engineering.     

Electrospun nanofibrous polycaprolactone scaffolds for tissue engineering of annulus fibrosus

The annulus fibrosus comprises concentric lamellae that can be damaged due to intervertebral disc degeneration; to provide permanent repair of these acquired structural defects, one solution is to fabricate scaffolds that are designed to support the growth of annulus fibrosus cells. In this study, electrospun nanofibrous scaffolds of polycaprolactone are fabricated in random, aligned, and round-end configurations. Primary porcine annulus fibrosus cells are grown on the scaffolds and evaluated for attachment, proliferation, and production of extracellular matrix. The scaffold consisting of round-end nanofibers substantially outperforms the random and aligned scaffolds on cell adhesion; additionally, the scaffold with aligned nanofibers strongly affects the orientation of cells.     

Incorporation of nanohydroxyapatite and vitamin D3 into electrospun PCL/Gelatin scaffolds: The influence on the physical and chemical properties and cell behavior for bone tissue engineering

Scaffold, an essential element of tissue engineering, should provide proper physical and chemical properties and evolve suitable cell behavior for tissue regeneration. Polycaprolactone/Gelatin (PCL/Gel)‐based nanocomposite scaffolds containing hydroxyapatite nanoparticles (nHA) and vitamin D3 (Vit D3) were fabricated using the electrospinning method. Structural and mechanical properties of the scaffold were determined by scanning electron microscopy (SEM) and tensile measurement. In this study, smooth and bead‐free morphology with a uniform fiber diameter and optimal porosity level with appropriate pore size was observed for PCL/Gel/nHA nanocomposite scaffold. The results indicated that adding nHA to PCL/Gel caused an increase of the mechanical properties of scaffold. In addition, chemical interactions between PCL, gelatin, and nHA molecules were shown with XRD and FT‐IR in the composite scaffolds. MG‐63 cell line has been cultured on the fabricated composite scaffolds; the results of viability and adhesion of cells on the scaffolds have been confirmed using MTT and SEM analysis methods. Here in this study, the culture of the osteoblast cells on the scaffolds showed that the addition of Vit D3 to PCL/Gel/nHA scaffold caused further attachment and proliferation of the cells. Moreover, DAPI staining results showed that the presence and viability of the cells were greater in PCL/Gel/nHA/Vit D3 scaffold than in PCL/Gel/nHA and PCL/Gel scaffolds. The results also approved increasing cell proliferation and alkaline phosphatase (ALP) activity for MG‐63 cells cultured on PCL/Gel/nHA/Vit D3 scaffold. The results indicated superior properties of hydroxyapatite nanoparticles and vitamin D3 incorporated in PCL/Gel scaffold for use in bone tissue engineering.     

Formation of functional nanofibrous electrospun polyurethane and murivenna oil with improved haemocompatibility for wound healing

Electrospinning is an emerging tool and promising method to fabricate polymer nanofibers. The aim of this work was to fabricate electrospun polyurethane mats reinforced with murivenna oil for wound dressings. The scanning electron microscopy (SEM) micrographs showed the fiber diameter of nanocomposites in the range of 740 ± 160 nm and found to be decreased compared to pure polyurethane. Surface of nanocomposites was analyzed by Fourier transform infrared spectroscopy (FTIR) insinuated the interactions between PU and murivenna oil by the formation of hydrogen bond and changes in the characteristics peaks. Contact angle of the PU incorporated murivenna oil showed a decrease in its value compared to pure PU indicating the increased wettability and hydrophilic nature. The thermal degradation and stability of fabricated composites was found be enhanced compared to pure PU. The surface morphology through atomic force microscopy (AFM) analysis showed a change in surface roughness due to presence of murivenna oil in the polymer matrix. In blood compatibility results, both activated partial thromboplastin time (APTT) and prothrombin time (PT) were delayed due to improved surface properties and the addition of murivenna oil in the PU matrix. Compared to pure PU, the hemolysis assay of the PU incorporated murivenna oil showed a significant decrease in the percentage of lysis of red blood cells indicating better blood compatibility. Following the results, it was confirmed that fabricated novel scaffolds having better physicochemical and enhanced blood compatibility properties may be utilized for wound dressing.     

Magnesium-containing silk fibroin/polycaprolactone electrospun nanofibrous scaffolds for accelerating bone regeneration

Bone tissue engineering has become one of the most effective methods for treating bone defects. In this study, an electrospun tissue engineering membrane containing magnesium was successfully fabricated by incorporating magnesium oxide (MgO) nanoparticles into silk fibroin and polycaprolactone (SF/PCL)-blend scaffolds. The release kinetics of Mg²⁺ and the effects of magnesium on scaffold morphology, and cellular behavior were investigated. The obtained Mg-functionalized nanofibrous scaffolds displayed controlled release of Mg²⁺, satisfactory biocompatibility and osteogenic capability. The in vivo implantation of magnesium-containing electrospun nanofibrous membrane in a rat calvarial defect resulted in the significant enhancement of bone regeneration twelve weeks post-surgery. This work represents a valuable strategy for fabricating functional magnesium-containing electrospun scaffolds that show potential in craniofacial and orthopedic applications.     

Optimization of fibrin gelation for enhanced cell seeding and proliferation in regenerative medicine applications

In order to improve the cell seeding efficiency and cell compatibility inside porous tissue scaffolds, a method of fibrin gel‐mediated cell encapsulation inside the scaffold was optimized. Disc‐type poly(d ,l ‐glycolic‐co‐lactic acid) (PLGA) scaffolds without a dense surface skin layer were fabricated using an established solvent casting and particulate leaching method as a model porous scaffold, which showed high porosity ranging from 90 ± 2% to 96 ± 2%. The thrombin and fibrinogen concentration as precursors of fibrin gel was varied to control the gelation kinetics as measured by rheology analysis, and optimized conditions were developed for a uniform fibrin gel formation with the target cells inside the porous PLGA scaffold. The fibroblast cell seeding accompanied by a uniform fibrin gel formation at an optimized gelation condition inside the PLGA scaffold resulted in an increase in cell seeding efficiency, a better cell proliferation, and an increase in final cell density inside the scaffold. Scanning electron microscopy images revealed that cells were better spread and grown by fibrin gel encapsulation inside scaffold compared with the case of bare PLGA scaffold. Copyright © 2016 John Wiley & Sons, Ltd.     

Cellulose nanocrystals as biobased nucleation agents in poly-l-lactide scaffold: Crystallization behavior and mechanical properties

The mechanical strength of polymer scaffold is closely related to its crystallinity. In this work, cellulose nanocrystals (CNC) were incorporated into poly-l-lactide (PLLA) scaffold which was fabricated by selective laser sintering, aiming to improve the mechanical properties. CNC possesses numerous hydroxyl groups which might form hydrogen bond with PLLA molecular chains. The hydrogen bond induces the ordered arrangement of PLLA chain by using CNC as heterogeneous nucleating agent, thereby increasing crystallization rate and crystallinity. Results showed that PLLA scaffolds with 3 wt% CNC resulted in 191%, 351%, 34%, 83.5%, 56% increase in compressive strength, compressive modulus, tensile strength, tensile modulus and Vickers hardness, respectively. Encouragingly, with the incorporation of hydrophilic CNC, the PLLA/CNC scaffolds showed not only better hydrophilicity, but also faster degradation than PLLA. In vitro cell culture studies proved that the PLLA/CNC scaffolds were biocompatible and capable of supporting cell adhesion, proliferation and differentiation. The above results indicated that the PLLA/CNC scaffolds may therefore be a potential replacement in bone repair.     

Dual-syringe reactive electrospinning of cross-linked hyaluronic acid hydrogel nanofibers for tissue engineering applications

A facile fabrication of a cross-linked hyaluronic acid (HA) hydrogel nanofibers by a reactive electrospinning method is described. A thiolated HA derivative, 3,3'-dithiobis(propanoic dihydrazide)-modified HA (HA-DTPH), and poly(ethylene glycol) diacrylate (PEGDA) are selected as the cross-linking system. The cross-linking reaction occurs simultaneously during the electrospinning process using a dual-syringe mixing technique. Poly(ethylene oxide) (PEO) is added into the spinning solution as a viscosity modifier to facilitate the fiber formation and is selectively removed with water after the electrospinning process. The nanofibrous structure of the electrospun HA scaffold is well preserved after hydration with an average fiber diameter of 110 nm. A cell morphology study on fibronectin (FN)-adsorbed HA nanofibrous scaffolds shows that the NIH 3T3 fibroblasts migrate into the scaffold through the nanofibrous network, and demonstrate an elaborate three-dimensional dendritic morphology within the scaffold, which reflects the dimensions of the electrospun HA nanofibers. These results suggest the application of electrospun HA nanofibrous scaffolds as a potential material for wound healing and tissue regeneration. [image: see text] Laser scanning confocal microscopy demonstrates that the NIH3T3 fibroblast develops an extended 3D dendritic morphology within the fibronectin-adsorbed electrospun HA nanofibrous scaffold.     

Thermally produced biodegradable scaffolds for cartilage tissue engineering

A novel process was developed to fabricate biodegradable polymer scaffolds for tissue engineering applications, without using organic solvents. Solvent residues in scaffolds fabricated by processes involving organic solvents may damage cells transplanted onto the scaffolds or tissue near the transplantation site. Poly(L-lactic acid) (PLLA) powder and NaCl particles in a mold were compressed and subsequently heated at 180 degrees C (near the PLLA melting temperature) for 3 min. The heat treatment caused the polymer particles to fuse and form a continuous matrix containing entrapped NaCl particles. After dissolving the NaCl salts, which served as a porogen, porous biodegradable PLLA scaffolds were formed. The scaffold porosity and pore size were controlled by adjusting the NaCl/PLLA weight ratio and the NaCl particle size. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting/particulate leaching (SC/PL) process, in terms of pore structure, pore-size distribution, and mechanical properties. A scanning electron microscopic examination showed highly interconnected and open pore structures in the scaffolds fabricated using the thermal process, whereas the SC/PL process yielded scaffolds with less interconnected and closed pore structures. Mercury intrusion porosimetry revealed that the thermally produced scaffolds had a much more uniform distribution of pore sizes than the SC/PL process. The utility of the thermally produced scaffolds was demonstrated by engineering cartilaginous tissues in vivo. In summary, the thermal process developed in this study yields tissue-engineering scaffolds with more favorable characteristics, with respect to, freedom from organic solvents, pore structure, and size distribution than the SC/PL process. Moreover, the thermal process could also be used to fabricate scaffolds from polymers that are insoluble in organic solvents, such as poly(glycolic acid). Cartilage tissue regenerated from thermally produced PLLA scaffold.     

In situ mineralization of hydroxyapatite on electrospun chitosan-based nanofibrous scaffolds

A biocomposite of hydroxyapatite (HAp) with electrospun nanofibrous scaffolds was prepared by using chitosan/polyvinyl alcohol (CS/PVA) and N-carboxyethyl chitosan/PVA (CECS/PVA) electrospun membranes as organic matrix, and HAp was formed in supersaturated CaCl2 and KH2PO4 solution. The influences of carboxylic acid groups in CECS/PVA fibrous scaffold and polyanionic additive poly(acrylic acid) (PAA) in the incubation solution on the crystal distribution of the HAp were investigated. Field-emission scanning electron microscopy (FE-SEM), energy-dispersive spectroscopy (EDS), wide-angle X-ray diffraction (WAXD), and Fourier transform infrared (FTIR) were used to characterize the morphology and structure of the deposited mineral phase on the scaffolds. It was found that addition of PAA to the mineral solution and use of matrix with carboxylic acid groups promoted mineral growth and distribution of HAp. MTT testing and SEM imaging from mouse fibroblast (L929) cell culture revealed the attachment and growth of mouse fibroblast on the surface of biocomposite scaffold, and that the cell morphology and viability were satisfactory for the composite to be used in bioapplications.     

Valve leaflet‐inspired elastomeric scaffolds with tunable and anisotropic mechanical properties

Fibrous scaffolds, which can mimic the elastic and anisotropic mechanical properties of native tissues, hold great promise in recapitulating the native tissue microenvironment. We previously fabricated electrospun fibrous scaffolds made of hybrid synthetic elastomers (poly(1,3‐diamino‐2‐hydroxypropane‐co‐glycerol sebacate)‐co‐poly (ethylene glycol) (APS‐co‐PEG) and polycaprolactone (PCL)) to obtain uniaxial mechanical properties similar to those of human aortic valve leaflets. However, conventional electrospinning process often yields scaffolds with random alignment, which fails to recreate the anisotropic nature of most of the soft tissues such as native heart valves. Inspired by the structure of native valve leaflet, we designed a novel valve leaflet‐inspired ring‐shaped collector to modulate the electrospun fiber alignment and studied the effect of polymer formulation (PEG amount [mole %] in APS‐co‐PEG; ratio between APS‐co‐PEG and PCL; and total polymer concentration) in tuning the biaxial mechanical properties of the fibrous scaffolds. The fibrous scaffolds collected on the ring‐shaped collector displayed anisotropic biaxial mechanical properties, suggesting that their biaxial mechanical properties are closely associated with the fiber alignment in the scaffold. Additionally, the scaffold stiffness was easily tuned by changing the composition and concentration of the polymer blend. Human valvular interstitial cells (hVICs) cultured on these anisotropic scaffolds displayed aligned morphology as instructed by the fiber alignment. Overall, we generated a library of biologically relevant fibrous scaffolds with tunable mechanical properties, which will guide the cellular alignment.     

Scaffold Design for Tissue Engineering

Tissue engineering has emerged as a promising alternative approach in the treatment of malfunctioning or lost organs. In this approach, a temporary scaffold is needed to serve as an adhesive substrate for the implanted cells and a physical support to guide the formation of the new organs. In addition to facilitating cell adhesion, promoting cell growth, and allowing the retention of differentiated cell functions, the scaffold should be biocompatible, biodegradable, highly porous with a large surface/volume ratio, mechanically strong, and malleable. A number of three‐dimensional porous scaffolds fabricated from various kinds of biodegradable materials have been developed. This paper reviews some of the advances in scaffold design focusing on the hybrid scaffolds recently developed in the authors' laboratory.     

Polycaprolactone-polymethyl methacrylate electrospun blends for biomedical applications

Electrospinning is one of most versatile process to fabricate porous scaffolds in biomedical field. Synthetic polymers such as polycaprolactone (PCL) and polymethyl methacrylate (PMMA) provide excellent properties for biomedical applications due to their biocompatibility and tunable mechanical properties. PCL-PMMA electrospun blends combine compressive/tensile properties of individual polymers as well as biocompatibility/biodegradability. Together with porosity of scaffold, drug/nutrient supply is required in tissue regeneration and healing. High pressure CO₂ has been investigated to plasticize many biopolymers and impregnate drugs in scaffolds. This study explores several compositions of PCL-PMMA electrospun scaffolds for morphological and mechanical properties. These scaffolds are impregnated with hydrophilic (Rhodamine B) and hydrophobic (Fluorescein) dyes using high pressure CO₂ and air plasma treatment. Furthermore, release profiles of dyes have been studied from thin films and porous scaffolds to understand several controlling factors for controlled release applications. Results show dye-polymer interactions, CO₂ impregnation and stress relaxation of electrospun fibers are key factors in release profile from electrospun fibers. This study is a step forward in developing PCL-PMMA based electrospun scaffolds for drug delivery and tissue engineering.