Simultaneous Delivery of Highly Diverse Bioactive Compounds from Blend Electrospun Fibers for Skin Wound Healing.

Blend emulsion electrospinning is widely perceived to destroy the bioactivity of proteins, and a blend emulsion of water-soluble and nonsoluble molecules is believed to be thermodynamically unstable to electrospin smoothly. Here we demonstrate a method to retain the bioactivity of disparate fragile biomolecules when electrospun. Using bovine serum albumin as a carrier protein; water-soluble vitamin C, fat soluble vitamin D3, steroid hormone hydrocortisone, peptide hormone insulin, thyroid hormone triiodothyronine (T3), and peptide epidermal growth factor (EGF) were simultaneously blend-spun into PLGA-collagen nanofibers. Upon release, vitamin C maintained the ability to facilitate Type I collagen secretion by fibroblasts, EGF stimulated skin fibroblast proliferation, and insulin potentiated adipogenic differentiation. Transgenic cell reporter assays confirmed the bioactivity of vitamin D3, T3, and hydrocortisone. These factors concertedly increased keratinocyte and fibroblast proliferation while maintaining keratinocyte basal state. This method presents an elegant solution to simultaneously deliver disparate bioactive biomolecules for wound healing applications.

Microstructure and properties of nano-fibrous PCL-b-PLLA scaffolds for cartilage tissue engineering.

Nano-fibrous scaffolds which could potentially mimic the architecture of extracellular matrix (ECM) have been considered a good candidate matrix for cell delivery in tissue engineering applications. In the present study, a semicrystalline diblock copolymer, poly(epsilon-caprolactone)-block-poly(L-lactide) (PCL-b-PLLA), was synthesized and utilized to fabricate nano-fibrous scaffolds via a thermally induced phase separation process. Uniform nano-fibrous networks were created by quenching a PCL-b-PLLA/THF homogenous solution to -20 degrees C or below, followed by further gelation for 2 hours due to the presence of PLLA and PCL microcrystals. However, knot-like structures as well as continuously smooth pellicles appeared among the nano-fibrous network with increasing gelation temperature. DSC analysis indicated that the crystallization of PCL segments was interrupted by rigid PLLA segments, resulting in an amorphous phase at high gelation temperatures. Combining TIPS (thermally induced phase separation) with salt-leaching methods, nano-fibrous architecture and interconnected pore structures (144+/-36 mm in diameter) with a high porosity were created for in vitro culture of chondrocytes. Specific surface area and protein adsorption on the surface of the nano-fibrous scaffold were three times higher than on the surface of the solid-walled scaffold. Chondrocytes cultured on the nano-fibrous scaffold exhibited a spherical condrocyte-like phenotype and secreted more cartilage-like extracellular matrix (ECM) than those cultured on the solid-walled scaffold. Moreover, the protein and DNA contents of cells cultured on the nano-fibrous scaffold were 1.2-1.4 times higher than those on the solid-walled scaffold. Higher expression levels of collagen II and aggrecan mRNA were induced on the nano-fibrous scaffold compared to on the solid-walled scaffold. These findings demonstrated that scaffolds with a nano-fibrous architecture could serve as superior scaffolds for cartilage tissue engineering.

Fabrication, mechanical property and in vitro evaluation of poly (L-lactic acid-co-ε-caprolactone) core-shell nanofiber scaffold for tissue engineering.

Coaxial electrospinning, in which Poly (L-lactic acid-co-ε-caprolactone) (PLC) with different Lactic acid (LA) to caprolactone (CL) ratio (75:25 and 50:50) were employed to electrospin core-shell nanofibers which could mimic the native extracellular matrix for tissue engineering applications. Core-shell nanofibrous scaffolds of PLC (50:50)/BSA (426 ±â€¯157 nm) and PLC (75:25)/BSA (427 ±â€¯197 nm) were fabricated and model drug bovine serum albumin (BSA) was entrapped in the core layer. The morphology, core-shell structure and sustained release behaviors were evaluated by Scanning electron microscopy (SEM), transmission electron microscopy (TEM), inverted fluorescence microscopy, water contact angle test and in vitro release test, respectively. The effect of core-shell structure and shell layer materials on the variation tendency of mechanical characterization in dry and wet situation were also investigated by tensile testing. The in vitro biocompatibility of scaffolds were investigated by growing human mesenchymal stem cells (hMSCs) on scaffolds surface and the proliferation of cells were evaluated with Alamar Blue tests. In vitro cultivations of hMSCs showed that PLC (50:50)/BSA scaffolds supported a significantly higher proliferation rate of seeded cells than scaffolds prepared by polymer PLC (75:25)/BSA. Overall, the PLC core-shell nanofibers possessed potentially regulable mechanical properties useful for tissue engineering as well as sustained release potential for medical applications.

Electrospun nanofiber scaffolds for rapid and rich capture of bone marrow-derived hematopoietic stem cells.

Interactions between bone marrow-derived hematopoietic stem cells (BM-HSCs) and their local microenvironment are an integral part of signaling control of BM-HSCs migration, proliferation and differentiation. We hypothesized that both substrate topographical and biochemical cues promote BM-HSCs adhesive behaviors, which are crucial for BM-HSCs' homing, self-renewal and lineage commitment within their microenvironment. We employed electrospinning technique to fabricate nanofiber scaffolds (NFS) with poly(dl-lactide-co-glycolide) blended with collagen I. NFS was further coated with E-selectin, a critical adhesive biomolecule. Capture efficiency study showed that blended NFS, after coated with E-selectin, significantly increased cell capture percentage from 23.40% to 67.41% within 30 min and from 29.44% to 70.19% within 60 min of incubation at room temperature. This study highlights the potential of using a biomimetic scaffold to design a site-specific niche-like unit for facilitating proliferation or differentiation functions of BM-HSCs.

Long-term viability of coronary artery smooth muscle cells on poly(L-lactide-co-epsilon-caprolactone) nanofibrous scaffold indicates its potential for blood vessel tissue engineering.

Biodegradable polymer nanofibres have been extensively studied as cell culture scaffolds in tissue engineering. However, long-term in vitro studies of cell-nanofibre interactions were rarely reported and successful organ regeneration using tissue engineering techniques may take months (e.g. blood vessel tissue engineering). Understanding the long-term interaction between cells and nanofibrous scaffolds (NFS) is crucial in material selection, design and processing of the tissue engineering scaffolds. In this study, poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (70:30) copolymer NFS were produced by electrospinning. Porcine coronary artery smooth muscle cells (PCASMCs) were seeded and cultured on the scaffold to evaluate cell-nanofibre interactions for up to 105 days. A favourable interaction between this scaffold and PCASMCs was demonstrated by cell viability assay, scanning electron microscopy, histological staining and extracellular matrix (ECM) secretion. Degradation behaviours of the scaffolds with or without PCASMC culture were determined by mechanical testing and gel permeation chromatography (GPC). The results showed that the PCASMCs attached and proliferated well on the P(LLA-CL) NFS. Large amount of ECM protein secretion was observed after 50 days of culture. Multilayers of aligned oriented PCASMCs were formed on the scaffold after two months of in vitro culture. In the degradation study, the PCASMCs were not shown to significantly increase the degradation rate of the scaffolds for up to 105 days of culture. The in vitro degradation time of the scaffold could be as long as eight months by extrapolating the results from GPC. These observations further supported the potential use of the P(LLA-CL) nanofibre in blood vessel tissue engineering.

Self-assembly of nano-hydroxyapatite on multi-walled carbon nanotubes.

Inspired by self-assembly of nano-hydroxyapatite (nHA) on collagen associated with the 67nm periodic microstructure of collagen, we used multi-walled carbon nanotubes (MWCNTs) with approximately 40nm bamboo periodic microstructure as a template for nHA deposition to form a nHA-MWCNT composite. The assembled apatite was analyzed by transmission electron microscopy and scanning electron microscopy. Defects that were analogous to edge dislocations along the carbon nanotubes' multi-walled surfaces were the nucleation sites for nHA after these defects had been functionalized principally into carboxylic groups. Spindle-shaped units consisting of an assembly of near parallel, fibril-like nHA polycrystals were formed and oriented at a certain angle to the long axis of the carbon nanotubes, unlike nHA-collagen in which the nHA is oriented along the longitudinal axis of the collagen molecule. One possible explanation for this difference is that there are more bonds for calcium chelation (-COOH, >CO) on the collagen fibril surface than on the surface of MWCNTs. Spindle-shaped units that are detached from the MWCNT template are able to maintain the ordered parallel structure of the nHA polycrystal fibril. We have thus created a self-assembled hydroxyapatite on MWCNTs.

Mechanical properties and biological behavior of carbon nanotube/polycarbosilane composites for implant materials.

Multiwalled carbon nanotube/polycarbosilane (MWCNT/PCS) composites were fabricated by the spark plasma sintering (SPS) method. The MWCNT/PCS composites consisted of MWCNTs and nanosized SiC particles pyrolyzed from PCS and possessing good mechanical properties for bone tissue repair or dental implantation. The MWCNT/PCS composites were implanted in the subcutaneous tissue and femur of rats at 1 and 4 weeks after implantation. Histological investigations showed that there was little inflammatory response in the subcutaneous tissue, and newly formed bone tissue was observed in the femur. These results indicated that the MWCNT/PCS composite had little prophlogistic effect and good osteoconductivity. The study suggested the possibility that the MWCNT/PCS composite could be a candidate bone-substitute and dental-implant material in the future.

The degradation of the three layered nano-carbonated hydroxyapatite/collagen/PLGA composite membrane in vitro.

OBJECTIVE: The purpose of this paper was to investigate the in vitro biodegradation of a guided tissue regeneration composite membrane, nano-carbonated hydroxyapatite/collagen/poly(lactic-co-glycolic acid) (nCHAC/PLGA). Especially for periodontal therapy, the functional graded material (FGM) nCHAC/PLGA membrane was prepared that consisted of three layers with 8 wt% nCHAC + PLGA/4 wt% nCHAC + PLGA/PLGA, where one face of the membrane is porous, thereby allowing cell growth thereon and the opposite face of the membrane smooth, thereby inhibiting cell adhesion. METHODS: For evaluation, in vitro degradation specimens of nCHAC/PLGA were immersed into artificial saliva solution at 37 degrees C for 1, 2, 4, 8 and 12 weeks to detect the weight loss over the period, and set pure PLGA membrane as control to compare the degraded behaviors. pH value and calcium concentration of the residual solution were measured, and morphology change was investigated by scanning electron microscopy (SEM). RESULTS: During the experimental period in vitro, the whole shape of the membrane could be kept for 4 weeks, after that it became powder at between 8 and 12 weeks. The results demonstrated that weight loss increased continuously with a reduction in mass of 23.1% after 4 weeks and 88% after 12 week for the nCHAC/PLGA three FGM layers composite membrane. The calcium concentration in the residual solution showed a significant increase after 4 weeks, which referred to the nano-carbonated hydroxyapatite degradation. Moreover, the pH value in the solution of the nCHAC/PLGA membrane was a little higher than that of the pure PLGA membrane, which demonstrated the possible neutralization effect from nCHAC composite for the acid outcome of PLGA in the solution. The pore structure of 8 wt% nCHAC + PLGA was enlarged on the porous surface, while the nonporous surface of pure PLGA also showed a small porous structure after increased time. SIGNIFICANCE: Degradation of the composite membrane is appropriate for practical periodontal repair. Moreover, the new mineral formation on the surface of the composite membrane referred to the possible positive effect in vivo for new bone tissue regeneration.

Human neutrophils reaction to the biodegraded nano-hydroxyapatite/collagen and nano-hydroxyapatite/collagen/poly(L-lactic acid) composites.

The impact of biodegraded nano-hydroxyapatite/collagen (nHAC) composite and nano-hydroxyapatite/collagen/poly(L-lactic acid) (nHAC/PLA) scaffold composite on neutrophils reaction was evaluated in vitro. Neutrophils were separated from human peripheral blood of healthy subjects. The nHAC and nHAC/PLA materials were immersed in the D-Hanks' Balanced Salt Solution (D-HBSS) for 1 day, 7 days and 2, 4, 8 weeks (37 degrees C) as testing solution, which mixed with the neutrophils for 1 h. Both of the nHAC and nHAC/PLA materials were shown the same cell survival rate as blank control, but the lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha) released from the neutrophils were increased significantly after the 2 weeks in nHAC sample. The possible reason relied on the high concentration of calcium due to the quick biodegradation of the nHAC material. Before 2 weeks, the LDH value of nHAC/PLA is higher than that of nHAC sample that corresponded to the initial PLA degradation in vitro. This study provided the biocompatibility test of neutrophils other than common methods, such as osteoblastic cells for biomimetic materials. Moreover, it demonstrated the calcium concentration stimulating effect for cytokine release from neutrophils.

A three-layered nano-carbonated hydroxyapatite/collagen/PLGA composite membrane for guided tissue regeneration.

Functional graded materials (FGM) provided us one new concept for guided tissue regeneration (GTR) membrane design with graded component and graded structure where one face of the membrane is porous thereby allowing cell growth thereon and the opposite face of the membrane is smooth, thereby inhibiting cell adhesion in periodontal therapy. The goal of the present study was to develop a three-layered graded membrane, with one face of 8% nano-carbonated hydroxyapatite/collagen/poly(lactic-co-glycolic acid) (nCHAC/PLGA) porous membrane, the opposite face of pure PLGA non-porous membrane, the middle layer of 4% nCHAC/PLGA as the transition through layer-by-layer casting method. Then the three layers were combined well with each other with flexibility and enough high mechanical strength as membrane because the three layers all contained PLGA polymer that can be easily used for practical medical application. This high biocompatibility and osteoconductivity of this biodegraded composite membrane was enhanced by the nCHAC addition, for the same component and nano-level crystal size with natural bone tissue. The osteoblastic MC3T3-E1 cells were cultured on the three-layered composite membrane, the primary result shows the positive response compared with pure PLGA membrane.

The preparation and characteristics of a carbonated hydroxyapatite/collagen composite at room temperature.

Nanocarbonated hydroxyapatite/collagen (nCHAC) composite was prepared at room temperature via biomimetic self-assembly method. X-ray diffraction (XRD), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) were performed. This composite shows the same inorganic phase of natural bone with nanosized level and low degree of crystallinity, and contains 2.8-14.7 wt % of carbonated content. TEM results confirm that the microstructure of this composite is the mineralized collagen fiber bundle like the hierarchical structure of natural bone. The diameter of a single mineralized collagen fiber is about 4 nm. Slightly different assembly units of the composite with different carbonates and collagen were demonstrated. The carbonated percentage affects the mineral crystal size and collagen fibril assembly. Because of the biomimetic component and microstructure, the use of nCHAC composite is promising for hard tissue therapy.

[Mineralized collagen based composite for bone tissue engineering].

OBJECTIVE: To construct a mineralized collagen based composite by biomimetic synthesis for bone tissue engineering. METHODS: Using the molecular collagen as the template, the calcium phosphate is deposited on it to produce a mineralized collagen based composite, then is combined with minute amount of poly lactic acid (PLA), the three-dimensional scaffold composite is prepared by liquid phase separation. Using osteoblast culture technique, the biocompatibility of this biomaterial in vitro is detected by x-ray diffraction, SEM, TEM, fluoroscopy and CLSM. RESULTS: Both degree and the size of crystals in the composite are low, which are similar to that of nature bone. It possesses porous structure and the porosity of the composite is high. The typical fibrillar microstructure is self-assembled of the collagen and the nano-crystal hydroxyapatite (HA) in the composite, moreover, the x-ray diffraction graphic of HA crystal shows the [002]-oriented. CONCLUSIONS: The biomimetic three-dimensional composite can serve as one of the optimal scaffold material for bone tissue engineering both on structure and on property.

Carbon nanotubes reinforced composites for biomedical applications.

This review paper reported carbon nanotubes reinforced composites for biomedical applications. Several studies have found enhancement in the mechanical properties of CNTs-based reinforced composites by the addition of CNTs. CNTs reinforced composites have been intensively investigated for many aspects of life, especially being made for biomedical applications. The review introduced fabrication of CNTs reinforced composites (CNTs reinforced metal matrix composites, CNTs reinforced polymer matrix composites, and CNTs reinforced ceramic matrix composites), their mechanical properties, cell experiments in vitro, and biocompatibility tests in vivo.

Injectable hydrogel incorporating with nanoyarn for bone regeneration.

Traditional bone grafting requires an open surgical approach to the graft application sites with the attendant complications of a large surgical scar, increased pain and a longer post-operative recovery. To overcome these limitations, there is a great need for the development of better bone graft substitutes. In this study, we developed a novel injectable system which was a biomimetic bone substitute consisted of Poly (L-lactide-co-ε-caprolactone) (P(LLA-CL)) nanoyarns suspended in type I collagen hydrogel (Col). A dynamic liquid support system was employed to fabricate continuous P(LLA-CL) nanoyarns. The electrospun long nanoyarns were chopped into short nanoyarns before they were incorporated into Col. The result of rheological evaluation showed that the mechanical property of Col was enhanced after the nanoyarns were incorporated into it. The mixture of Col and nanoyarn could be smoothly injected out of 16 gauge needle. In vitro study showed that human mesenchymal stem cells (hMSCs) proliferated well on Col with nanoyarns. Alkaline phosphatase activity and osteocalcin expression of hMSCs on hydrogel with nanoyarns were much higher than those on control groups. This study highlights the potential of using a novel injectable biomimetic scaffold for bone regeneration.

Efficacy of cold light bleaching using different bleaching times and their effects on human enamel.

This study investigated the efficacy of cold light bleaching using different bleaching times and the effects thereof on tooth enamel. Before and after bleaching, stained tooth specimens were subjected to visual and instrumental colorimetric assessments using Vita Shade Guide and spectrophotometric shade matching. Enamel surface alterations were examined using scanning electron microscopy (SEM) to analyze surface morphology, surface microhardness (SMH) measurement to determine changes in mechanical properties, and X-ray diffraction (XRD) to characterize post-bleaching enamel composition. Cold light bleaching successfully improved tooth color, with optimal efficacy when bleaching time was beyond 10 min. Significant differences in surface morphology were observed among the different bleaching times, but no significant differences were observed for enamel composition and surface microhardness among the different bleaching times. Results of this study revealed an association between the bleaching time of cold light bleaching and its whitening efficacy. Together with the results on enamel surface changes, this study provided positive evidence to support cold light bleaching as an in-office bleaching treatment.

Surface modification of PLLA nano-scaffolds with laminin multilayer by LbL assembly for enhancing neurite outgrowth.

In this study, PLLA nanofibers are fabricated by electrospinning and their surfaces are modified by laminin/chitosan (LN/CS) polyelectrolyte multilayer. Surface C/N ratio determined by XPS analysis quantitatively indicates of discrete coating layers on the nanofibers. The amount of LN deposited sustainably increases with LbL assembly processing, approximately 60 ng mm(-2) LN per cycle of LN/CS deposition. The LN-modified PLLA scaffolds significantly induce neurite outgrowth of DRG neurons and NSC compared to the pure PLLA nanofibrous scaffolds. Furthermore, higher amounts of LN adsorbed assist in promoting cell proliferation than PLLA as-spun nanofibers. Therefore, a facile and efficient method to modify nano-scaffolds for the construction of a biomimetic scaffold to promote highly efficient neurite outgrowth is presented.

Electrospun poly(L-lactic acid) nanofibres loaded with dexamethasone to induce osteogenic differentiation of human mesenchymal stem cells.

Dexamethasone (Dex), a synthetic corticosteroid, was loaded into poly(L-lactic acid) (PLLA) nanofibrous scaffolds with a concentration of 0.333 wt% by electrospinning. The Dex-loaded PLLA nanofibres increased the mechanical strength in comparison with pure PLLA nanofibres. A sustained release profile for over 2 months with an initial burst release after 12 h of 17% was shown. Importantly, the amounts of Dex released from the PLLA nanofibres every 3 days were close to the ones used for the standard osteogenic medium. The sustained osteoinductive environment created by released Dex strongly differentiated human mesenchymal stem cells (hMSCs) cultured in the Ost(-Dex) medium. ALP activity, BSP expression and calcium deposition were significantly higher than those of the cells cultured on the PLLA scaffolds without Dex. A large amount of hydroxyapatite-like minerals was observed on the Dex-loaded PLLA scaffolds after 21 days culture. The cells on these scaffolds also indicated an osteoblastic morphology on the 14th day. Besides, these scaffolds slightly increased the cell proliferation comparing to the scaffolds without Dex. As such, the PLLA nanofibres loaded with 0.333 wt% Dex was an effective osteoinductive scaffold which acts as a promising strategy for bone treatment.

Biomimetic surface modification of titanium surfaces for early cell capture by advanced electrospinning.

The time required for osseointegration with a metal implant having a smooth surface ranges from three to six months. We hypothesized that biomimetic coating surfaces with poly(lactic-co-glycolic acid) (PLGA)/collagen fibers and nano-hydroxyapatite (n-HA) on the implant would enhance the adhesion of mesenchymal stem cells. Therefore, this surface modification of dental and bone implants might enhance the process of osseointegration. In this study, we coated PLGA or PLGA/collagen (50:50 w/w ratio) fiber on Ti disks by modified electrospinning for 5 s to 2 min; after that, we further deposited n-HA on the fibers. PLGA fibers of fiber diameter 0.957 ± 0.357 µm had a contact angle of 9.9 ± 0.3° and PLGA/collagen fibers of fiber diameter 0.378 ± 0.068 µm had a contact angle of 0°. Upon n-HA incorporation, all the fibers had a contact angle of 0° owing to the hydrophilic nature of n-HA biomolecule. The cell attachment efficiency was tested on all the scaffolds for different intervals of time (10, 20, 30 and 60 min). The alkaline phosphatase activity, cell proliferation and mineralization were analyzed on all the implant surfaces on days 7, 14 and 21. Results of the cell adhesion study indicated that the cell adhesion was maximum on the implant surface coated with PLGA/collagen fibers deposited with n-HA compared to the other scaffolds. Within a short span of 60 min, 75% of the cells adhered onto the mineralized PLGA/collagen fibers. Similarly by day 21, the rate of cell proliferation was significantly higher (p ⩽ 0.05) on the mineralized PLGA/collagen fibers owing to enhanced cell adhesion on these fibers. This enhanced initial cell adhesion favored higher cell proliferation, differentiation and mineralization on the implant surface coated with mineralized PLGA/collagen fibers.

Photosensitive materials and potential of photocurrent mediated tissue regeneration.

Photocurrent therapy with participation of light and electrical stimulations could be an innovative and promising approach in regenerative medicine, especially for skin and nerve regeneration. Photocurrent is generated when light irradiates on a photosensitive device, and with more and more types of photosensitive materials being synthesized, photocurrent could be applied for enhanced regeneration of tissue. Photosensitive scaffolds such as composite poly (3-hexylthiophene)/polycaprolactone (P3HT/PCL) nanofibers are fabricated by electrospinning process in our lab for skin regeneration in presence of applied photocurrent. This review article discuss on the various in vitro, in vivo and clinical studies that utilized the principle of 'electrotherapy' and 'phototherapy' for regenerative medicine and evaluates the potential application of photocurrent in regenerative medicine. We conclude that photocurrent therapy will play an important role in regenerative medicine.

The role of nanofibrous structure in osteogenic differentiation of human mesenchymal stem cells with serial passage.

UNLABELLED: Using scaffolds with autologous stem cells is a golden strategy for the treatment of bone defects. In this strategy, human mesenchymal stem cells (hMSCs) have often been isolated and expanded in vitro on a plastic surface to obtain a sufficient cell number before seeding on a suitable scaffold. MATERIALS & METHODS: Investigating the influence of serial passages (from passage two to passage eight) on the abilities of proliferation and osteogenic differentiation of hMSCs on 24-well tissue culture polystyrene plates and poly L-lactic acid electrospun nanofibrous scaffolds was performed to determine how prolonged culture affected these cellular abilities and how the nanofibrous scaffolds supported the osteogenic differentiation potential of hMSCs. RESULTS & CONCLUSION: Serial passage caused adverse changes in hMSCs characteristics, which were indicated by the decline in both proliferation and osteogenic differentiation abilities. Interestingly, the poly L-lactic acid nanofibrous scaffolds showed a significant support in recovering the osteogenic abilities of hMSCs, which had been severely affected by prolonged culture.