Tissue engineering and peripheral nerve regeneration (III)

The biodegradation rate and biocompatibility of poly (d, / -lactide) (PDLLA)in vivo were evaluated. The aim of this study was to establish a nerve guide constructed by the PDLLA with 3-D microenvironment and to repair a 10 mm of sciatic nerve gap in rats. The process of the nerve regeneration was investigated by histological assessment, electrophysiological examination, and determination of wet weight recovery rate of the gastrocnemius muscle. After 3 weeks, the nerve guide had changed from a transparent to an opaque status. The conduit was degraded and absorbed partly and had lost their strength with breakage at the 9th week of postoperation. At the conclusion of 12 weeks, proximal and distal end of nerves were anastomosed by nerve regeneration and the conduit vanished completely. The results suggest that PDLLA conduits may serve for peripheral nerve regeneration and PDLLA is a sort of hopeful candidate for tissue engineering.     

Tissue engineering and peripheral nerve regeneration (Ⅲ)——Sciatic nerve regeneration with PDLLA nerve guide

The biodegradation rate and biocompatibility of poly(d, / -lactide) (PDLLA) in vivo were evaluated. The aim of this study was to establish a nerve guide constructed by the PDLLA with 3-D microenvironment and to repair a 10 mm of sciatic nerve gap in rats. The process of the nerve regeneration was investigated by histological assessment, electrophysiological examination, and determination of wet weight recovery rate of the gastrocnemius muscle. After 3 weeks, the nerve guide had changed from a transparent to an opaque status. The conduit was degraded and absorbed partly and had lost their strength with breakage at the 9th week of postoperation. At the conclusion of 12 weeks, proximal and distal end of nerves were anastomosed by nerve regeneration and the conduit vanished completely. The results suggest that PDLLA conduits may serve for peripheral nerve regeneration and PDLLA is a sort of hopeful candidate for tissue engineering.     

In vivo evaluation of a biodegradable EDC/NHS-cross-linked gelatin peripheral nerve guide conduit material

Peripheral nerve regeneration has been evaluated using a biodegradable nerve conduit, which is made of a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross-linked gelatin. The EDC/NHS crosslinked gelatin (ENG) conduit is brownish in appearance, and is concentric and round with a smooth outer surface and inner lumen. After subcutaneous implantation on the dorsal side of a rat, the degraded ENG conduit only evoked a mild tissue response, with the formation of a thin tissue capsule surrounding the conduit. Biodegradability of the ENG conduit and its effectiveness as a guidance channel has been examined by its use to repair a 10 mm gap in the rat sciatic nerve. As a result, the tubes degraded throughout the implantation period, but still remained circular with a thin round lumen until they were completely integrated with the enclosed nerves. Successful regeneration through the gap occurred in all the conduits over the three experimental periods of 4, 8, and 12 weeks. Histological observation showed that numerous myelinated axons had crossed through the gap region even at the shortest implantation period of 4 weeks. Peak amplitude, area under the muscle action potential curve, and nerve conductive velocity all showed an increase as a function of the recovery period, which indicates that the nerve had undergone adequate regeneration. These results indicate the superiority of the ENG materials and suggest that the novel ENG conduits provide a promising tool for neuro-regeneration.     

Materials for peripheral nerve regeneration

Recent efforts in scientific research in the field of peripheral nerve regeneration have been directed towards the development of artificial nerve guides. We have studied various materials with the aim of obtaining a biocompatible and biodegradable two layer guide for nerve repair. The candidate materials for use as an external layer for the nerve guides were poly(caprolactone) (PCL), a biosynthetic blend between PCL and chitosan (CS) and a synthesised poly(ester-urethane) (PU). Blending PCL, which is a biocompatible synthetic polymer, with a natural polymer enhanced the system biocompatibility and biomimetics, fastened the degradation rates and reduced the production costs. Various novel block poly(ester-urethane)s are being synthesised by our group with tailored properties for specific tissue engineering applications. One of these poly(ester-urethane)s, based on a low molecular weight poly(caprolactone) as the macrodiol, cycloesandimethanol as the chain extender and hexamethylene diisocyanate as the chain linker, was investigated for the production of melt extruded nerve guides. We studied natural polymers such as gelatin (G), poly(L-lysine) (PL) and blends between chitosan and gelatin (CS/G) as internal coatings for nerve guides. In vitro and in vivo tests were performed on PCL guides internally coated either with G or PL to determine the differences in the quality of nerve regeneration associated with the type of adhesion protein. CS/G natural blends combined the good cell adhesion properties of the protein phase with the ability to promote nerve regeneration of the polysaccharide phase. Natural blends were crosslinked both by physical and chemical crosslinking methods. In vitro neuroblast adhesion tests were performed on CS/G film samples, PCL/CS and PU guides internally coated with G to evaluate the ability of such materials towards nerve repair.     

Biodegradable glutaraldehyde-crosslinked casein conduit promotes regeneration after peripheral nerve injury in adult rats

In this study, GCC protein was used for the first time to construct a biodegradable conduit for peripheral nerve repair. The GCC was highly stable with a sufficiently high level of mechanical properties and it was non-toxic and non-apoptotic which could maintain the survival and outgrowth of Schwann cells. Noninvasive bioluminescence imaging accompanied with histochemical assessment showed the GCC was highly biocompatible after subcutaneous implantation in transgenic mice. Electrophysiology, labeling of calcitonin gene-related peptide in the lumbar spinal cord and histology analysis also showed a rapid morphological and functional recovery for disrupted rat sciatic nerves repaired with the GCC conduits. Therefore, we conclude that the GCC can offer great nerve regeneration characteristics and can be a promising material for the successful repair of peripheral nerve defects.     

Improvement of mechanical properties of poly(dl-lactide) films by blending of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(dl-lactide) (PDLLA) were blended at different ratios in an attempt to form a biomaterial with suitable properties for nerve regeneration. FT-IR and X-ray analysis showed that the blending of the PDLLA component did not alter the helical structure of PHBHHx, but did lead to a reduction of crystallinity. Differential scanning calorimetry (DSC) analysis indicated that the two polymers were immiscible in the melted state. The mechanical properties of certain composite films were more desirable than those of unblended PDLLA films. Blends consisting of PDLLA and PHBHHx at ratios of 2:1 and 1:2 exhibited a lower elastic modulus and a higher elongation at break compared to unblended PDLLA. ELISA results indicated that the amount of fibronectin adsorbed on composite films was much higher than the amount adsorbed on PDLLA film. The results of this study demonstrate the feasibility of using PDLLA/PHBHHx blended materials for biomedical applications.     

Degradation of covalently cross-linked carboxymethyl chitosan and its potential application for peripheral nerve regeneration

Chitosan has been widely used in a variety of biomedical applications including peripheral nerve repair because of its excellent mechanical properties and biocompatibility. However, chitosan itself has a very slow degradation rate, and its molecules degrade in an uncontrollable manner. We hypothesized that the cross-linking of carboxymethyl chitosan (CM-chitosan), which is soluble in water, would result in a higher degradation rate in lysozyme solutions, while retaining its excellent mechanical properties and nerve cell affinity. In this study, we characterized the constructed matrix formed using a combination of carboxymethylation of chitosan chains and thereafter 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross-linking. Specifically, after EDC cross-linking, the hydrophilicity and elastic modulus of the CM-chitosan films decreased. These changes are beneficial in the application of chitosan derivatives for nerve repair. The porous conduits degraded to 30% in weight during eight weeks of incubation in lysozyme solution (pH 7.4, 37 °C). In addition, the cross-linked CM-chitosan films enhanced the spread of Neuro-2a cells and provided a good proliferation substratum for Neuro-2a cells, as compared to chitosan films. Therefore, cross-linking with EDC is a promising way to modify chitosan derivatives for peripheral nerve regeneration.     

Photocured biodegradable polymer substrates of varying stiffness and microgroove dimensions for promoting nerve cell guidance and differentiation

Photocross-linkable and biodegradable polymers have great promise in fabricating nerve conduits for guiding axonal growth in peripheral nerve regeneration. Here, we photocross-linked two poly(ε-caprolactone) triacrylates (PCLTAs) with number-average molecular weights of ～7000 and ～10?000 g mol(-1) into substrates with parallel microgrooves. Cross-linked PCLTA7k was amorphous and soft, while cross-linked PCLTA10k was semicrystalline with a stiffer surface. We employed different dimensions of interests for the parallel microgrooves, that is, groove widths of 5, 15, 45, and 90 μm and groove depths of 0.4, 1, 5, and 12 μm. The behaviors of rat Schwann cell precursor line (SpL201) cells with the glial nature and pheochromocytoma (PC12) cells with the neuronal nature were studied on these microgrooved substrates, showing distinct preference to the substrates with different mechanical properties. We found different threshold sensitivities of the two nerve cell types to topographical features when their cytoskeleton and nuclei were altered by varying the groove depth and width. Almost all of the cells were aligned in the narrowest and deepest microgrooves or around the edge of microgrooves. Oriented SpL201 cell movement had a higher motility as compared to unaligned ones. After forskolin treatment, SpL201 cells demonstrated significantly upregulated S-100 and O4 on stiffer substrates or narrower microgrooves, suggesting more differentiation toward early Schwann cells (SCs). PC12 neurites were oriented with enhanced extension in narrower microgrooves. The present results not only improve our fundamental understanding on nerve cell-substrate interactions, but also offer useful conduit materials and appropriate feature dimensions to foster guidance for axonal growth in peripheral nerve regeneration.     

Poly(ester urethane) guides for peripheral nerve regeneration

A biocompatible and elastomeric PU was synthesized from low-molecular-weight PCL as macrodiol, CMD as chain extender and HDI as chain linker for applications in the field of peripheral nerve repair. PU cast films supported in vitro attachment and proliferation of NOBEC. The in vitro adhesion and proliferation of S5Y5 neuroblastoma cells on the inner surface of uncoated, gelatin- and PL-coated PU guides were compared. Due to their superior in vitro performance, PL-coated PU guides were tested in vivo for the repair of 1.8 cm-long defects in rat sciatic nerves. The progressive regeneration was confirmed by EMG and histological analysis showing the presence of regenerating fibers in the distal stumps.     

Biofabrication of a flexible and conductive 3D polymeric scaffold for neural tissue engineering applications; physical,chemical, mechanical,and biological evaluations

Tissue engineering approach aims to overcome the transplant drawbacks and facilitate tissue repair and regeneration. Here, a new conductive, highly porous, and flexible polycaprolactone/gelatin/polypyrrole/graphene 3D scaffolds for nerve tissue repair is presented. A simple and efficient porogen leaching fabrication method is applied to create a 3D network with a pore radius of 3.8 ± 0.7 to 4.2 ± 0.8 μm with an exceptional uniform circular porous structure. The conductivity of the polymeric scaffold without graphene, in wet conditions, was found to be 0.78 ± 0.1 S.m⁻¹ and it increased to 3.3 ± 0.2 S.m⁻¹ for the optimized sample containing 3wt% graphene (G3). Tensile strength was measured at 163 KPa for the base sample (without graphene) and improved to 526 KPa for G3 sample. Following 42 days of incubation in PBS, 32.5% degradation for the base sample (without graphene) was observed. The cell study demonstrated a non-cytotoxic nature of all scaffolds tested and the cells had mostly stretched and covered the surface. Overall, the sum of results presented in this study demonstrate a simple fabrication platform with extraordinary aspects that can be utilized to mimic the native conductive tissue properties, and also because of its flexibility it can easily be rolled into a nerve conduit to fill gaps in nerve tissue regeneration.     

Synthesis, characterization and biodegradation of butanediamine-grafted poly(dl-lactic acid)

Novel butanediamine-grafted poly(dl-lactic acid) polymers (BDPLAs) were synthesized via a series of chemical bulk modifications in this study. Briefly, maleic anhydride (MAH) was first grafted onto the side chain of poly(dl-lactic acid) (PDLLA) molecules via melt free radical copolymerization using benzoyl peroxide (BPO) as initiator to get maleic anhydride-grafted PDLLA polymers (MPLAs); thereafter butanediamine (BDA) was immobilized onto grafted anhydride groups in MPLAs via N-acylation reaction to obtain the desired BDPLAs. Gel permeation chromatography with multi-angle laser light scattering (GPC-MALLS), FT-IR, ¹³C NMR and XPS were employed to qualitatively characterize these synthesized polymers. Rhodamine-carboxyl interaction method and ninhydrin reaction were further used to quantitatively determine the graft ratio of MAH (MAH%) in MPLAs and the graft ratio of BDA (BDA%) in BDPLAs, respectively. The degradations of BDPLAs, PDLLA and MPLAs were investigated by observation of the changes of the pH value of incubation medium, molecular weight and weight loss ratio for a time interval of 12 weeks in vitro, respectively. The results revealed that grafting butanediamine onto PDLLA has weakened or neutralized the acidity of PDLLA degradation products. A uniform degradation of BDPLAs was observed in comparison with an acidity-induced auto-accelerating degradation featured by PDLLA and MPLAs. The biodegradation behaviors of BDPLAs are tunable by controlling the content of BDA. BDPLAs might be a new derivative of PDLLA-based biodegradable materials for medical applications without acidity-caused irritations and acidity-induced auto-accelerating degradation behavior as that of PDLLA.     

Superiority of poly(L-lactic acid) microspheres as dermal fillers

The demand for injectable dermal filler has unde rgone significant growth with the rapid development of the beauty industry.Poly(lactic acid)(PLA) as a benefit of excellent biocompatibility and long-term promotion of collagen regeneration has been favored as a commonly used filler.However,the effects of chirality and particle size of PLA on the efficacy of dermal filler have not been studied.In this study,we prepared three kinds of microspheres(MSs) consisting of poly(D-lactic acid)(PDLA MS),poly(L-lactic acid)(PLLA MS),or meso-PLA(PDLLA MS)at 5,10 and 20 μmto reveal the different biological functions as dermal filler.Following intradermal injection into guinea pig,it was found that PLLA MS induced the slightest inflammation,and the level of pro-inflammatory cytokine IL-1β induced by PLLA MS is only 0.3 or 0.7-fold of that induced by PDLA or PDLLA MS,respectively.More importantly,PLLA MS significantly stimulated the regeneration of collagen,which was 1.4 or 1.1 times higher than those stimulated by PDLA MS or PDLLA MS,respectively.The size of PLA MSs did not affect the levels of inflammation and collagen regeneration.The results confirmed the superiority of PLLA as a dermal filler.     

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.     

聚左旋乳酸/聚消旋乳酸共混物的结晶行为

非晶态聚消旋乳酸(PDLLA)对PLLA的结晶行为有较大的影响。本文利用差示扫描量热仪(DSC)和偏光显微镜(POM)对不同分子量PLLA、PDLLA按不同比例制得的共混物结晶进行了系统研究。结果表明随PDLLA含量的增大PLLA冷结晶温度升高,且越接近熔融温度。PDLLA分子量较小时PLLA球晶特征被明显破坏,PDLLA分子量较大时PLLA更易形保持球晶特征且易形成环带球晶形貌,这与结晶速率与非晶组分的扩散速率匹配程度有关。低分子量的PDLLA使PLLA的最大生长速率对应的温度出现在较低温度。     

Effects of mechanical load on the degradation of poly(d,l-lactic acid) foam

Degradable behaviors of polymer for implantation in body should be evaluated before clinical application. The effect of continuous mechanical load on the degradation progress of poly(d,l-lactic acid) (PDLLA) foam gasket was investigated in detail by specially designed load-providing devices. While PDLLA degraded in the PBS solution (pH, 7.4) at 37 °C for 3 months, the changes of surface morphology, molecular weight, elastic modulus, tensile strength and mass loss were recorded. The results revealed that the degradation rates of PDLLA under continuous loads were obviously quicker than those without load. Moreover, the influence of tensile plus compressive load was larger than that of tensile load. It was indicated that in vivo degradation of PDLLA would not only be influenced by the local solution, but also by the surrounding load. When regulating the degradation rate of bioabsorbable polymer, one should consider the indispensable effect of load where implanted.     

Methylcobalamin‐Loaded PLCL Conduits Facilitate the Peripheral Nerve Regeneration

The feasible fabrication of nerve guidance conduits (NGCs) with good biological performance is important for translation in clinics. In this study, poly(d ,l ‐lactide‐co‐caprolactone) (PLCL) films loaded with various amounts (wt; 5%, 15%, 25%) of methylcobalamin (MeCbl) are prepared, and are further rolled and sutured to obtain MeCbl‐loaded NGCs. The MeCbl can be released in a sustainable manner up to 21 days. The proliferation and elongation of Schwann cells, and the proliferation of Neuro2a cells are enhanced on these MeCbl‐loaded films. The MeCbl‐loaded NGCs are implanted into rats to induce the regeneration of 10 mm amputated sciatic nerve defects, showing the ability to facilitate the recovery of motor and sensory function, and to promote myelination in peripheral nerve regeneration. In particular, the 15% MeCbl‐loaded PLCL conduit exhibits the most satisfactory recovery of sciatic nerves in rats with the largest diameter and thickest myelinated fibers.     

Systematically engineered electrospun conduit based on PGA/collagen/bioglass nanocomposites: The evaluation of morphological,mechanical, and bio‐properties

Peripheral nerve injury can considerably affect the daily life of affected people through reduced function and permanent deformation of the nerve. One of the conventional treatments used for the management of the disease is the application of autograft, which is recognized as a golden standard method; however, the process of gaining access to autograft has posed a significant challenge to its use. Nerve guidance channels (conduits), which are made in different methods, can act as an alternative therapy for patients that have undergone nerve injury; but, achieving these conduits has always been a major dilemma to be applied for patients with nerve injury. In this study, a novel conduit based on polymer blend nanocomposites of polyglycolic acid (PGA), collagen, and nanobioglass (NBG) were prepared by electrospinning technique and then compared with PGA/collagen and PGA conduits that were made in previous studies. Additionally, their various properties were characterized by scanning electron microscopy (SEM), X‐ray diffraction (XRD), contact angle, dynamic mechanical thermal analysis (DMTA), tensile strength, Fourier‐transform infrared (FTIR), and the porosity and degradation. The results showed that the mechanical, chemical, biocompatibility, and biodegradability properties of PGA/collagen/NBG conduits were more favorable in comparison with other materials. According to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and 4′,6‐diamidino‐2‐phenylindole (DAPI) staining technique, nanofibrous electrospun PGA/collagen/NBG conduits are more suitable for cell adhesion and proliferation in comparison with either PGA or PGA/collagen conduits and can have potential for nerve regeneration.     

Synthesis and characterization of biodegradable network hydrogels having both hydrophobic and hydrophilic components with controlled swelling behavior

A new class of biodegradable hydrogels, consisting of hydrophobic poly(D ,L )lactic acid (PDLLA) and hydrophilic dextran segments with a polymer network structure, was synthesized with UV photopolymerization. Unsaturated vinyl groups first were introduced onto the PDLLA and dextran polymer backbones, then followed by a crosslinking reaction of diacrylate-terminated PDLLA and dextran acrylate. The chemical crosslinking forced the hydrophobic PDLLA and hydrophilic dextran segments to mix with each other in the network hydrogels. The new polymers were characterized by standard polymer characterization methods such as NMR, Fourier transform infrared spectroscopy, and gel permeation chromatography. The effects of reaction time, temperature, and molar ratio of the reactants on the incorporation of acrylate onto the polymer backbone were examined. A series of hydrogels with different dextran/PDLLA composition ratios was prepared, and their swelling behaviors were studied. These new bicomponent network hydrogels had a wide range of hydrophilicity to hydrophobicity that was difficult to achieve in totally hydrophilic hydrogels. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 4554–4569, 1999     

Comparison between heparin-conjugated fibrin and collagen sponge as bone morphogenetic protein-2 carriers for bone regeneration

Bone morphogenetic protein-2 (BMP-2) is used to promote bone regeneration. However, the bone regeneration ability of BMP-2 relies heavily on the delivery vehicle. Previously, we have developed heparin- conjugated fibrin (HCF), a vehicle for long-term delivery of BMP-2 and demonstrated that long-term delivery of BMP-2 enhanced its osteogenic efficacy as compared to short-term delivery at an equivalent dose. The aim of this study was to compare the bone-forming ability of the BMP-2 delivered by HCF to that delivered by clinically utilized BMP-2 delivery vehicle collagen sponge. An in vitro release profile of BMP-2 showed that HCF released 80% of the loaded BMP-2 within 20 days, whereas collagen sponge released the same amount within the first 6 days. Moreover, the BMP-2 released from the HCF showed significantly higher alkaline phosphatase activity than the BMP-2 released from collagen sponge at 2 weeks in vitro. Various doses of BMP-2 were delivered with HCF or collagen sponge to mouse calvarial defects. Eight weeks after the treatment, bone regeneration was evaluated by computed tomography, histology, and histomorphometric analysis. The dose of BMP-2 delivered by HCF to achieve 100% bone formation in the defects was less than half of the BMP-2 dose delivered by collagen sponge to achieve a similar level of bone formation. Additionally, bone regenerated by the HCF-BMP-2 had higher bone density than bone regenerated by the collagen sponge-BMP-2. These data demonstrate that HCF as a BMP-2 delivery vehicle exerts better osteogenic ability of BMP-2 than collagen sponge, a clinically utilized delivery vehicle.     

A lab-on-a-chip for detection of nerve agent sarin in blood

Sarin (C(4)H(10)FO(2)P,O-isopropyl methylphosphonofluoridate) is a colourless, odourless and highly toxic phosphonate that acts as a cholinesterase inhibitor and disrupts neuromuscular transmission. Sarin and related phosphonates are chemical warfare agents, and there is a possibility of their application in a military or terrorist attack. This paper reports a lab-on-a-chip device for detecting a trace amount of sarin in a small volume of blood. The device should allow early detection of sarin exposure during medical triage to differentiate between those requiring medical treatment from mass psychogenic illness cases. The device is based on continuous-flow microfluidics with sequential stages for lysis of whole blood, regeneration of free nerve agent from its complex with blood cholinesterase, protein precipitation, filtration, enzyme-assisted reaction and optical detection. Whole blood was first mixed with a nerve gas regeneration agent, followed by a protein precipitation step. Subsequently, the lysed product was filtered on the chip in two steps to remove particulates and fluoride ions. The filtered blood sample was then tested for trace levels of regenerated sarin using immobilised cholinesterase on the chip. Activity of immobilised cholinesterase was monitored by the enzyme-assisted reaction of a substrate and reaction of the end-product with a chromophore. Resultant changes in chromophore-induced absorbance were recorded on the chip using a Z-shaped optical window. Loss of enzyme activity obtained prior and after passage of the treated blood sample, as shown by a decrease in recorded absorbance values, indicates the presence of either free or regenerated sarin in the blood sample. The device was fabricated in PMMA (polymethylmethacrylate) using CO(2)-laser micromachining. This paper reports the testing results of the different stages, as well as the whole device with all stages in the required assay sequence. The results demonstrate the potential use of a field-deployable hand-held device for point-of-care triage of suspected nerve agent casualties.