Bone marrow stromal cells-loaded chitosan conduits promote repair of complete transection injury in rat spinal cord

In this study, a chitosan conduit loaded with bone marrow stromal cells (BMSCs) was developed to bridge the gap in the transected spinal cord of adult rats, and the nerve repair outcomes were evaluated by functional and histological techniques at 12 weeks after implantation. As compared to chitosan conduits alone, incorporation of BMSCs within chitosan conduits yielded additional improving effects on nerve regeneration and function restoration. The measurements with the Basso, Beattie and Bresnahan locomotor rating scale or of motor evoked potentials indicated that motor functional recovery was enhanced; retrograde tracing confirmed that the ascending tract was regenerated and the neural pathway was established; and histological analyses revealed that axon growth and remyelination in the regenerated nerve was promoted. The three-dimensional reconstruction showed that the chitosan conduit loaded with BMSCs significantly reduced the spinal cord cavity volume at the injured site. Taken together, the results collectively suggest that implantation with BMSCs-loaded chitosan conduits may become a promising approach to the repair of spinal cord injury.     

Use of chitosan conduit combined with bone marrow mesenchymal stem cells for promoting peripheral nerve regeneration

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test chitosan conduit to use in peripheral nerve reconstruction, either alone or combined with bone marrow mesenchymal stem cells (BMSCs). In this study, the roles of the degree of deacetylation (DD) and molecular weight of chitosan on some biological properties of chitosan films, including hydrophilicity, degradation and BMSCs affinity were investigated. The molecular weight of Chitosans used are 5 × 10⁴ Da, 2 × 10⁵ Da, 5 × 10⁵ Da, 1 × 10⁶ Da, the deacetylation degrees are 85, 95%, respectively. The affinity of eight kinds of Chitosans to the BMSCs was assessed by MTT assay, the contact angle and the degradation time of the materials in vivo were also measured. Chitosans with the molecular weight of 1 × 10⁶ Da and DD of 95% can significantly promote the survival and outgrowth of cells, which have better hydrophilicity and can remain integrity even after 8 to 16 weeks, all of above meet the requirement of nerve engineering. The BMSCs we transplanted can differentiate into neural stem cells in vivo, and the materials we selected combined with BMSCs can bridge 8-mm-long neural gap better resulting from the differentiation effects of the BMSCs.     

LvBMP-2 gene-modified BMSCs combined with calcium phosphate cement scaffolds for the repair of calvarial defects in rats

The study aims to evaluate the effect of bone marrow stromal cells (BMSCs) expressing bone morphogenic protein-2 (BMP-2) mediated by lentiviral (Lv) gene transduction combined with calcium phosphate cement (CPC) scaffolds for the repair of critical size calvarial defects in rats. BMSCs derived from Fisher 344 rats were transduced with LvBMP-2 or lentivirus encoding enhanced green fluorescent protein (LvEGFP) in vitro. Obvious osteogenic differentiation of BMSCs in the LvBMP-2 group was demonstrated by alkaline phosphatase staining and alizarin red staining. Enzyme-linked immunosorbent assay results show that LvBMP-2 gene expression in vitro can last for at least 8 weeks. Gene-transduced or untransduced BMSCs were seeded onto CPC scaffolds to repair rat calvarial defects with a diameter of 5 mm. Scanning electron microscope analysis indicated that porous CPC scaffolds facilitated initial adhesion and spreading of BMSCs onto its surface. Calvarial defects were successfully repaired with LvBMP-2-transduced BMSCs/CPC constructs 8 weeks postoperatively. The percentage of new bone formation in the LvBMP-2 group was significantly higher than in other control groups. Lentiviral mediated BMP-2 gene therapy together with CPC scaffolds can be used successfully in calvarial repair and bone regeneration.     

Preparation and evaluation of novel nano-bioglass/gelatin conduit for peripheral nerve regeneration

Peripheral nerves are exposed to physical injuries usually caused by trauma that may lead to a significant loss of sensory or motor functions and is considered as a serious health problem for societies today. This study was designed to develop a novel nano bioglass/gelatin conduit (BGGC) for the peripheral nerve regeneration. The bioglass nanoparticles were prepared by sol–gel technique and characterized using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analysis. The interfacial bonding interaction between the nano-bioglass and gelatin in the developed conduits was assessed by FTIR. The surface morphology and pore size of the nanocomposite were investigated through scanning electron microscopy with the pore size of the conduits being 10–40 μm. Biocompatibility was assessed by MTT assay which indicated the BGGC to have good cytocompatibility. The guidance channel was examined and used to regenerate a 10 mm gap in the right sciatic nerve of a male Wistar rat. Twenty rats were randomly divided into two experimental groups, one with the BGGC and the other being normal rats. The gastrocnemius muscle contractility was also examined at one, two and three months post-surgery in all groups using electromyography (EMAP). Histological and functional evaluation and the results obtained from electromyography indicated that at three months, nerve regeneration of the BGGC group was statistically equivalent to the normal group (p > 0.05). Our result suggests that the BGGC can be a suitable candidate for peripheral nerve repair.     

A silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee

Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded in a three-dimensional scaffold of silk fibroin (SF) and chitosan (CS) to repair cartilage defects in the rabbit knee. Totally 54 rabbits were randomly assigned to BMSCs + SF/CS scaffold, SF/CS scaffold and control groups. A cylindrical defect was created at the patellofemoral facet of the right knee of each rabbit and repaired by scaffold respectively. Samples were prepared at 4, 8 and 12 weeks post-surgery for gross observation, hematoxylin–eosin and toluidine blue staining, type II collagen immunohistochemistry, Wakitani histology. The results showed that differentiated BMSCs proliferated well in the scaffold. In the BMSCs + SF/CS scaffold group, the bone defect was nearly repaired, the scaffold was absorbed and immunohistochemistry was positive. In the SF/CS scaffold alone group, fiber-like tissues were observed, the scaffold was nearly degraded and immunohistochemistry was weakly positive. In the control group, the defect was not well repaired and positive immunoreactions were not detected. Modified Wakitani scores were superior in the BMSCs + SF/CS scaffold group compared with those in other groups at 4, 8 and 12 weeks (P < 0.05). A SF/CS scaffold can serve as carrier for stem cells to repair cartilage defects and may be used for cartilage tissue engineering.     

Rapid continuous 3D printing of customizable peripheral nerve guidance conduits

Engineered nerve guidance conduits (NGCs) have been demonstrated for repairing peripheral nerve injuries. However, there remains a need for an advanced biofabrication system to build NGCs with complex architectures, tunable material properties, and customizable geometrical control. Here, a rapid continuous 3D-printing platform was developed to print customizable NGCs with unprecedented resolution, speed, flexibility, and scalability. A variety of NGC designs varying in complexity and size were created including a life-size biomimetic branched human facial NGC. In vivo implantation of NGCs with microchannels into complete sciatic nerve transections of mouse models demonstrated the effective directional guidance of regenerating sciatic nerves via branching into the microchannels and extending toward the distal end of the injury site. Histological staining and immunostaining further confirmed the progressive directional nerve regeneration and branching behavior across the entire NGC length. Observational and functional tests, including the von Frey threshold test and thermal test, showed promising recovery of motor function and sensation in the ipsilateral limbs grafted with the 3D-printed NGCs.     

Comparison of morphology and biocompatibility of acellular nerve scaffolds processed by different chemical methods

To investigate the morphological differences among acellular rat nerve scaffolds processed by different chemical methods and compare the biocompatibility between rat nerve grafts processed by different chemical methods and rat adipose-derived stem cells in vitro. Acellular rat sciatic nerve scaffolds processed by two different chemical methods (the Sondell method and the optimized method) and normal rat sciatic nerves were used as control. The structure and components of nerve scaffold were observed under microscopy, the degrees of decellularization and demyelination of nerve scaffold and integrity of nerve fiber tubes were assessed. The rat adipose-derived stem cells growth and adherence on scaffold were studied by scanning electron microscopy, the activity and adhesive ratio of rat adipose-derived stem cells in the nerve scaffold were compared. The basal lamina tubes and the extracellular matrix in the epineurium and perineurium in the nerve graft of optimized method were better preserved than the nerve graft of the Sondell method. After co-cultured with scaffolds, the difference of cell activity between three groups (two cell–scaffold combinations and control group) at the same observation time were not statistically significant (P > 0.05),the adhesive ratio of rat adipose-derived stem cells in the scaffold of the optimized method was better than that of the Sondell method. The scaffold of the optimized method is more effective than the scaffold of the Sondell method for peripheral nerve tissue engineering.     

Chitosan/silk fibroin-based tissue-engineered graft seeded with adipose-derived stem cells enhances nerve regeneration in a rat model

Sciatic nerve injury presents an ongoing challenge in reconstructive surgery. Local stem cell application has recently been suggested as a possible novel therapy. In the present study we evaluated the potential of a chitosan/silk fibroin scaffold serving as a delivery vehicle for adipose-derived stem cells and as a structural framework for the injured nerve regeneration. The cell-loaded scaffolds were used to regenerate rat sciatic nerve across a 10 mm surgically-induced sciatic nerve injury. The functional nerve recovery was assessed by both walking track and histology analysis. Results showed that the reconstruction of the injured sciatic nerve had been significantly enhanced with restoration of nerve continuity and function recovery in the cell-loaded scaffold groups, and their target skeletal muscle had been extensively reinnervated. This study raises a potential possibility of using the newly developed nerve grafts as a promising alternative for nerve regeneration.     

Poloxamer-based hydrogels hardening at body core temperature as carriers for cell based therapies: in vitro and in vivo analysis

Cell-based regenerative therapies for bone defects usually employ bone precursor cells seeded on solid scaffolds. Thermosensitive hydrogels that harden at body core temperature are promising alternative cell carriers as they are applicable minimally invasively. We modified Pluronic^® P123 with different chain extenders and assessed rheology and biocompatibility of the resulting hydrogels. The best candidate was tested in a rat’s femoral defect model. All gels hardened above 25 °C with butane-diisocyanate-hydrogels (BDI-gels) displaying the highest storage moduli. BDI-gels showed the most favourable biocompatibility and did not affect cellular adipogenic or osteogenic differentiation in vitro. Implantation of BDI-hydrogel into femoral defects did not impede bone healing in vivo as evidenced by μCT and histological analysis. We conclude that thermosensitive BDI-gels are promising alternative cell carriers. The gels harden upon injection in vivo without interfering with bone metabolism. Further experiments will assess the gels’ capacity to effectively transport living cells into bone defects.     

The controlling biodegradation of chitosan fibers by N-acetylation in vitro and in vivo

Aim In the present study, we investigated the biodegradation of the fibers of chitosan and its acetylated derivatives in vitro and in vivo. Methods A series of chitosan fibers, with acetylation degrees of 7.7%, 21.6%, 40.9%, 61.2%, 82.5% and 93.4%, were obtained by acetylating chitosan filament with acetic anhydride, and were investigated by FT-IR analysis, elemental analysis and scanning electron microscopy analysis. Results The in vitro experimental data indicated that the degradation rate of chitosan fiber was strongly dependent on the degree of acetylation, and the degradation rate increased with an enhancement of the acetylation degree of chitosan fibers. In vivo degradation experiment evaluated by light microscopy as well as scanning electron microscopy, was studied by implanting the fibers between the two nerve stumps of the rat sciatic nerve gap (6 months). The findings demonstrated that acetylation degree could influence the degradation rate of chitosan fibers in vivo. Conclusion These results suggested that acetylated chitosan (chitin) fibers were more biodegradable than chitosan and the biodegradation rate of chitin fiber can be controlled to desirable extent by the variation of acetylation degree.     

Development of the chitosan tube prepared from crab tendon for nerve regeneration

Crystalline chitosan was successfully prepared from crab tendons with aligned chitin molecules. Chitosan obtained preserved the tube structure with a suitable size for nerve conduits (t-chitosan tube). The chitosan tubes were compounded with hydroxy apatite (HAp) using an alternate soaking method (t-chitosan/HAp tube) to enhance the strength of the tube walls. HAp crystals formed in the walls of the chitosan tubes, and their c-axis aligned well in parallel with the chitosan molecules. The growth of the HAp crystals was found to occur at the nucleation sites, most probably by forming complexes with amino groups on chitosan mediated by Ca ions. Furthermore, these tubes were treated at 120 °C to prevent from swelling. These treatments preserved well the hollow nature of the tubes. And the mechanical test showed that the force was significantly higher in the t-chitosan/HAp tube when compared to the circular or triangular t-chitosan tube at each strain.Bridge grafting (15 mm) into the sciatic nerve of SD rats was carried out using the t-chitosan tubes having either a circular or triangular cross-section as well as t-chitosan/HAp tubes (N=18 in each group). Specimens were taken after two, four, six and eight weeks for histology (N=3 in each group). And nerve regeneration was evaluated histologically after 12 weeks (N=6 in each group). The t-chitosan/HAp tubes, demonstrated preferable biodegradation and biocompatibility. In addition to the mechanical properties of the tubes, the results of histological findings suggest that a triangular shape of the tube's cross-section and HAp coating may benefit nerve regeneration.     

Electroactive chitosan-aniline pentamer hydrogel for peripheral nerve regeneration

Electroactive hydrogels could guide the regeneration of nerves and promote their functional recovery. An aniline pentamer-crosslinked chitosan (CS-AP) hydrogel with better electroactivity and degradation was fabricated by the carbodiimide method, and then injected into the repair site of sciatic nerve damage, with its gelation time, tensile strength, and conductivity reaching 35 min, 5.02−6.69 MPa, and from 2.97 × 10⁻⁴ to 3.25 × 10⁻⁴ S·cm⁻¹, respectively, due to the cross-linkage and well-distribution of AP. There was better cytocompativility of CS-AP hydrogel on nerve cells. The results of the in vivo repair indicated that CS-AP10 hydrogel induced the capillaries formation and the repair of sciatic nerve defect, and re-innervated gastrocnemius muscle in the CS-AP10 group were obviously better than other experimental groups, due to the electroactivity of CS-AP and its degradation into fragments. These results indicated the potential application of CS-AP hydrogel in the regeneration and function recovery of peripheral nerve injury.     

Regeneration of peripheral nerves by bioabsorbable polymer tubes with fibrin gel

We developed two types of nerve conduits, straight tubes, and bellows tubes, for peripheral nerve regeneration with bioabsorbable polymer membranes. Mechanical properties of these straight and bellows tubes were analyzed. 30 straight tubes and 30 bellows tubes were implanted to a nerve defect made in a rat sciatic nerve and the nerve regeneration in the tube was investigated. A half of these tubes were utilized alone and the others were filled with fibrin gel made from coagulated plasma and implanted. Bellows tubes were superior in mechanical characteristics to straight tubes and the inner cavities of the bellows tubes were suitably maintained after implantation. At 4 weeks after operations, remyelinations were observed in the regenerated tissue at the location of the middle parts of the tubes filled with fibrin gel whereas no remyelinations in the tubes without fibrin gel. The use of fibrin gel as filling materials within the tubes significantly enhanced the nerve regeneration. The fibrin gel might be soft nanomaterials significantly enhance the regeneration of the peripheral nerves. We concluded that the developed bioabsorbable bellows tubes filled with fibrin gel were effective for peripheral nerve regeneration.     

The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model

The amnion muscle combined graft (AMCG) conduits showed good clinical results in peripheral nerves gap repair. It combines the human amniotic membrane with autologous skeletal muscle fibres. These results seem attributable to the biological characteristics of human amniotic membrane: Pluripotency, anti-inflammatory and low immunogenicity.We here evaluate the final outcome of nerve regeneration morphologically and functionally, across the AMCG compared to nerve autograft. Fourteen Wistar rats were divided into two groups: In Group A, including 6 rats, the left forelimb was treated performing a 1.5?cm length gap on median nerve that was then reconstructed with a reverse autograft. In Group B, including 8 rats, the gap was reconstructed with AMCG. Functional results were evaluated at 30, 60 and 90 days performing grasping tests. Morphological and stereological analyses were performed at T90 using high-resolution light microscopy and design-based stereology. The AMCG conduits revealed nerve fibres regeneration and functional recovery. Functional recovery was observed in both groups with AMCG conduits group showing lower values and a regeneration of median nerves with more myelinated fibres with the same axon size, but thinner myelin than the autograft group. Though the autograft remains the gold standard to restore wide nerve gaps, the AMCG conduit has proved to be effective in enabling nerve regeneration through a critical rat’s nerve gap of 15?mm. These findings empirically support the great clinical results obtained using AMCG conduit to restore traumatic nerve’s gap from 3 to 6?cm of mixed forearm nerves.

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Optimization of fully aligned bioactive electrospun fibers for “in vitro” nerve guidance

Complex architecture of natural tissues such as nerves requires the use of multifunctional scaffolds with peculiar topological and biochemical signals able to address cell behavior towards specific events at the cellular (microscale) and macromolecular (nanoscale) level. In this context, the electrospinning technique is useful to generate fiber assemblies having peculiar fiber diameters at the nanoscale and patterned by unidirectional ways, to facilitate neurite extension via contact guidance. Following a bio-mimetic approach, fully aligned polycaprolactone fibers blended with gelatin macromolecules have been fabricated as potential bioactive substrate for nerve regeneration. Morphological and topographic aspects of electrospun fibers assessed by SEM/AFM microscopy supported by image analyses elaboration allow estimating an increase of fully aligned fibers from 5 to 39 % as collector rotating rate increases from 1,000 to 3,000 rpm. We verify that fully alignment of fibers positively influences in vitro response of hMSC and PC-12 cells in neurogenic way. Immunostaining images show that the presence of topological defects, i.e., kinks—due to more frequent fiber crossing—in the case of randomly organized fiber assembly concurs to interfere with proper neurite outgrowth. On the contrary, fully aligned fibers without kinks offer a more efficient contact guidance to direct the orientation of nerve cells along the fibers respect to randomly organized ones, promoting a high elongation of neurites at 7 days and the formation of bipolar extensions. So, this confirms that the topological cue of fully alignment of fibers elicits a favorable environment for nerve regeneration.     

Evaluation of a multi-layer microbraided polylactic acid fiber-reinforced conduit for peripheral nerve regeneration

We evaluated peripheral nerve regeneration using a biodegradable multi-layer microbraided polylactic acid (PLA) fiber-reinforced conduit. Biodegradability of the PLA conduit and its effectiveness as a guidance channel were examined as it was used to repair a 10 mm gap in the rat sciatic nerve. As a result, tube fragmentation was not obvious and successful regeneration through the gap occurred in all the conduits at 8 weeks after operation. These results indicate the superiority of the PLA materials and suggest that the multi-layer microbraided PLA fiber-reinforced conduits provide a promising tool for neuro-regeneration. Ming-Chin Lu, Chun-Hsu Yao, and Yueh-Sheng Chen are contributed equally to this work.     

Application potential of bone marrow mesenchymal stem cell (BMSCs) based tissue-engineering for spinal cord defect repair in rat fetuses with spina bifida aperta

Spina bifida aperta are complex congenital malformations resulting from failure of fusion in the spinal neural tube during embryogenesis. Despite surgical repair of the defect, most patients who survive with spina bifida aperta have a multiple system handicap due to neuron deficiency of the defective spinal cord. Tissue engineering has emerged as a novel treatment for replacement of lost tissue. This study evaluated the prenatal surgical approach of transplanting a chitosan–gelatin scaffold seeded with bone marrow mesenchymal stem cells (BMSCs) in the healing the defective spinal cord of rat fetuses with retinoic acid induced spina bifida aperta. Scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMSCs. After transplantation of the scaffold combined with BMSCs, the defective region of spinal cord in rat fetuses with spina bifida aperta at E20 decreased obviously under stereomicroscopy, and the skin defect almost closed in many fetuses. The transplanted BMSCs in chitosan–gelatin scaffold survived, grew and expressed markers of neural stem cells and neurons in the defective spinal cord. In addition, the biomaterial presented high biocompatibility and slow biodegradation in vivo. In conclusion, prenatal transplantation of the scaffold combined with BMSCs could treat spinal cord defect in fetuses with spina bifida aperta by the regeneration of neurons and repairmen of defective region.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

Transverse elasticity of rabbit sciatic nerves tested by in vitro compression

Abstract

The Young's modulus of a rabbit sciatic nerve under stepwise in vitro linear compression test was estimated. Segments of rabbit sciatic nerves were compressed in the transverse direction with a custom‐made parallel compression apparatus. Digital images of the cross‐sectional face were taken simultaneously by using a camera mounted on a microscope. The applied force and the gap between the parallel plates of the compression apparatus were measured. Digitized images of the nerve crosssections were used to construct two‐dimensional models of the nerves. The parallel compression results showed that the mean Young's modulus of rabbit sciatic nerves was 41.6±5.0 kPa. By direct visual inspection and by comparing the finite element model simulation results and experimental data, we may suggest that the large fascicle is the main load‐bearing component while the small fascicle and the loose connective tissues like the epineurium bear less load in the parallel compression process.     

Comparative study of three types of polymer materials co-cultured with bone marrow mesenchymal stem cells for use as a myocardial patch in cardiomyocyte regeneration

The purpose of this study was to investigate the most suitable polymer material for supporting stem cell growth as a myocardial patch. After cell isolation and expansion of mouse bone marrow mesenchymal stem cells (BMSC), the cells were induced to differentiate into cardiomyocytes with 5-azacytidine to determine their differentiation potential. BMSCs were also seeded onto three types of polymer material film, including polyurethane (PU), 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)], and polypropylene carbonate (PPC). The results revealed that cell numbers were more abundant on both the PU and P(3HB-co-4HB) material surfaces. Conversely, the surface of PPC was smooth with only cell lysate debris observed. The average cell counts were as follows: 143.78 ± 38.38 (PU group), 159.50 ± 33.07 [P(3HB-co-4HB) group], and 1.40 ± 0.70 (PPC group). There was no statistically significant difference in cell numbers between the PU and P(3HB-co-4HB) groups. A statistically significant difference was identified between the PPC group and both the PU (P1) and P(3HB-co-4HB) groups (P2). Polymer biomaterial patches composed of PU and P(3HB-co-4HB) permit good stem cell growth. P(3HB-co-4HB) has the potential for development as a clinical alternative to current treatment methods for the regeneration of cardiomyocytes in patients with myocardial infarction.