Allotransplanted neurons used to repair peripheral nerve injury do not elicit overt immunogenicity

A major problem hindering the development of autograft alternatives for repairing peripheral nerve injuries is immunogenicity. We have previously shown successful regeneration in transected rat sciatic nerves using conduits filled with allogeneic dorsal root ganglion (DRG) cells without any immunosuppression. In this study, we re-examined the immunogenicity of our DRG neuron implanted conduits as a potential strategy to overcome transplant rejection. A biodegradable NeuraGen® tube was infused with pure DRG neurons or Schwann cells cultured from a rat strain differing from the host rats and used to repair 8 mm gaps in the sciatic nerve. We observed enhanced regeneration with allogeneic cells compared to empty conduits 16 weeks post-surgery, but morphological analyses suggest recovery comparable to the healthy nerves was not achieved. The degree of regeneration was indistinguishable between DRG and Schwann cell allografts although immunogenicity assessments revealed substantially increased presence of Interferon gamma (IFN-γ) in Schwann cell allografts compared to the DRG allografts by two weeks post-surgery. Macrophage infiltration of the regenerated nerve graft in the DRG group 16 weeks post-surgery was below the level of the empty conduit (0.56 fold change from NG; p<0.05) while the Schwann cell group revealed significantly higher counts (1.29 fold change from NG; p<0.001). Major histocompatibility complex I (MHC I) molecules were present in significantly increased levels in the DRG and Schwann cell allograft groups compared to the hollow NG conduit and the Sham healthy nerve. Our results confirmed previous studies that have reported Schwann cells as being immunogenic, likely due to MHC I expression. Nerve gap injuries are difficult to repair; our data suggest that DRG neurons are superior medium to implant inside conduit tubes due to reduced immunogenicity and represent a potential treatment strategy that could be preferable to the current gold standard of autologous nerve transplant.     

Nerve conduit based on HAP/PDLLA/PRGD for peripheral nerve regeneration with sustained release of valproic acid

The nerve conduits have been developed for nerve defect repair. However, no artificial conduits have obtained comparable results to autografts to bridge the large gaps. A possible reason for this poor performance may be a lack of sustainable neurotrophic support for axonal regrowth. Previous studies suggested nanocomposite conduits can be used as a carrier for valproic acid (VPA), a common drug that can produce effects similar to the neurotrophic factors. Here, we developed the novel bioabsorbable conduits based on hydroxyapatite/poly d -l -lactic acid (PDLLA)/poly{(lactic acid)-co-[(glycolic acid)-alt-(l -lysine)]} with sustained release of VPA. Firstly, the sustained release of VPA in this conduit was examined by high-performance liquid chromatography. Then Schwann cells were treated with the conduit extracts. The cell metabolic activity and proliferation were assayed by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide and bromodeoxyuridine staining. A 10-mm segment of rat sciatic nerve was resected and then repaired, respectively, using the VPA conduit (Group A), the PDLLA conduit (Group B), or the autografts (Group C). Nerve conduction velocities (NCVs), compound muscle action potentials (CMAPs), and histological staining were assayed following the surgery. The cell metabolic activity and proliferation were significantly increased (p < .05) by the extracts from VPA-conduit extract compared to others. NCVs and CMAPs were significantly higher in Groups A and C than Group B (p < .05). The nerve density of Groups A and C was higher than Group B. There was no significant difference between Groups A and C. Taken together, this study suggested the sustained-release VPA conduit promoted peripheral nerve regeneration that was comparable to the autografts. It holds potential for future use in nerve regeneration.     

Development of a bioartificial nerve graft. II. Nerve regeneration in vitro

A promising alternative for the repair of peripheral nerve injuries is the bioartificial nerve graft, or BNG, comprised of a tubular conduit preseeded with Schwann cells, which are an effective substrate for enhancing nerve regeneration. The physical properties of the conduit, porosity and wall thickness, as well as the Schwann cell seeding density, were tested for their effect on axon growth using rat dorsal root ganglia. These parameters can influence the amount of nutrients and growth factors made available to the neural tissue. Results show that a greater wall thickness and lower porosities have a detrimental effect on the growth of the axons. Over a four week period, axons extended 3.2 mm for the optimum case (DeltaR = 0.82 mm, epsilon = 0.75) compared to 1.8 and 1.6 mm for a lower porosity (0.55) and a greater wall thickness (1.4 mm), respectively. A maximum in the growth rate occurs at a porosity of 75% for Schwann cell seeded conduits but not for unseeded ones. When compared to mass transfer predictions, the results suggest that, at higher porosities, more growth factors diffuse out of the conduit, while at low porosities there is competition for nutrients. Increasing the Schwann cell seeding density enhances growth but also leads to an increase in the number of axons along the length of the conduit. This is indicative of branching of the axons, which requires additional resources to maintain and can lead to painful neuroma formation. Wall thickness and porosity were found not to have any significant effect on the axon number sprouting from the dorsal root ganglia and the mean diameter (p > 0.05). Considerations need to be made, not just on the polymer used, but also on its porosity, wall thickness, and Schwann cell seeding density. These parameters can be adjusted to create a bioartificial nerve graft that provides the optimal environment for nerve growth.     

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase

In spite of the extensive research using induced pluripotent stem (iPS) cells, the therapeutic potential of iPS cells in the treatment of peripheral nerve injury is largely unknown. In this study, we repaired peripheral nerve gaps in mice using tissue-engineered bioabsorbable nerve conduits coated with iPS cell-derived neurospheres. The secondary neurospheres derived from mouse iPS cells were suspended in each conduit (4000,000 cells per conduit) and cultured in the conduit in three-dimensional (3D) culture for 14 days. We then implanted them in the mouse sciatic nerve gaps (5 mm) (iPS group; n=10). The nerve conduit alone was implanted in the control group (n=10). After 4, 8 and 12 weeks, motor and sensory functional recovery in mice were significantly better in the iPS group. At 12 weeks, all the nerve conduits remained structurally stable without any collapse and histological analysis indicated axonal regeneration in the nerve conduits of both groups. However, the iPS group showed significantly more vigorous axonal regeneration. The bioabsorbable nerve conduits created by 3D-culture of iPS cell-derived neurospheres promoted regeneration of peripheral nerves and functional recovery in vivo. The combination of iPS cell technology and bioabsorbable nerve conduits shows potential as a future tool for the treatment of peripheral nerve defects.     

成年大鼠雪旺细胞的快速扩增

采用接种雪旺细胞的可降解导管修复外周神经损伤是一种有望替代自体神经移植的方法。如何在短期内利用病人少量的神经碎片获得大量雪旺细胞是该方法用于临床的关键。以大鼠坐骨神经为模型,利用雪旺细胞增殖的内在机制,探索出一种快速增殖成年雪旺细胞的方法。采用预变性7d的坐骨神经,用酶消化分离出雪旺细胞,接种在层粘连蛋白包被的培养瓶中,经过7d的培养,获得纯度为96％、细胞密度为600个／mm^2的雪旺细胞,雪旺细胞的纯度和密度明显高于对照的新鲜神经。未使用霍乱毒素、毛喉素等促有丝分裂剂和抑制成纤维细胞的基因毒素,符合临床使用要求。结果表明,可以利用少量的损伤神经碎片在短期内获得大量可用于临床的雪旺细胞。     

In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.     

A nanofibrous PHBV tube with Schwann cell as artificial nerve graft contributing to Rat sciatic nerve regeneration across a 30-mm defect bridge

Abstract

A nanofibrous PHBV nerve conduit has been used to evaluate its efficiency based on the promotion of nerve regeneration in rats. The designed conduits were investigated by physical, mechanical and microscopic analyses. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently high mechanical properties to serve as a nerve guide. The results demonstrated that in the nanofibrous graft with cells, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. For the grafts especially the nanofibrous conduits with cells, muscle cells of gastrocnemius on the operated side were uniform in their size and structures. This study proves the feasibility of artificial conduit with Schwann cells for nerve regeneration by bridging a longer defect in a rat model.     

Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions

In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.     

Behavioral evaluation of regenerated rat sciatic nerve by a nanofibrous PHBV conduit filled with Schwann cells as artificial nerve graft

Abstract

The aim of this study is to develop a nanofibrous polymeric nerve conduit with Schwann cells (SCs) and to evaluate its efficiency on the promotion of functional and locomotive activities in rats. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the rats were monitored and evaluated by behavioral analyses such as toe out angle, toe spreading analysis, walking track analysis, extensor postural thrust, open-field analysis, swimming test and nociceptive function, four months post surgery. Four months post-operatively, the results from behavioral analyses demonstrated that in the grafted groups especially in the grafted group with SCs, the rat sciatic nerve trunk had been reconstructed with functional recovery such as walking, swimming and recovery of nociceptive function. This study proves the feasibility of artificial conduit with SCs for nerve regeneration by bridging a longer defect in the rat model.     

Polyglycolic acid filaments guide Schwann cell migration in vitro and in vivo

Nerve conduits filled with longitudinal aligned filaments have demonstrated a better regenerative outcome for bridging large peripheral nerve gaps than hollow nerve conduits. In the present study, we investigated the in vitro and in vitro cellular behavior of Schwann cells on polyglycolic acid (PGA) filaments by immunocyto/histochemistry and light/electron microscopy. After 1-3-week culture of rat dorsal root ganglia (DRGs) onto PGA filaments, Schwann cells from rat DRGs adhered to and migrated along PGA filaments. Twenty-four rats received implantation of chitosan conduits inserted with PGA filaments to bridge 10-mm-long sciatic nerve gaps. At 1, 2, 3 and 4 weeks post-implantation (n = 6, each time point), Schwann cells were found to migrate along PGA filaments and form cell columns resembling bands of Büngner. These results suggest that PGA filaments may play a contact guidance role in Schwann cell migration and thus serve as a promising conduit-filling material to facilitate peripheral nerve repair.     

嗅鞘细胞复合PLGA导管修复周围神经缺损的研究

探讨嗅鞘细胞(OECs)复合聚乳酸-聚羟基乙酸共聚物(PLGA)导管对大鼠坐骨神经缺损的修复作用。方法:SD大鼠80只,随机分成4组,切除右侧部分神经干造成10mm的神经缺损。OECs PLGA组用充满细胞外基质凝胶和OECs悬液(CM-DiI预标记)的PLGA导管桥接坐骨神经缺损;OECs 硅胶管组用含相同内容物的硅胶管桥接;PLGA组和硅胶管组则分别用充满细胞外基质凝胶和DMEM/F12培养基的PLGA导管和硅胶管桥接。术后每周进行感觉运动功能检测,8周时行腓肠肌湿重恢复率、乙酰胆碱脂酶(AChE)染色、电生理和组织形态学分析等检测,同时移植细胞的两组每周进行细胞示踪观察。结果:移植细胞沿神经纵轴分布;除坐骨神经功能指数(SFI)指标外,OECs PLGA组的各项再生功能指标均优于其它三组。结论:OECs复合PLGA导管能够促进再生神经的成熟和靶组织功能的恢复,二者联合移植是一种有效的周围神经缺损修复方法。     

Perfusion seeding of channeled elastomeric scaffolds with myocytes and endothelial cells for cardiac tissue engineering

The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% ± 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% ± 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The Effects of Controlled Release of Neurotrophin-3 from PCLA Scaffolds on the Survival and Neuronal Differentiation of Transplanted Neural Stem Cells in a Rat Spinal Cord Injury Model

Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2±4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8±15.1%) and neuronal differentiation (21.5±5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.     

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的:探讨应用改进静电纺丝技术一次成型制备三维(3D)取向聚乳酸与聚羟基乙酸共聚物(PLGA)纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法:应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组(每组12只),A组:非取向PLGA神经导管组(阴性对照);B组:取向PLGA神经导管组,C组:自体神经移植组(阳性对照),于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果:神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异(P0.05),均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论:无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

三维取向PLGA神经导管促进坐骨神经再生的实验研究

目的：探讨应用改进静电纺丝技术一次成型制备三维（3D）取向聚乳酸与聚羟基乙酸共聚物（PLGA）纳米神经导管的可行性,检测其对坐骨神经再生的促进作用。方法：应用改进的静电纺丝技术制备无缝取向PLGA纳米神经导管,通过扫描电镜和透射电镜检测支架的纳米结构;分别制备取向和非取向纳米纤维支架修复13mm坐骨神经缺损模型。36只成年SD大鼠随机分为3组（每组12只）,A组：非取向PLGA神经导管组（阴性对照）;B组：取向PLGA神经导管组,C组：自体神经移植组（阳性对照）,于术后3月通过大体观察、行走足印分析、腓肠肌萎缩率、电生理检测、组织形态学检测、透射电镜检测及图像分析,评价无缝取向PLGA纳米神经导管修复坐骨神经缺损的效果。结果：神经导管修复神经缺损三月后,大体观察显示神经导管结构完整,无坍塌和断裂;各组再生神经均有通过神经导管长入远端。B组与C组的腓肠肌萎缩率和神经电传导速度无统计学差异（P〈0.05）,均优于A组。B组与C组再生神经纤维数量及成熟程度均要明显优于A组。结论：无缝取向PLGA纳米神经导管能够诱导并促进神经再生,提高坐骨神经再生的质量,有望成为自体神经移植的替代物。     

Drug-delivering nerve conduit improves regeneration in a critical-sized gap

Autologous nerve grafts are the current “gold standard” for repairing large nerve gaps. However, they cause morbidity at the donor nerve site and only a limited amount of nerve can be harvested. Nerve conduits are a promising alternative to autografts and can act as guidance cues for the regenerating axons, without the need to harvest donor nerve. Separately, it has been shown that localized delivery of GDNF can enhance axon growth and motor recovery. FK506, an FDA approved small molecule, has also been shown to enhance peripheral nerve regeneration. This paper describes the design of a novel hole-based drug delivery apparatus integrated with a polytetrafluoroethylene (PTFE) nerve conduit for controlled local delivery of a protein such as GDNF or a small molecule such as FK506. The PTFE devices were tested in a diffusion chamber, and the bioactivity of the released media was evaluated by measuring neurite growth of dorsal root ganglions (DRGs) exposed to the released drugs. The drug delivering nerve guide was able to release bioactive concentrations of FK506 or GDNF. Following these tests, optimized drug releasing nerve conduits were implanted across 10 mm sciatic nerve gaps in a BL6 yellow fluorescent protein (YFP) mouse model, where they demonstrated significant improvement in muscle mass, compound muscle action potential, and axon myelination in vivo as compared with nerve conduits without the drug. The drug delivery nerve guide could release drug for extended periods of time and enhance axon growth in vitro and in vivo.     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。     

Physical and biological performance of a novel block copolymer nerve guide

Although the ability to regenerate is evident in the nervous system, lesioned neurites are unable to cross gaps in neuronal pathways. In order to bridge gaps, guiding cues are essential to direct neurite regrowth. To overcome many of the shortcomings of polymer-based nerve guides, we developed a bioresorbable nerve guide composed of a novel trimethylene carbonate-caprolacton block copolymer (TMC-CL). Pore formation was controlled by using special solvent/precipitation media compositions in combination with the pore forming agent poly ethylene glycol (PEG). NMR spectroscopy, shear force-, compression-, and permeation assays were used for conduit characterization. The polymer conduit has a semipermeable wall with submicron pores to allow free metabolite/drug exchange. In order to investigate the principle of temporally controlled expression of therapeutic proteins in nerve guides, Neuro-2a cells were genetically engineered to express the reporter gene product green fluorescent protein (GFP) under the control of the Tet-On system. When these transduced cells were encapsulated in nerve guides, GFP expression could be induced for days by adding the antibiotic tetracycline derivative doxycycline to the nerve guide environment. Furthermore, encapsulated dorsal root ganglia (DRG) produced long neurites in vitro. In subsequent in vivo experiments, nerve guides filled with Schwann cells (SC) were implanted into lesioned spinal cords of adult rats. Regeneration of spinal cord axons into nerve guides was promoted by co-implanted Schwann cells. The data suggest that the novel TMC-CL nerve guides provide a promising tool for neuroregeneration.     

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three‐dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long‐term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform‐sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live‐dead assay, and real‐time RT‐PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell‐seeded scaffold product for applications in regenerative medicine.     

Lubricated biodegradable polymer networks for regulating nerve cell behavior and fabricating nerve conduits with a compositional gradient

We present a method of tuning surface chemistry and nerve cell behavior by photo-cross-linking methoxy poly(ethylene glycol) monoacrylate (mPEGA) with hydrophobic, semicrystalline poly(ε-caprolactone) diacrylate (PCLDA) at various weight compositions of mPEGA (?(m)) from 2 to 30%. Improved surface wettability is achieved with corresponding decreases in friction, water contact angle, and capability of adsorbing proteins from cell culture media because of repulsive PEG chains tethered in the network. The responses of rat Schwann cell precursor line (SpL201), rat pheochromocytoma (PC12), and E14 mouse neural progenitor cells (NPCs) to the modified surfaces are evaluated. Nonmonotonic or parabolic dependence of cell attachment, spreading, proliferation, and differentiation on ?(m) is identified for these cell types with maximal values at ?(m) of 5-7%. In addition, NPCs demonstrate enhanced neuronal differentiated lineages on the mPEGA/PCLDA network at ?(m) of 5% with intermediate wettability and surface energy. This approach lays the foundation for fabricating heterogeneous nerve conduits with a compositional gradient along the wall thickness, which are able to promote nerve cell functions within the conduit while inhibiting cell attachment on the outer wall to prevent potential fibrous tissue formation following implantation.