Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment.

Introduction: Forming a bridge made of functional axons to span the lesion is essential to reconstruct the motor circuitry following spinal cord injury (SCI). Dorsal root ganglion (DRG) axons are robust in axon growth and have been proved to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. However, whether DRG transplantation affects the axon outgrowth of spinal motor neurons (SMNs) that play crucial roles in motor circuitry remains unclear. Methods: We investigated the axonal growth patterns of co-cultured DRGs and SMN aggregates (SMNAs) taking advantage of a well-designed 3D-printed in vitro system. Chondroitin sulphate proteoglycans (CSPG) induced inhibitory matrix was introduced to imitate the inhibitory environment following SCI. Axonal lengths of DRG, SMNA or DRG & SMNA cultured on the permissive or CSPG induced inhibitory matrix were measured and compared. Results: Our results indicated that under the guidance of full axonal connection generated from two opposing populations of DRGs, SMNA axons were growth-enhanced and elongated along the DRG axon bridge to distances that they could not otherwise reach. Quantitatively, the co-culture increased the SMNA axonal length by 32.1 %. Moreover, the CSPG matrix reduced the axonal length of DRGs and SMNAs by 46.2 % and 17.7 %, respectively. This inhibitory effect was antagonized by the co-culture of DRGs and SMNAs. Especially for SMNAs, they extended the axons across the CSPG-coating matrix, reached the lengths close to those of SMNAs cultured on the permissive matrix alone. Conclusions: This study deepens our understanding of axon-facilitated reconstruction of the motor circuitry. Moreover, the results support SCI treatment utilizing the enhanced outgrowth of axons to restore functional connectivity in SCI patients.

Histological and functional outcomes in a rat model of hemisected spinal cord with sustained VEGF/NT-3 release from tissue-engineered grafts.

Microvascular disturbance, excessive inflammation and gliosis are key pathophysiologic changes in relation to functional status following the traumatic spinal cord injury (SCI). Continuous release of vascular endothelial growth factor (VEGF) to the lesion site was proved be able to promote the vascular remodelling, whereas the effects on reduction of inflammation and gliosis remain unclear. Currently, aiming at exploring the synergistic roles of VEGF and neurotrophin-3 (NT-3) on angiogenesis, anti-inflammation and neural repair, we developed a technique to co-deliver VEGF165 and NT-3 locally with a homotopic graft of tissue-engineered acellular spinal cord scaffold (ASCS) in a hemisected (3 mm in length) SCI model. As the potential in secretion of growth factors (GFs), bone mesenchymal stem cells (BMSCs) were introduced with the aim to enhance the VEGF/NT-3 release. Our data demonstrate that sustained VEGF/NT-3 release from ASCS significantly increases the local levels of VEGF/NT-3 and angiogenesis, regardless of whether it is in combination with BMSCs transplantation that exhibits positive effects on anti-inflammation, axonal outgrowth and locomotor recovery. This study verifies that co-delivery of VEGF/NT-3 reduces inflammation and gliosis in the hemisected spinal cord, promotes axonal outgrowth and results in better locomotor recovery, while the BMSCs transplantation facilitates these functions limitedly.

Dorsal root ganglion axons facilitate and guide cortical neural outgrowth: In vitro modeling of spinal cord injury axonal regeneration.

BACKGROUND: Spinal cord injury (SCI) patients represent a heterogeneous group, with injuries ranging from partial compression to complete transection. Patients with complete injuries are unlikely to exhibit recovery and suffer from paralysis as well as the loss of bowel and bladder function. One treatment option is the formation of a bridge through a lesion site, whereby transplanted cells or biocompatible scaffolds guide the regenerating axons across the site of injury. Moreover, the viability of transplanted dorsal root ganglia (DRGs) into rat spinal cord has been previously demonstrated. OBJECTIVE: We aim to demonstrate the feasibility of using DRG axons as a bridging tool to help guide the axonal growth of cortical neurons. METHODS: Cortical neurons were isolated from embryonic rats and two aggregated populations were cultured at increasing distances in isolation and in a co-culture with DRG explants. Growth rates of the sprouting axons and connections between the two populations were observed over a period of twelve days. RESULTS: DRG explants demonstrated the ability to grow robust axonal connections that can connect two explants separated by up to 10 mm, however, CNAs could not achieve connections in distances greater than 2 mm. The co-culture of CNAs with DRG explants facilitated axonal growth between two populations of CNAs at distances they cannot otherwise traverse. CONCLUSIONS: Our findings support the use of DRG axons to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. We believe these results could have implications for the treatment of SCI.

Transplantation of a Peripheral Nerve with Neural Stem Cells Plus Lithium Chloride Injection Promote the Recovery of Rat Spinal Cord Injury.

Transplantation of neural stem cells (NSCs) holds great potential for the treatment of spinal cord injury (SCI). However, transplanted NSCs poorly survive in the SCI environment. We injected NSCs into tibial nerve and transplanted tibial nerve into a hemisected spinal cord and investigated the effects of lithium chloride (LiCl) on the survival of spinal neurons, axonal regeneration, and functional recovery. Our results show that most of the transplanted NSCs expressed glial fibrillary acidic protein, while there was no obvious expression of nestin, neuronal nuclei, or acetyltransferase found in NSCs. LiCl treatment produced less macrosialin (ED1) expression and axonal degeneration in tibial nerve after NSC injection. Our results also show that a regimen of LiCl treatment promoted NSC differentiation into NF200-positive neurons with neurite extension into the host spinal cord. The combination of tibial nerve transplantation with NSCs and LiCl injection resulted in more host motoneurons surviving in the spinal cord, more regenerated axons in tibial nerve, less glial scar area, and decreased ED1 expression. We conclude that lithium may have therapeutic potential in cell replacement strategies for central nervous system injury due to its ability to promote survival and neuronal generation of grafted NSCs and reduced host immune reaction.

Acellular Spinal Cord Scaffold Implantation Promotes Vascular Remodeling with Sustained Delivery of VEGF in a Rat Spinal Cord Hemisection Model.

BACKGROUND: Promoting angiogenesis provides a possible therapeutic approach in treating spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a pro-angiogenic substance that is involved in endothelial cell (EC) proliferation, migration, and survival. Exogenous administration of VEGF to the lesion epicenter of the spinal cord has been recently revealed as a potential method for promoting the blood vessel sprouting. METHODS: Spinal cord hemisection in a rat model was established and angiogenesis was studied through implant of an acellular spinal cord scaffold (ASCS) with sustained delivery of VEGF₁₆₅. The poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating VEGF₁₆₅ were fabricated on basis of an emulsion and solvent evaporation method and conjugated to ASCS by a Genipin (GP) crosslinking technology. The resultant scaffolds were marked as V-ASCS. VEGF₁₆₅ entrapment efficiency (EE) and released kinetics were determined by an ultraviolet absorption measurement. Angiogenesis and vascular remodeling were observed via a high-resolution micro-CT and analyzed quantitatively by vascular morphometric parameters. Spinal cord histology and Basso, Beattie, and Bresnahan (BBB) locomotor rating scale were further studied. RESULTS: VEGF₁₆₅ was entrapped with high efficiency (90.8±3.1) %. In vitro VEGF₁₆₅ release kinetics study showed an initial burst of 1.966 µg mg NPs-1 and 1.045µg mg V-ASCS-1 respectively in the first 24 hours. In the phase of sustained release, approximately 0.040µg mg NPs-1 and 0.022µg mg V-ASCS-1 per day was on-going until 720h. In the rat spinal cord hemisection model, implant of V-ASCS at the injured site showed a promotion of angiogenesis and vascular remodeling following SCI. A better outcome can be confirmed histologically. However, functional improvement is limited in the animal model. CONCLUSION: The results indicate that progress of vascular reconstruction is accelerated in the V-ASCS implanted SCI rats.

Effect of melatonin on the proliferation and differentiation of chondrocytes from rat vertebral body growth plate in vitro.

PURPOSE: Abnormal growth of vertebral body growth plate (VBGP) is considered as one of the etiologic factors in the adolescent idiopathic scoliosis (AIS). It was well-known that melatonin was correlated with the emergence and development of AIS. This study aimed to investigate the effect of melatonin on rat VBGP chondrocytes in vitro. METHODS: Chondrocytes were isolated from rat VBGP, and treated with or without melatonin. Cell proliferation was measured by the Alamar Blue assay. Gene expression of collagen type II and aggrecan were evaluated by real-time PCR. Expression of the melatonin receptors (MT1, MT2), proliferating cell nuclear antigen (PCNA, a cell proliferation marker), Sox9 (a chondrocytic differentiation marker) and Smad4 (a common mediator in regulating the proliferation and differentiation of chondrocytes) were detected by Western blotting. RESULTS: Expression of melatonin receptors (MT1, MT2) were detected in the rat VBGP chondrocytes. Melatonin, at 10 and 100 µg/mL concentration, significantly inhibited the proliferation of VBGP-chondrocytes and the gene expression of collagen type II and aggrecan, and down-regulated the protein expression of PCNA, Sox9 and Smad4. In addition, the inhibitory effect of melatonin was reversed by luzindole, a melatonin receptor antagonist. CONCLUSIONS: These results suggest that melatonin at high concentrations can inhibit the proliferation and differentiation of VBGP chondrocytes, which might give some new insight into the pathogenic mechanism of AIS.

Repair of critical size bone defects with porous poly(D,L-lactide)/nacre nanocomposite hollow scaffold.

OBJECTIVE: To generate a novel porous poly(D,L-lactide)/nacre nanocomposite hollow scaffold. METHODS: This study was performed in the Department of Spine Surgery, Southern Medical University, Guangzhou, China from September 2010 to September 2011. Nacre nanoparticles were prepared using a physical process and identified by x-ray diffraction and transmission electron microscopy, to generate a novel scaffold though the salt leaching processing technique. The morphology and structure properties of this scaffold were further investigated under scanning electron microscope and mechanical property testing. Additionally, the biological characteristics were evaluated by cell culture experiments in vitro. Thirty-six rabbits were randomly divided into 3 groups. The defects were implanted with/without poly(D,L-lactide)/nacre scaffold or poly(D,L-lactide) scaffold. The results were assessed by radiographs and bone mineral density to monitor bone repairing. RESULTS: The nacre nanoparticles were spherical in shape, with a diameter range from 45-95 nm. The scaffolds possessed an interconnected porous structure with an average pore size of 322.5+/-50.8 µm, and exhibited a high porosity (82.5 +/-0.8%), as well as good compressive strength of 4.5+/-0.25 Mpa. Primary biocompatibility experiments in vitro showed that cells adhered and proliferated well on the scaffolds. The animal study further demonstrated that the scaffolds could repair the critical size segmental bone defects in 12 weeks. CONCLUSION: Newly established scaffolds may serve as a promising biomaterial for bone tissue engineering.

Does whole-body vibration with alternative tilting increase bone mineral density and change bone metabolism in senior people?

BACKGROUND AND AIMS: Whole-body vibration (WBV) presents as osteogenic in animal models and young patients, but the effect remains unclear in senior people. The use of alternative tilting during WBV to ameliorate bone mass and bone metabolism, particularly in senior people, has not previously been reported. This study assessed changes in bone mineral density (BMD) and bone metabolism in senior people after six-month treatment of whole-body vibration with alternative tilting (WBVAT). METHODS: Fifty-three senior people (11M/42F, >65 yrs, mean age 77) and 15 adults (4M/11F, 50-60 yrs, mean age 53) were enrolled and assigned randomly to WBVAT (senior: n=27; adult: n=7) and control groups (senior: n=26; adult: n=7), respectively. The WBVAT groups were subjected to vertical vibration (0.5-0.8 g, 45-55 Hz) and alternative tilting (2° tilting angle or 8 mm displacement at 0.4 Hz) 20 minutes per day, 3 days a week, for 6 months. BMD in the lumbar spine and femoral neck was measured at 0, 3 and 6 months, respectively, as well as biochemical markers of bone metabolism, including serum calcium, phosphorus, alkaline phosphatase (ALP), osteocalcin and tartrate resistance acid phosphatase at 0, 1, 3 and 6 months, respectively. RESULTS: After 6-month WBVAT treatment, BMD in the lumbar spine and femoral neck increased significantly by 2.52% and 3.22% for senior people, and 1.63% and 2.05% for adults, respectively. The 6-month WBVAT treatment increased BMD in the senior people, both with and without osteoporosis (OP) and in both men and women, but led to a BMD gain greater in people with OP (p<0.01) and women (p<0.01), respectively. The serum ALP level increased significantly by a net 24.4% in seniors after WBVAT treatment at 3 months; other biochemical markers showed non-significant differences between the WBVAT and control groups. CONCLUSIONS: WBVAT treatment may increase BMD in senior people, particularly those with OP and women. Changes in bone metabolism after WBVAT treatment were not observed in most cases.

Amide-linkage formed between ammonia plasma treated poly(D,L-lactide acid) scaffolds and bio-peptides: enhancement of cell adhesion and osteogenic differentiation in vitro.

The surface characteristics of scaffolds for bone tissue engineering must support cell adhesion, migration, proliferation, and osteogenic differentiation. In the study, poly(D,L-lactide acid) (PDLLA) scaffolds were modified by combing ammonia (NH(3) ) plasma pretreatment with Gly-Arg-Gly-Asp-Ser (GRGDS)-peptides coupling technologies. The x-ray photoelectron spectroscopy (XPS) survey spectra showed the peak of N1s at the surface of NH(3) plasma pretreated PDLLA, which was further raised after GRGDS conjugation. Furthermore, N1s and C1s in the high-resolution XPS spectra revealed the presence of -C=N(imine), -C-NH-(amine), and -C=O-NH- (amide) groups. The GRGDS conjugation increased amide groups and decreased amine groups in the plasma-treated PDLLA. Confocal microscope and high performance liquid chromatography verified the anchored peptides after the conjugation process. Bone marrow mesenchymal stem cells were co-cultured with scaffolds. Fluorescent microscope and scanning electron microscope photographs revealed the best cell adhesion in NH(3) plasma pretreated and GRGDS conjugated scaffolds, and the least attachment in unmodified scaffolds. Real-time PCR demonstrated that expression of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, type I collagen, bone morphogenetic protein-2 and osteopontin, was upregulated in the single NH(3) plasma treated and NH(3) plasma pretreated scaffolds following GRGDS conjugation. The results show that NH(3) plasma treatment promotes the conjugation of GRGDS peptides to the PDLLA scaffolds via the formation of amide linkage, and combination of NH(3) plasma treatment and peptides conjugation may enhance the cell adhesion and osteogenic differentiation in the PDLLA scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 682-694, 2011.

[Effects of bioactive modification of poly-D,L-lactide acid scaffolds on the biological behaviors of the seed cells].

OBJECTIVE: To study the changes in the biological behavior of bone marrow mesenchymal stem cells (BMSCs) transfected with red fluorescent protein by lentivirus (RFP-BMSCs) seeded on in poly-D, L-lactide acid (PDLLA) scaffolds with bioactive modification by ammonia plasma and Gly-Arg-Gly-Asp-Ser (GRGDS) in vitro. METHODS: Circular sheets of PDLLA scaffolds (8 mm in diameter and 1 mm in thickness) were prepared and aminated with PDLLA (group A) or modified with the peptide conjugate A/PDLLA (group PA), with untreated PDLLA as the control (group P). The RFP-BMSCs were seeded on the scaffold materials and their proliferation and metabolic activity were detected using CyQuant NF and Alamar blue staining. The mineralization on the scaffolds was observed using calcein fluorescent dye under a fluorescent microscope. The adhesion and proliferation of RFP-BMSCs were observed by fluorescent microscope, and scanning electron microscope (SEM) was used to confirm the observed adhesion of the seed cells. RESULTS: The RFP-BMSCs seeded on the 3 scaffolds all showed proliferative activity at different time points after cell seeding, and the cell numbers decreased significantly in the order of PA>A>P (P<0.001). The cell number was significantly greater in group PA than in group A at all the time points except for days 10 (P=0.077) and 12 (P=0.491), and gradually became similar with the passage of time. The metabolic changes of the cells follow a similar pattern of cell proliferation. RFP-BMSCs showed more active proliferation in group A and group PA than in group P. On days 14 and 21, the intensity of green fluorescence decreased in the order of group PA, A and P. The RFP-BMSCs showed better adhesion in group PA than in group A, and the cells in group P appeared more scattered under scanning electron microscope. CONCLUSION: Bioactive modification of PDLLA by ammonia treatment and conjugation with GRGDS peptides may promotes the adhesion, proliferation, metabolism and mineralization of RFP-BMSCs seeded on PDLLA scaffolds.

[Histology and proliferative capability of thoracic vertebral body growth plates of rats at different ages].

OBJECTIVE: To compare the histological features of the thoracic vertebral body growth plates (VBGPs) of rats at different ages and assess their proliferative capability. METHODS: The thoracic VBGPs obtained from rats aged 1 day and 1, 4, 8, 16 and 28 weeks were identified using safranin O-fast green staining, and the height of the hypertrophic zone, proliferative zone, and resting zone were measured. The chondrocytes were isolated from these VBGPs with a modified trypsin-collagenase type II digestion method for primary culture in vitro. The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein was detected by real time-PCR and Western blotting, respectively. RESULTS: The 1-day- and 1-week-old rats showed significantly greater hypertrophic zone and proliferative zone in the VBGPs than older rats (P<0.01); the proliferative zone was significantly greater in rats aged 4 weeks than in those aged 28 weeks (P<0.05). The resting zone was obviously greater in rats aged 1 day and 1 week than in older rats (P<0.05), and also greater in rats aged 4 weeks than in those aged 16 and 28 weeks (P<0.05). Obvious ossification in the resting zone occurred at 16 weeks, and most of the resting zone became ossified at 28 weeks. The expression of PCNA decreased at both the mRNA and protein levels as the rats grew. CONCLUSION: The 3 zones of VBGPs are greater in rats aged 1 day and 1 week than in older ones. Ossification in the resting zone begins at 16 weeks, and till 28 weeks, most of the resting zone is ossified. The proliferation ability of VBGP chondrocytes decreases with the increase of age of the rats.

Growth/differentiation factor-5 induces osteogenic differentiation of human ligamentum flavum cells through activation of ERK1/2 and p38 MAPK.

Ossification of ligamentum flavum (OLF) is a pathological ectopic ossification in the spinal ligament, leading to spinal canal stenosis, but little was known about its pathogenesis. A previous study has found growth/differentiation factor (GDF)-5 expression at ossified sites of the ligaments from OLF patients. This study aimed to investigate the osteogenic effects of GDF-5 on cultured human ligamentum flavum cells (LFCs). LFCs were isolated from human spinal ligamentum flavum, and treated with or without recombinant human (rh) GDF-5. Alkaline phosphatase (ALP) activity was measured. Expression of osteocalcin was assessed by reverse transcriptase-PCR, Western blotting and immunofluorescence. Matrix mineralization was assessed by alizarin red staining. Activation of mitogen-activated protein kinases (MAPK) ERK1/2, p38 and JNK were detected by Western blotting. We found that rhGDF-5 treatment increased ALP activity and osteocalcin expression in a time- and dose-dependent manner, and induced mineralized nodule form. In addition, rhGDF-5 challenge mediated the ERK1/2 and p38 activation but not JNK. Inhibiting this activation pharmacologically, using U0126, a ERK1/2 inhibitor, or SB203580, a p38 inhibitor, resulted in significantly lower ALP activity and osteocalcin protein expression. The present study shows that rhGDF-5 induces osteogenic differentiation of human LFCs through activation of ERK1/2 and p38 MAPK. These findings give some new insight into the pathogenesis of OLF.

[Experimental study of reservation flaval ligament on prevention of peridural adhesion after operation on lumbar disc].

OBJECTIVE: Through an experiment in rabbits, to evaluate the role of reservation flaval ligament using different methods to prevent the epidural adhesion by observing the morphological changes and provide experiment evidences for clinic. METHODS: 45 adult rabbits were divided into three groups: Group A (treated with no reservation of flaval ligament), Group B (treated with reserving half flaval ligament), and Group C (treated with reserving the complete flaval ligament). Every group has 15 rabbits. At the 2nd, 4th and 8th weeks, the samples were obtained for gross observation, histological examinations, and the images were analysed by computer. The data were processed using SPSS11.5. RESULT: Gross Observation and Histological Examinations, at the 2nd, 4th and 8th postoperative week, there was significant difference between the group A, B and C (gross observation: F2w=4.660, F4w=8.591, F8w=31.858, P<0.05; histological examinations: F2w=5.190, F4w=9.561, F8w=34.658, P<0.05). Images analysis: the difference was very significant in the groups at 2nd, 4th and 8th weeks (F2w=30.783, F4w=47.350, F8w=25.544, P<0.01). With time passing, the ratio became larger and larger in the same group. CONCLUSIONS: Reserving the complete flaval ligament can significantly prevent the peridural adhesion from the back as a barrier, and can reduce the adhesion formed.

[Prevention of dura adherence in spinal canal after microendoscopic discectomy by different methods: a clinical study of 165 cases].

OBJECTIVE: To evaluate the effects of different methods in prevention of post-operational scar formation and dura adherence in the spinal canal after microendoscopic discectomy (MED). METHODS: 165 patients undergoing MED were randomly divided into 3 equal groups: Group A, with the yellow ligament dissected and with the space between vertebral laminae sprinkled with sodium hyaluronate before the closing of the incision; Group B, with the yellow ligament reserved; and Group C, with the yellow ligament reserved and with the space between vertebral laminae sprinkled with sodium hyaluronate before the closing of the incision. All the patients were followed up 2, 4, and 8 weeks, and 1 and 2 years after the operation. Japanese Orthopedic Association (JOA) scoring system was used to evaluate the outcomes. RESULTS: The JOA scores 2 weeks after MED were not significantly different among the 3 groups; and from then on the JOA scores of Groups B and C were all higher than those of group A (A and B: t(4w) = 0.602, t(8w) = 0.701, t(1y) = 0.623, t(2y) = 0.654; A and C: t(4w) = 0.833, t(8w) = 0.759, t(1y) = 0.714, t(2y) = 0.771, all P < 0.05), however, there were not significantly differences at all time points between Groups B and C (B and C: t(2w) = 0.041, t(4w) = 0.135, t(8w) = 0.980, t(1y) = 0.530, t(2y) = 0.103, all P > 0.05). CT showed that a great amount of scar was seen, surrounding the dura mater sac and nerve roots in Group A, and there was a great amount of scar outside the yellow ligament and no remarkable compression of dura mater sac and nerve roots in Groups B and C. CONCLUSION: Reservation of yellow ligament effectively prevents scar adhesion inside the vertebral canal after MED. Sprinkling of sodium hyaluronate is also effective, however, its effect only lasts a short time.