Ming-Hong Chen

Neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering.

Brain injury is almost irreparable due to the poor regenerative capability of neural tissue. Nowadays, new therapeutic strategies have been focused on stem cell therapy and supplying an appropriate three dimensional (3D) matrix for the repair of injured brain tissue. In this study, we specifically linked laminin-derived IKVAV motif on the C-terminal to enrich self-assembling peptide RADA(16) as a functional peptide-based scaffold. Our purpose is providing a functional self-assembling peptide 3D hydrogel with encapsulated neural stem cells to enhance the reconstruction of the injured brain. The physiochemical properties reported that RADA(16)-IKVAV can self-assemble into nanofibrous morphology with bilayer β-sheet structure and become gelationed hydrogel with mechanical stiffness similar to brain tissue. The in vitro results showed that the extended IKVAV sequence can serve as a signal or guiding cue to direct the encapsulated neural stem cells (NSCs) adhesion and then towards neuronal differentiation. Animal study was conducted in a rat brain surgery model to demonstrate the damage in cerebral neocortex/neopallium loss. The results showed that the injected peptide solution immediately in situ formed the 3D hydrogel filling up the cavity and bridging the gaps. The histological analyses revealed the RADA(16)-IKVAV self-assembling peptide hydrogel not only enhanced survival of encapsulated NSCs but also reduced the formation of glial astrocytes. The peptide hydrogel with IKVAV extended motifs also showed the support of encapsulated NSCs in neuronal differentiation and the improvement in brain tissue regeneration after 6 weeks post-transplantation.

Control of three-dimensional substrate stiffness to manipulate mesenchymal stem cell fate toward neuronal or glial lineages

The unlimited self-renewal and multipotency of stem cells provide great potential for applications in tissue engineering and regenerative medicine. The differentiation of stem cells can be induced by multiple factors including physical, chemical and biological cues. The fate of stem cells can be manipulated by deliberately controlling the interaction between stem cells and their microenvironment. The purpose of this study is to investigate the change in matrix stiffness under the influence of neurogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, three-dimensional (3-D) porous scaffolds were synthesized by type I collagen (Col) and hyaluronic acid (HA). The elastic modulus of the 3-D substrates was modified by adjusting the concentration of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. The mechanical properties of Col-HA scaffolds were evaluated and the induction and characterization of hMSC differentiation toward neural lineages on substrates with different stiffnesses were studied. Using EDC of different concentrations for crosslinking, the stiffness of the matrices can be controlled in the range of 1-10 kPa for soft to stiff substrates, respectively. The results showed that MSCs were likely to differentiate into neuronal lineage in substrate at 1 kPa, while they transformed into glial cells in matrix at 10 kPa. The morphology and proliferation behavior of hMSCs responded to the different stiffnesses of substrates. Using this modifiable matrix, we can investigate the relationship between stem cell behavior and substrate mechanical properties in extracellular matrix-based biomimetic 3-D scaffolds. A substrate with controllable stiffness capable of inducing hMSCs specifically toward neuronal differentiation may be very useful as a tissue-engineered construct or substitute for delivering hMSCs into the brain and spinal cord.

Multichanneled Nerve Guidance Conduit with Spatial Gradients of Neurotrophic Factors and Oriented Nanotopography for Repairing the Peripheral Nervous System

Peripheral nerve injuries, causing sensory and motor impairment, affect a great number of patients annually. It is therefore important to incorporate different strategies to promote nerve healing. Among the treatment options, however, the efficacy of nerve conduits is often compromised by their lack of living cells, insufficient growth factors, and absence of the extracellular matrix (ECM)-like structure. To improve the functional recovery, we aimed to develop a natural biodegradable multichanneled scaffold characterized with aligned electrospun nanofibers and neurotrophic gradient (MC/AN/NG) to guide axon outgrowth. The gelatin-based conduits mimicked the fascicular architecture of natural nerve ECM. The multichanneled (MC) scaffolds, cross-linked with microbial transglutaminase, possessed sustainable mechanical stability. Meanwhile, the release profile of dual neurotrophic factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), exhibited a temporal-controlled manner. In vitro, the differentiated neural stem cells effectively extended their neurites along the aligned nanofibers. Besides, in the treated group, the cell density increased in high NGF concentration regions of the gradient membrane, and the BDNF significantly promoted myelination. In a rabbit sciatic nerve transection in vivo model, the MC/AN/NG scaffold showed superior nerve recovery and less muscle atrophy comparable to autograft. By integrating multiple strategies to promote peripheral nerve regeneration, the MC/AN/NG scaffolds as nerve guidance conduits showed promising results and efficacious treatment alternatives for autologous nerve grafts.

Ibuprofen-conjugated hyaluronate/polygalacturonic acid hydrogel for the prevention of epidural fibrosis:

The formation of fibrous tissue is part of the natural healing response following a laminectomy. Severe scar tissue adhesion, known as epidural fibrosis, is a common cause of failed back surgery syndrome. In this study, by combining the advantages of drug treatment with a physical barrier, an ibuprofen-conjugated crosslinkable polygalacturonic acid and hyaluronic acid hydrogel was developed for epidural fibrosis prevention. Conjugation was confirmed and measured by 1D 1H NMR spectroscopy. In vitro analysis showed that the ibuprofen-conjugated polygalacturonic acid–hyaluronic acid hydrogel showed low cytotoxicity. In addition, the conjugated ibuprofen decreased prostaglandin E2 production of the lipopolysaccharide-induced RAW264.7 cells. Histological data in in vivo studies indicated that the scar tissue adhesion of laminectomized male adult rats was reduced by the application of our ibuprofen-conjugated polygalacturonic acid-hyaluronic acid hydrogel. Its use also reduced the population of giant cells and collagen deposition of scar tissue without inducing extensive cell recruitment. The results of this study therefore suggest that the local delivery of ibuprofen via a polygalacturonic acid-hyaluronic acid-based hydrogel reduces the possibility of epidural fibrosis.

An injectable extracellular matrix for the reconstruction of epidural fat and the prevention of epidural fibrosis.

Extensive epidural fibrosis is a common complication following spinal surgery and can cause pain and limited mobility. In the present study, a novel biomimetic approach was developed to prevent postsurgical adhesion of the dura. We aimed to reconstruct epidural fat, which prevents scar-tissue adhesion, through the development of an injectable decellularized adipose matrix (DAM)-containing hyaluronic acid (HA) hydrogel loaded with adipose stromal cells (ASCs). Injectable DAM was prepared from porcine adipose tissue by four freeze-thaw cycles with subsequent pepsin digestion. Residual analyses confirmed the efficacy of detergent-free decellularization, while most sulfated glycosaminoglycans and collagen were preserved. The Transwell migration assay demonstrated the anti-infiltrative property of the DAM-containing HA hydrogel. After 14 d of 3D culture, the DAM-containing HA hydrogel showed inductive potential in the adipogenic differentiation of ASCs. For an in vivo study, the ASC-loaded DAM-containing HA hydrogel (DAM/ASC-incorporated HA hydrogel) was injected into adult laminectomized male rats, and the results were assessed by microscopic histological examination. The in vivo data indicated that HA hydrogel, DAM, and ASCs were all required for the ability of the engineered fat tissue to block the invasion of the fibrous tissue. Our results suggested that this injectable DAM/ASC-incorporated HA hydrogel has potential applications in minimally invasive surgery for soft-tissue reconstruction and epidural fibrosis prevention.

Development of biomimetic micro-patterned device incorporated with neurotrophic gradient and supportive Schwann cells for the applications in neural tissue engineering

In these years, the artificial nerve guidance conduit (NGC) has been developed as an alternative way to repair peripheral nerve injury. Unlike autologous nerve graft, the artificial NGC without proper stimulating factors and guidance cues still cannot obtain satisfactory prognosis for clinical patients. In this study, a biodegradable polymer-based implantable device has been developed and characterized. By incorporating three stimulating factors: (1) micro-patterned surface that can directionally guide the axon as physical cue; (2) neurotrophic gradient membrane that can continually attract axon outgrowth from the proximal to distal stump as chemical cue; (3) Schwann cells (SCs) that can support the growth of neurite and form myelin sheath around axon as biological cue, we expect that this construct can be used as a promising NGC for peripheral nerve regeneration. The results showed that the micro-patterned surface with specific dimension of channels and chambers can be precisely fabricated by laser ablation. Attachment and directional extension of differentiated neural stem cells (NSCs) were observed in micro-channels. The gradient distribution of nerve growth factor 7S on gelatin membrane was successfully achieved. Significant improvement in neurite length and increase in neuronal gene expressions were also noticed in higher concentration region. When co-culturing with SCs, NSCs can differentiate toward neuronal cells with strong expression of mature neuronal markers: βIII tubulin and microtubule-associated protein-2 (Map 2). Meanwhile, myelin basic protein was also observed, suggesting that SCs can provide biological support to neuronal cells in vitro. In the future, this advanced artificial NGC may be used as implantable prosthesis for the treatment of peripheral nerve injury with better functional recovery.

A mutation of the Col2a1 gene (G1170S) alters the transgenic murine phenotype and cartilage matrix homeostasis

BACKGROUND/PURPOSE Genomic studies have revealed that there is a significant association between a point mutation of the human Col2A1 gene (G1170S) and several hip disorders. The purpose of the study was to explore the phenotype and altered cartilage matrix homeostasis of transgenic mice carrying this mutated Col2a1 gene. METHODS Wild-type and transgenic mice were used as the control and study groups, respectively. Body weight measurement, radiographic analysis, and histological analysis of the mice were carried out to describe differences between the wild-type and transgenic mice at different ages. Cartilage metabolism studies were also carried out, including an MTT assay of cellular proliferation and nitric oxide and glycosaminoglycan assays. Allelic expression levels of the mutant A allele and the normal G allele were established by TaqMan assay. Cytokine and protease gene expression were measured. RESULTS Transgenic mice had a lower mean body weight, a deformed skeletal structure, and abnormal cartilage histomorphology. Chondrocyte proliferation was significantly compromised and this was linked to significantly higher NO secretion and less soluble glycosaminoglycan formation. TNF-α and IL-1β gene expression was significantly upregulated, while MMP-13 gene expression was significantly downregulated. CONCLUSION The mutant G1170S Col2a1 gene in mice clearly alters the transgenic murine phenotype and cartilage matrix homeostasis.

Elastin-derived peptides induce inflammatory responses through the activation of NF-κB in human ligamentum flavum cells.

The formation of fibrotic tissue in the ligamentum flavum (LF) is usually preceded by breakdown of elastic fibers. Elastin-derived peptides (EDPs) from breakdown of elastic fibers display a wide range of biological activities in a variety of cells, but there is minimal information regarding the involvement in the processes of LF hypertrophy. The aim of this study is to elucidate the effects of EDPs on cultured human LF cells and to investigate their molecular and biochemical mechanisms. Human LF cells were obtained from 18 patients who underwent lumbar spine surgery. After treatment with different concentrations of EDPs with or without specific inhibitors in culture medium, the viability and proliferation of LF cells, genes expression, and the signaling pathways were evaluated and analyzed. It was found that 50 μg/ml EDPs significantly increased cell proliferation and synthesis of prostaglandin E2. The gene expression and protein production of proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1β, and IL-6, were also upregulated. The levels of p-ERK (extracellular signal-regulated kinase) and NF-κB increased immediately following EDP treatment and sustained up to 90 min. It was also found that NF-κB inhibitor, but not ERK1/2 inhibitor, attenuated EDP-dependent induction of IL-1α, IL-1β, and IL-6 expression, indicating that NF-κB pathways are required for EDP-induced IL-1α, IL-1β, and IL-6 gene expression in human LF cells. The results of this in vitro experiment suggest that EDPs do induce inflammatory responses in human LF cells and plays the key role in the development of LF hypertrophy.

Gelatin-tricalcium phosphate membrane modified with ngf and cultured schwann cells for peripheral nerve repair : A tissue engineering approach

This study attempted to enhance the efficacy of peripheral nerve regeneration using our previously developed gelatin-tricalcium phosphate (GTG) conduits by incorporating them with nerve growth factors and cultured Schwann cells. The nerve growth factors were covalently immobilized onto the GTG conduits (GEN) using carbodiimide. Schwann cells were harvested from neonatal Lewis rats, cultured for seven days and injected into the GEN conduits. The experiment was performed in three groups: GTG conduits, GEN conduits and GEN conduits with Schwann cells injected (GEN+Sc). The effects of different conduits (GTG, GEN and GEN with Schwann cells) on the peripheral nerve regeneration were evaluated in rat sciatic nerve repair model. 24 weeks after implantation of conduits, degradation of the conduits in all groups was illustrated by the fragmentation of the conduits. All conduits were well tolerated by the host tissue. Under microscopic evaluations, regenerated nerve tissue with myelinated and unmyelinated axons presented in all groups. Histomorphometrically, the total nerve area of GEN+Sc group was significantly higher than GTG group. Conversely, the autotomy score evaluated 12 weeks after nerve repair showed better results for GTG group. Besides, GEN+Sc group had the highest average recovery index of compound muscle action potential, but the difference among each group did not reach statistical significance. Although the electrophysiological recovery of nerve was not significantly improved with GEN+Sc conduit, nerve repair using tissue engineered conduits still provided better histological results. However, it should be noticed that autotomy may be the price paid for enhanced peripheral nerve.

THE EVALUATION OF THERMAL PROPERTIES AND IN VITRO TEST OF CARBODIIMIDE OR GLUTARALDEHYDE CROSS-LINKED GELATIN FOR PC 12 CELLS CULTURE

The thermal and degradable properties of carbodiimide (EDC) or glutaraldehyde (GTA) cross-linked gelatin membranes have been investigated in order to evaluate the effects of different concentrations of two kinds of cross-linking reagent on the stability of membranes. In the thermogram recorded from a gelatin membrane cross-linked with EDC solution, the endothermic peak of 0.8% EDC cross-linking gelatin was centered at about 61°C that was higher than other samples treated with EDC solutions. Denaturation temperature (Td) of gelatin samples increased on increasing EDC concentration (0.2% to 0.8%), in agreement with the simultaneous increased of the extent of cross-linking. But increasing GTA concentration from 0.05% to 0.6%, the Td values of gelatin samples were decreased from 66.2°C to 56.3°C . In addition, two endothermic peaks were observed in 0.4% and 0.6% GTA cross-linking groups because of the GTA concentration was too high to complete cross-linking reaction. Therefore, partial of gelatin membrane was cross-linked completely but others were not. In the thermogravimetric analysis, the proportion of cracking endothermic peak of 0.6% GTA cross-linking gelatin (g15G0.6) was higher than the peak of 0.6% EDC cross-linking gelatin (g15C0.6). Therefore, g15G0.6 cracked to smaller molecules has to absorb more calorific capacity than g15C0.6. The increase in the strength of covalent binding on increasing the proportion of endothermic peak was evident. The results of degradable rate were in agreement with the lower concentration of cross-linked reagent the faster degraded rate of gelatin membrane. The MTT assay showed that 15% gelatin cross-linked by 0.8% EDC has the least cytotoxicity, and cell activity of this group was similar to control group (blank dish). As the concentration of GTA in gelatin membranes was down to 0.05% or 0.1% the cell viability was returned to approach the value of control group.