Qin Shi

Collagen scaffolds modified with collagen-binding bFGF promotes the neural regeneration in a rat hemisected spinal cord injury model

Nerve conduit is one of strategies for spine cord injury (SCI) treatment. Recently, studies showed that biomaterials could guide the neurite growth and promote axon regeneration at the injury site. However, the scaffold by itself was difficult to meet the need of SCI functional recovery. The basic fibroblast growth factor (bFGF) administration significantly promotes functional recovery after organ injuries. Here, using a rat model of T9 hemisected SCI, we aimed at assessing the repair capacity of implantation of collagen scaffold (CS) modified by collagen binding bFGF (CBD-bFGF). The results showed that CS combined with CBD-bFGF treatment improved survival rates after the lateral hemisection SCI. The CS/CBD-bFGF group showed more significant improvements in motor than the simply CS-implanted and untreated control group, when evaluated by the 21-point Basso-Beattie-Bresnahan (BBB) score and footprint analysis. Both hematoxylin and eosin (H&E) and immunohistochemical staining of neurofilament (NF) and glial fibrillary acidic protein (GFAP) demonstrated that fibers were guided to grow through the implants. These findings indicated that administration of CS modified with CBD-bFGF could promote spinal cord regeneration and functional recovery.

Sensitization of osteosarcoma cell line SaOS-2 to chemotherapy by downregulating survivin.

BACKGROUND AND AIMS Osteosarcoma is the most frequent malignant bone tumor with a peak incidence in the second and third decades of life. Survivin, a member of the IAP family of proteins, is overexpressed in osteosarcomas and plays an important role in protecting cells from apoptosis. Here we investigated the anti-cancer effects of downregulating survivin by shRNA vector pSUPER-sh in combination with chemotherapeutic drugs on human osteosarcoma cells. METHODS Expression of survivin was detected by Western blot. The effects of pSUPER-sh and chemotherapeutic drugs on osteosarcoma cell lines Saos-2 and U2OS by cell viability assay and its underlying mechanisms were analyzed by flow cytometry and caspase-3 activity assay. RESULTS Downregulated survivin could significantly induce apoptosis of osteosarcoma cell lines Saos-2 and U2OS. The effect probably resulted from downregulation of survivin induced by pSUPER-sh. Importantly, we found that the downregulation of survivin by pSUPER-sh could enhance the anticancer effects of chemotherapies such as etoposide, cisplatin and doxorubicin through decreasing mitochondrial membrane potentials and increasing caspase-3 activity. CONCLUSIONS Downregulated survivin by pSUPER-sh could markedly induce apoptosis of osteosarcoma cells lines and pSUPER-sh may be a promising adjuvant in osteosarcoma chemotherapy.

One-year clinical study of NeuroRegen scaffold implantation following scar resection in complete chronic spinal cord injury patients

The objective of this clinical study was to assess the safety and feasibility of the collagen scaffold, NeuroRegen scaffold, one year after scar tissue resection and implantation. Scar tissue is a physical and chemical barrier that prevents neural regeneration. However, identification of scar tissue is still a major challenge. In this study, the nerve electrophysiology method was used to distinguish scar tissue from normal neural tissue, and then different lengths of scars ranging from 0.5–4.5 cm were surgically resected in five complete chronic spinal cord injury (SCI) patients. The NeuroRegen scaffold along with autologous bone marrow mononuclear cells (BMMCs), which have been proven to promote neural regeneration and SCI recovery in animal models, were transplanted into the gap in the spinal cord following scar tissue resection. No obvious adverse effects related to scar resection or NeuroRegen scaffold transplantation were observed immediately after surgery or at the 12-month follow-up. In addition, patients showed partially autonomic nervous function improvement, and the recovery of somatosensory evoked potentials (SSEP) from the lower limbs was also detected. The results indicate that scar resection and NeuroRegen scaffold transplantation could be a promising clinical approach to treating SCI.

Bone marrow mesenchymal stem cells slow intervertebral disc degeneration through the NF-κB pathway

BACKGROUND CONTEXT Previous studies have demonstrated the use of bone marrow mesenchymal stem cells (BMSCs) in tissue-engineering treatments to slow or reverse diseased intervertebral discs. Several approaches have successfully used the coculturing of stem cells with disc-native nucleus pulposus cells (NPCs) with the evidence of transformed BMSCs into NP-like cells, increased activity and matrix production by NPCs, or elements of both. The influence of the cytokine transforming growth factor-beta (TGF-β) in the differentiation of BMSCs into NP-like cells and its upregulation in coculture to increase matrix production are well established. However, the role of the inflammatory signaling molecule nuclear factor kappa B (NF-κB) in intervertebral disc degeneration is far less clear, although there is some existing evidence suggesting its role in the pathogenesis and progression of disc disease. A limited number of studies in other pathologies have alluded to the antagonistic relationship between both proteins. To date, there is no such investigation of their dynamic role in coculture of BMSCs and NPCs. PURPOSE The purpose of this study was to investigate the relationship of the regenerative effects of BMSCs cocultured with NPCs. The authors hypothesized that as levels of TGF-β increase in the coculture, the levels of NF-κB will concomitantly decrease. This would in turn be reflected by an increase in the expression of messenger RNA markers of the nucleus pulposus matrix that includes aggrecan, Type II collagen (CII), and SOX-9 and an increase in the cellular proliferation. STUDY DESIGN/SETTING This study is based on a coculture with the contact of rabbit NPCs and BMSCs. METHODS Bone marrow mesenchymal stem cells were cocultured with NPCs at a ratio of 1:1 and compared with BMSC and NPC controls cultured alone. Cell proliferation was evaluated by Cell Counting Kit-8 from 3 to 9 days. Gene expressions of aggrecan, CII, and SOX-9 was assayed by reverse transcription-polymerase chain reaction from 5 to 14 days. Detection of TGF-βl and NF-κB was determined by enzyme-linked immunosorbent assay, and immunohistochemical staining was carried out to evaluate CII synthesis. RESULTS After 3 days, cellular proliferation of the cocultured group exceeded that of controls. After 11 days, the expression of SOX-9 in the cocultured group had also exceeded controls. Furthermore, after 14 days, expressions of aggrecan and CII significantly exceeded controls. Immunohistochemical stains of CII in the NPC control group were positive at each point in time and demonstrated strongest expression at 14 days. Coculturing BMSCs with NPCs, therefore, seems to have resulted in the promotion of aggrecan, CII, and SOX-9 gene expressions. Finally, after 11 days, TGF-βl content of the cocultured group significantly exceeded control levels, whereas NF-κB content had significantly lowered. CONCLUSIONS Coculture of BMSCs may be able to delay NPC matrix degeneration potentially through the concomitant upregulation of TGF-β and the downregulation of NF-κB pathway.

A novel monoclonal antibody against human CD80 and its immune protection in a mouse lupus-like disease.

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), is an attractive means to induce antigen-specific peripheral tolerance in autoimmune disease and organ transplantation. In this study, we generated and characterized a monoclonal antibody (Clone 4E5) against human CD80. 4E5 could recognize both human and mouse CD80 and suppress mixed lymphocyte reaction in vitro. To investigate their potency for clinical use, we further administrated 4E5 to a mouse lupus-like disease model (C57BL/J6) induced by Pristane. 4E5 could inhibit the immune response and attenuate the severity of lupus-like disease. The data showed 4E5 function and suggested that blockade of CD80/CD28 co-stimulatory signal pathway with 4E5 is a promising strategy to decelerate the progression of lupus-like disease and other autoimmune diseases.

Accelerated Postero-Lateral Spinal Fusion by Collagen Scaffolds Modified with Engineered Collagen-Binding Human Bone Morphogenetic Protein-2 in Rats

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role in bone regeneration and repair. However, its distribution and side effects are major barriers to its success as therapeutic treatment. The improvement of therapy using collagen delivery matrices has been reported. To investigate a delivery system on postero-lateral spinal fusion, both engineered human BMP-2 with a collagen binding domain (CBD-BMP-2) and collagen scaffolds were developed and their combination was implanted into Sprague-Dawley (SD) rats to study Lumbar 4–5 (L4–L5) posterolateral spine fusion. We divided SD rats into three groups, the sham group (G1, n = 20), the collagen scaffold-treated group (G2, n = 20) and the BMP-2-loaded collagen scaffolds group (G3, n = 20). 16 weeks after surgery, the spines of the rats were evaluated by X-radiographs, high-resolution micro-computed tomography (micro-CT), manual palpation and hematoxylin and eosin (H&E) staining. The results showed that spine L4–L5 fusions occurred in G2(40%) and G3(100%) group, while results from the sham group were inconsistent. Moreover, G3 had better results than G2, including higher fusion efficiency (X score, G2 = 2.4±0.163, G3 = 3.0±0, p<0.05), higher bone mineral density (BMD, G2: 0.3337±0.0025g/cm3, G3: 0.4353±0.0234g/cm3. p<0.05) and more bone trabecular formation. The results demonstrated that with site-specific collagen binding domain, a dose of BMP-2 as low as 0.02mg CBD-BMP-2/cm3 collagen scaffold could enhance the posterolateral intertransverse process fusion in rats. It suggested that combination delivery could be an alternative in spine fusion with dramatically decreased side effects caused by high dose of BMP-2.

Demineralized Bone Matrix Scaffolds Modified by CBD-SDF-1α Promote Bone Regeneration via Recruiting Endogenous Stem Cells

The reconstruction of bone usually depends on substitute transplantation, which has drawbacks including the limited bone substitutes available, comorbidity, immune rejection, and limited endogenous bone regeneration. Here, we constructed a functionalized bone substitute by combining application of the demineralized bone matrix (DBM) and collagen-binding stromal-cell-derived factor-1α (CBD-SDF-1α). DBM was a poriferous and biodegradable bone substitute, derived from bovine bone and consisting mainly of collagen. CBD-SDF-1α could bind to collagen and be controllably released from the DBM to mobilize stem cells. In a rat femur defect model, CBD-SDF-1α-modified DBM scaffolds could efficiently mobilize CD34+ and c-kit+ endogenous stem cells homing to the injured site at 3 days after implantation. According to the data from micro-CT, CBD-SDF-1α-modified DBM scaffolds could help the bone defects rejoin with mineralization accumulated and bone volume expanded. Interestingly, osteoprotegerin (OPG) and osteopontin (OPN) were highly expressed in CBD-SDF-1α group at an early time after implantation, while osteocalcin (OCN) was more expanded. H&E and Massons trichrome staining showed that the CBD-SDF-1α-modified DBM scaffold group had more osteoblasts and that the bone defect rejoined earlier. The ultimate strength of the regenerated bone was investigated by three-point bending, showing that the CBD-SDF-1α group had superior strength. In conclusion, CBD-SDF-1α-modified DBM scaffolds could promote bone regeneration by recruiting endogenous stem cells.

Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2.

Osteoporosis is a serious health problem worldwide. MicroRNA is a post-transcriptional regulator of gene expression by either promoting mRNA degradation or interfering with mRNA translation of specific target genes. It plays a significant role in the pathogenesis of osteoporosis. Here, we first demonstrated that miR-106b (miR-106b-5p) negatively regulated osteogenic differentiation of mesenchymal stem cells in vitro. Then, we found that miR-106b expression increased in C57BL/6 mice with glucocorticoid-induced osteoporosis (GIOP), and that silencing of miR-106b signaling protected mice against GIOP through promoting bone formation and inhibiting bone resorption. At last, we showed that miR-106b inhibited osteoblastic differentiation and bone formation partly through directly targeting bone morphogenetic protein 2 (BMP2) both in vitro and in the GIOP model. Together, our findings have identified the role and mechanism of miR-106b in negatively regulating osteogenesis. Inhibition of miR-106b might be a potential new strategy for treating osteoporosis and bone defects.

Acceleration of healing of traumatic tympanic membrane perforation in rats by implanted collagen membrane integrated with collagen-binding basic fibroblast growth factor

Traumatic tympanic membrane (TM) perforation is very common in clinical practice. Several biomaterials have been reported to play a role in TM reparation, whereas their functional recovery is limited when used alone. Meanwhile, the administration of biofactors could promote functional recovery, but rapid distribution and short half-time obstruct their application. To study the effect of traumatic TM regeneration, we prepared collagen membrane (CM) integrated with collagen-binding basic fibroblast growth factor (CBD-bFGF) and implanted into the injury site of perforated TM in Sprague-Dawley rats. The study on CBD-bFGF in vitro showed that CBD-bFGF accelerated the proliferation of human fibroblast cell HS-865 biologically and was released from CM gradually. In vivo study, through the gross anatomy, auditory brainstem responses assay, histological staining, and transmission electron microscopy observation at d7, d14, and d28 after the acute TM perforation, we found that CBD-bFGF-integrated CM promoted the healing rate at an early stage (∼7 days), reduced the healing time of perforated TM, and notably retrieved the structure and hearing of TM. These findings suggest that CM modified with CBD-bFGF could be therapeutically appropriate for the treatment of TM perforation.

Effect of natural hirudin on random pattern skin flap survival in a porcine model.

Objective: The effect of local administration of hirudin on random pattern skin flap survival was investigated in a porcine model. Methods: Three random pattern skin flaps (4 × 14 cm) were created on each flank of five Chinese minipigs. The experimental group (10 flaps) received 20 antithrombin units of hirudin, injected subdermally into the distal half immediately after surgery and on days 1 and 2; a control group (10 flaps) was injected with saline and a sham group (10 flaps) was not injected. All flaps were followed for 10 days postoperatively. Results: Macroscopically, the congested/necrotic length in the experimental group was significantly decreased compared with the other two groups by day 3. Histopathological evaluation revealed venous congestion and inflammation in the control and sham groups from day 1, but minimal changes in the experimental group. By day 10, the mean ± SD surviving area was significantly greater in the experimental group (67.6 ± 2.1%) than in the control (45.2 ± 1.4%) or sham (48.3 ± 1.1%) groups. Conclusions: Local administration of hirudin can significantly increase the surviving area in overdimensioned random pattern skin flaps, in a porcine model.