Hongbin Fan

Anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold in large animal model

Although in vivo studies in small animal model show the ligament regeneration by implanting mesenchymal stem cells (MSCs) and silk scaffold, large animal studies are still needed to evaluate the silk scaffold before starting a clinical trial. The aim of this study is to regenerate anterior cruciate ligament (ACL) in pig model. The micro-porous silk mesh was fabricated by incorporating silk sponges into knitted silk mesh with lyophilization. Then the scaffold was prepared by rolling the micro-porous silk mesh around a braided silk cord to produce a tightly wound shaft. In vitro study indicated that MSCs proliferated profusely on scaffold and differentiated into fibroblast-like cells by expressing collagen I, collagen III and tenascin-C genes in mRNA level. Then the MSCs-seeded scaffold was implanted in pig model to regenerate ACL. At 24 weeks postoperatively, the MSCs in regenerated ligament exhibited fibroblast morphology. The key ligament-specific extracellular matrix components were produced prominently and indirect ligament-bone insertion with three zones (bone, Sharpeys fibers and ligament) was observed. Although there was remarkable scaffold degradation, the maximum tensile load of regenerated ligament could be maintained after 24 weeks of implantation. In conclusion, the results imply that silk-based material has great potentials for clinical applications.

In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold

Although most in vitro studies indicate that silk is a suitable biomaterial for ligament tissue engineering, in vivo studies of implanted silk scaffolds for ligament reconstruction are still lacking. The objective of this study is to investigate anterior cruciate ligament (ACL) regeneration using mesenchymal stem cells (MSCs) and silk scaffold. The scaffold was fabricated by incorporating microporous silk sponges into knitted silk mesh, which mimicked the structures of ligament extracellular matrix (ECM). In vitro culture demonstrated that MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of ligament-specific genes also increased with time. Then MSCs/scaffold was implanted to regenerate ACL in vivo. After 24 weeks, histology observation showed that MSCs were distributed throughout the regenerated ligament and exhibited fibroblast morphology. The key ligament ECM components including collagen I, collagen III, and tenascin-C were produced prominently. Furthermore, direct ligament-bone insertion with typical four zones (bone, mineralized fibrocartilage, fibrocartilage, ligament) was reconstructed, which resembled the native structure of ACL-bone insertion. The tensile strength of regenerated ligament also met the mechanical requirements. Moreover, its histological grading score was significantly higher than that of control. In conclusion, the results imply that silk scaffold has great potentials in future clinical applications.

Electrospun sulfated silk fibroin nanofibrous scaffolds for vascular tissue engineering

One of the major downfalls of tissue-engineered small-diameter vascular grafts is the inability to obtain a confluent endothelium on the lumenal surface. Loosely attached endothelial cells (ECs) are easily separated from the vessel wall when exposed to the in vivo vascular system. Thus any denuded areas on the lumenal surface of vascular grafts may lead to thrombus formation via platelet deposition and activation. If the denuded areas could express anticoagulant activity until the endothelial cell lining is fully achieved, it may greatly improve the chances of successful vascular reconstruction. In this study, we fabricate sulfated silk fibroin nanofibrous scaffolds (S-silk scaffolds) and assess the anticoagulant activity and cytocompatibility of S-silk scaffolds in vitro in order to improve the antithrombogenicity and get some insights into its potential use for vascular tissue engineering. Sulfated silk fibroin was prepared by reaction with chlorosulphonic acid in pyridine, and then was developed to form an S-silk scaffold by electrospinning technique. FTIR analyses identified the successful incorporation of sulfate groups in silk fibroin molecules. It was found that the anticoagulant activity of S-silk scaffolds was significantly enhanced compared with silk fibroin nanofibrous scaffolds (Silk scaffolds). Vascular cells, including ECs and smooth muscle cells (SMCs), demonstrated strong attachment to S-silk scaffolds and proliferated well with higher expression of some phenotype-related marker genes and proteins. Overall, the data in this study suggest the suitability of S-silk scaffolds used along with vascular cells for the development of tissue-engineered vascular grafts.

Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized β-tricalcium phosphate scaffold and mesenchymal stem cells.

Although vascularized tissue-engineered bone grafts (TEBG) have been generated ectopically in several studies, the use of prevascularized TEBG for segmental bone defect repair are rarely reported. In current study, we investigated the efficacy of prevascularized TEBG for segmental defect repair. The segmental defects of 15 mm in length were created in the femurs of rabbits bilaterally. In treatment group, the osteotomy site of femur was implanted with prevascularized TEBG, which is generated by seeding mesenchymal stem cells (MSCs) into β-TCP scaffold, and prevascularization with the insertion of femoral vascular bundle into the side groove of scaffold; whereas in the control group, only MSC mediated scaffolds (TEBG) were implanted. The new bone formation and vascularization were investigated and furthermore, the expression of endogenous vascular endothelial growth factor (VEGF) which might express during defect healing was evaluated, as well. At 4, 8, and 12 weeks postoperatively, the treatment of prevascularized TEBG led to significantly higher volume of regenerated bone and larger amount of capillary infiltration compared to non-vascularized TEBG. The expression of VEGF in mRNA and protein levels increased with implantation time and peaked at 4 weeks postoperatively, followed by a slow decrease, however, treatment group expressed a significant higher level of VEGF than control group throughout the whole study. In conclusion, this study demonstrated that prevascularized TEBG by insertion of vascular bundle could significantly promote the new bone regeneration and vascularization compared to non-vascularized TEBG, which could be partially explained by the up-regulated expression of VEGF.

Gelatin Microspheres Containing TGF-β3 Enhance the Chondrogenesis of Mesenchymal Stem Cells in Modified Pellet Culture

The study is to investigate the chondrogenesis of a kind of modified cell pellet formed using mesenchymal stem cells (MSCs) and gelatin microspheres containing transforming growth factor beta3 (TGF-beta3). The gelatin microspheres loaded with TGF-beta3 (MS-TGF) were prepared and showed the controlled release of cytokine in a biphasic fashion. Then the mixture of MSCs and MS-TGF was centrifuged to form pellet. The pellet was cultured over 4 weeks to determine the effects of MS-TGF on cartilage matrix production by biochemical analysis, immunohistochemistry staining, and Western blot test. The transcription level of cartilage-related genes was also evaluated by real-time quantitative RT-PCR assay. After 4 weeks of culture, the MSCs were distributed uniformly in the pellet and had good viability. Cells showed faster proliferation and higher DNA content compared to MSCs in a conventional pellet. The production of collagen and glycosaminoglycan also increased significantly. The immunohistochemistry staining and alcian blue staining confirmed the synthesis of cartilage extracellular matrix (ECM). Furthermore, the differentiated MSCs located in lacunae within the metachromatic staining matrix exhibited the typical chondrocyte morphology. The chondrogenic differentiation of MSCs was proved by the expression of collagen II gene in mRNA and protein level. The results indicate that MS-TGF can induce chondrogenic differentiation of MSCs and increase cartilage ECM production, which result in a bigger cartilage pellet. In conclusion, this modified pellet culture can provide an easy and effective way to construct the tissue-engineered cartilage in vitro.

Reconstruction of goat tibial defects using an injectable tricalcium phosphate/chitosan in combination with autologous platelet-rich plasma

Injectable scaffolds held great promise for the reconstruction of bone defects. We prepared an injectable composite named PTC by combining TCP/chitosan (TC) with platelet-rich plasma (PRP). The objective of this study was to investigate the composites mechanical and biological properties. First, we found that the introduction of PRP in TC showed no adverse effect on mechanical strength and that there were no significant differences in compressive strength between PTC and TC (P>0.05). In cell culture experiments, both cell count and alkaline phosphatase (ALP) activity measurements of PTC were higher than those of TC. The high levels of Cbfa1 and TGF-beta were detected early in PTC-induced MSCs by reverse transcriptase polymerase chain reaction. Bone formation following expression of collagen type I, osteocalcin, osteonectin and calcium nodules was also observed in PRP-induced MSCs. Finally, this composite was injected into the tibial bone defect in a goat model, and its ability to induce bone regeneration was observed. Sixteen weeks after the implantation of this composite, the tibial defects had completely recuperated, with significantly better formation of mature bone and less residual material than in the control. These results demonstrate that our composite, with its concomitant mechanical strength, biocompatibility, and osteoinductive properties, has significant potential as an injectable material for the treatment of bone defects.

TGF‐β3 immobilized PLGA‐gelatin/chondroitin sulfate/hyaluronic acid hybrid scaffold for cartilage regeneration

Although most in vitro studies indicate that transforming growth factor β3 (TGF-β3) immobilized scaffold is suitable for cartilage tissue engineering, in vivo studies of implanting immobilized scaffold for chondral defect repair are still lacking. This study is to evaluate the potentials of TGF-β3 immobilized poly-(lactic-co-glycolic acid)-gelatin/chondroitin sulfate/hyaluronic acid (PLGA-GCH) hybrid scaffold for cartilage regeneration. The scaffold was fabricated by incorporating GCH micro-sponges into PLGA frameworks and then crosslinked with TGF-β3 to mimic natural cartilaginous extra cellular matrix (ECM). In vitro study demonstrated that MSCs proliferated vigorously and produced abundant ECM on scaffold. The immunohistochemistry staining and alcian blue staining confirmed the cartilaginous ECM production. The chondrogenic differentiation of MSCs on scaffold was proved by the expression of collagen II gene in mRNA and protein level. Then MSCs/TGF-β3 immobilized scaffolds were implanted in rabbits for chondral defects repair. After eight weeks, histological observation showed that differentiated MSCs were located in lacunae within the metachromatic staining matrix and exhibited typical chondrocyte morphology. Histological grading scores also indicated the congruent cartilage was regenerated. In conclusion, the TGF-β3 immobilized PLGA-GCH hybrid scaffold has great potential in constructing the tissue-engineered cartilage.

Development of a silk cable-reinforced gelatin/silk fibroin hybrid scaffold for ligament tissue engineering.

The objective of this study was to develop a silk cable-reinforced gelatin/silk fibroin hybrid scaffold for ligament tissue engineering. The scaffold was fabricated by lyophilizing the cross-linked gelatin and silk fibroin mixture with braided silk cables. Scanning electronic microscopy (SEM) observation showed that microporous gelatin/silk fibroin sponges formed around silk cables mimicked the microstructures of ligament extracellular matrix (ECM). The silk cables significantly increased the tensile strength of the scaffold to meet the mechanical requirements for ligament tissue engineering. The scaffold possessed good cell adhesion property, and when mesenchymal stem cells (MSCs) were seeded on it, cells proliferated profusely. After 2 weeks of culture, seeded MSCs were distributed uniformly throughout the scaffold and were highly viable. Occurrence of cell death during culture was not significant. Deposition of collagen on the scaffold was found to increase with time. Differentiation of MSCs into ligament fibroblasts was verified by expressions of ligament ECM specific genes including collagen type I, collagen type III, and tenascin-C in mRNA and protein level. Immunohistochemistry stains also confirmed the production of key ligament ECM components on the scaffold. The results demonstrate that silk cable-reinforced gelatin/silk fibroin scaffold possesses the appropriate mechanical properties and has enlarged surface area. It is also capable of supporting cell proliferation and differentiation for ligament tissue engineering.

Comparison of chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells.

The purpose of this study was to compare chondral defects repair with in vitro and in vivo differentiated mesenchymal stem cells (MSCs). A novel PLGA-gelatin/chondroitin/hyaluronate (PLGA-GCH) hybrid scaffold with transforming growth factor-β1 (TGF-β1)-impregnated microspheres (MS-TGF) was fabricated to mimic the extracellular matrix. MS-TGF showed an initial burst release (22.5%) and a subsequent moderate one that achieved 85.1% on day 21. MSCs seeded on PLGA-GCH/MS-TGF or PLGA-GCH were incubated in vitro and showed that PLGA-GCH/MS-TGF significantly augmented proliferation of MSCs and glycosaminoglycan synthesis compared with PLGA-GCH. Then MSCs seeded on PLGA-GCH/MS-TGF were implanted and differentiated in vivo to repair chondral defect on the right knee of rabbit (in vivo differentiation repair group), while the contralateral defect was repaired with in vitro differentiated MSCs seeded on PLGA-GCH (in vitro differentiation repair group). The histology observation demonstrated that in vivo differentiation repair showed better chondrocyte morphology, integration, and subchondral bone formation compared with in vitro differentiation repair 12 and 24 weeks postoperatively, although there was no significant difference after 6 weeks. The histology grading score comparison also demonstrated the same results. The present study implies that in vivo differentiation induced by PLGA-GCH/MS-TGF and the host microenviroment could keep chondral phenotype and enhance repair. It might serve as another way to induce and expand seed cells in cartilage tissue engineering.

Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction

The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL) reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM) showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3) gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM) components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.