Yunyu Hu

Layered manufacturing of tissue engineering scaffolds via multi-nozzle deposition

The fabrication technology of Multi-nozzle Deposition Manufacturing (MDM) was proposed to fabricate porous tissue engineering scaffolds. This digital forming technology was developed based on the layer-by-layer manufacturing principle of Solid Freeform Fabrication (SFF). A four-nozzle MDM system was designed to carry out MDM processes. Bone tissue engineering scaffolds with different properties were fabricated by single-nozzle deposition process, bi-nozzle deposition process and tri-nozzle deposition process, respectively, in the MDM system. The bone scaffolds made by the single-nozzle deposition process in the MDM system have good biocompatibility and bone conductive property as a molecular scaffold for bone morphogenic protein (BMP) in the implantation experiment of repairing segment defect of rabbit radius.

Effects of different cross-linking conditions on the properties of genipin-cross-linked chitosan/collagen scaffolds for cartilage tissue engineering

A cross-linking reagent is required to improve mechanical strength and degradation properties of biopolymers for tissue engineering. To find the optimal preparative method, we prepared diverse genipin-cross-linked chitosan/collagen scaffolds using different genipin concentrations and various cross-linking temperatures and cross-linking times. The compressive strength increased with the increasing of genipin concentration from 0.1 to 1.0%, but when concentration exceeded 1.0%, the compressive strength decreased. Similarly, the compressive strength increased with the increasing of temperature from 4 to 20°C, but when temperature reached 37°C, the compressive strength decreased. Showing a different trend from the above two factors, the effect of cross-linking time on the compressive strength had a single increasing tendency. The other results also demonstrated that the pore size, degradation rate and swelling ratio changed significantly with different cross-linking conditions. Based on our study, 1.0% genipin concentration, 20°C cross-linking temperature and longer cross-linking time are recommended.

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.

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.

Skeletal Repair in Rabbits Using a Novel Biomimetic Composite Based on Adipose-Derived Stem Cells Encapsulated in Collagen I Gel with PLGA-β-TCP Scaffold

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose‐derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA‐β‐TCP scaffold (rASCs‐COL/PLGA‐β‐TCP, group A), the combination of rASCs and PLGA‐β‐TCP (rASCs/PLGA‐β‐TCP, group B), the combination of collagen I gel and PLGA‐β‐TCP (COL/PLGA‐β‐TCP, group C), and PLGA‐β‐TCP scaffold (group D). The composites were implanted into a 15‐mm length critical‐sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA‐β‐TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs‐based bone tissue engineering.

Collagen I Gel Can Facilitate Homogenous Bone Formation of Adipose-Derived Stem Cells in PLGA-β-TCP Scaffold

Cell-based tissue engineering is thought to be a new therapy for treatment of bone defects and nonunions after trauma and tumor resection. In this study, we explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricated by using collagen I gel to suspend rabbit adipose-derived stem cells (rASCs) into a porous poly(lactic-co-glycolic)acid-β-tricalcium phosphate (PLGA-β-TCP) scaffold (rASCs-COL/PLGA-β-TCP). In vitro and in vivo studies of the rASCs-COL/PLGA-β-TCP composite (group A) were carried out compared with the single combination of rASCs and PLGA-β-TCP (rASCs/PLGA-β-TCP; group B), the combination of acellular collagen I gel and PLGA-β-TCP (COL/PLGA-β-TCP; group C), and the PLGA-β-TCP scaffold (group D). Composites of different groups were cultured in vitro for 2 weeks in osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. After 2 weeks of in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in group A were significantly higher than in group B (p < 0.01, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in group A, whereas no apparent calcification was observed in groups B, C and D (n = 4). In group A, woven bone with a trabecular structure was formed, while in group B, only osteoid tissue was observed. Meanwhile, the bone-forming area in group A was significantly higher than in group B (p < 0.01, n = 4). No bone formation was observed in groups C or D (n = 4). In conclusion, by using collagen I gel to suspend rASCs into porous PLGA-β-TCP scaffold, osteogenic differentiation of rASCs can be improved and homogeneous bone tissue can be successfully formed in vivo.

Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2

BACKGROUND Adipose tissue has been demonstrated to contain a population of progenitor cells that can differentiate into bone and cartilage. Studies have suggested that adipose-derived adult stem (ADAS) cells can be induced to differentiate into chondrocytes by transforming growth factor-beta (TGF-beta). In this study, we examined whether bone morphogenetic protein-2 (BMP-2), as a member of the TGF-beta superfamily, could regulate ADAS cells to differentiate into a chondrolineage. METHODS ADAS cells were isolated and induced by rhBMP-2. These cells were cultured in pellets for 2 weeks, and the chondrogenic phenotype was observed in vitro and in vivo. ADAS cells cultured without BMP-2 were used as controls. RESULTS After 2 weeks of culture, the differentiated ADAS cells reacted positively to Alcian blue and collagen II, and the content of collagen II protein was obviously up-regulated at day 14. Glycosaminoglycan (GAG) content gradually increased from day 2 to day 14 (P < 0.05). However, H&E staining and collagen II expression were weak, and there was a little collagen II protein and GAG detected in the control group. Additionally, the pellets of ADAS cells induced by rhBMP-2 were transplanted into BALB/C nude mice and formed cartilage lacuna at week 8 in vivo. DISCUSSION These data demonstrate that rhBMP-2 induce ADAS cells to differentiate into chondrocytes in vitro and in vivo. This is useful for basic and clinical studies aimed at repairing cartilage damage. But in a control group, ADAS cells tended towards differentiation into chondrocytes, which was affected by ITS. We will be exploring the mechanism further.

A study on a tissue-engineered bone using rhBMP-2 induced periosteal cells with a porous nano-hydroxyapatite/collagen/poly(L-lactic acid) scaffold

We investigated the in vivo osteogenic ability of rhBMP-2 induced periosteal cells in a new porous scaffold, nano-hydroxyapatite (nano-HA)/collagen/poly(L-lactic acid) (PLA). The nano-HA/collagen/PLA composites were utilized as an extracellular matrix for a cell-based strategy of bone tissue engineering. Periosteal cells were cultivated with 500 ng ml(-1) rhBMP-2, followed by seeding into prewet nano-HA/collagen/PLA scaffolds. The cell-scaffold constructs were then subcutaneously implanted in nude mice compared to controls with cell suspension and scaffold alone. Scanning electron microscopy examination proved that the scaffold supported adhesion and proliferation of periosteal cells. Histological bone formation was observed only in experimental groups with cell transplants 8 weeks post-implantation. The animals of the control groups did not show bone formation. The results strongly encourage the approach of the transplantation of rhBMP-2 induced periosteal cells within a suitable carrier structure for bone regeneration.

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.

Adipose-derived stem cells and chondrogenesis.

Cartilage has only a very limited capacity to renew its original structure. Stem cells have been used to repair damaged cartilage, and recent studies have indicated that stem cells from adipose tissue are attractive cell sources that have the capacity of multipotentiality to differentiate into osteogenic, chondrogenic, myogenic, neurogenic and endothelial cells. Adipose-derived stem cells (ASC) have unique characteristics compared with stem cells from BM. At present, ASC have been studied to promote chondrogenesis. This review discusses the application of ASC to cartilage formation.