Guangpeng Liu

A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and β-TCP ceramics

This study investigated the in vitro effects of akermanite, a new kind of Ca-, Mg-, Si-containing bioceramic, on the attachment, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). Parallel comparison of the cellular behaviors of hASCs on the akermanite was made with those on beta-tricalcium phosphate (beta-TCP). Scanning electron microscope (SEM) observation and fluorescent DiO labeling were carried out to reveal the attachment and growth of hASCs on the two ceramic surfaces, while the quantitative assay of cell proliferation with time was detected by DNA assay. Osteogenic differentiation of hASCs cultured on the akermanite and beta-TCP was assayed by ALP expression and osteocalcin (OCN) deposition, which was further confirmed by Real-time PCR analysis for markers of osteogenic differentiation. It was shown that hASCs attached and spread well on the akermanite as those on beta-TCP, and similar proliferation behaviors of hASCs were observed on the two ceramics. Both of them exhibited good compatibility to hASCs with only minor cytotoxicity as compared with the tissue culture plates. Interestingly, the osteogenic differentiation of hASCs could be enhanced on the akermanite compared with that on the beta-TCP when the culture time was extended to approximately 10 days. Thus, it can be ascertained that akermanite ceramics may serve as a potential scaffold for bone tissue engineering.

Repair of articular cartilage defect in non-weight bearing areas using adipose derived stem cells loaded polyglycolic acid mesh

The current study was designed to observe chondrogenic differentiation of adipose derived stem cells (ASCs) on fibrous polyglycolic acid (PGA) scaffold stabilized with polylactic acid (PLA), and to further explore the feasibility of using the resulting cell/scaffold constructs to repair full thickness articular cartilage defects in non-weight bearing area in porcine model within a follow-up of 6 months. Autologous ASCs isolated from subcutaneous fat were expanded and seeded on the scaffold to fabricate ASCs/PGA constructs. Chondrogenic differentiation of ASCs in the constructs under chondrogenic induction was monitored with time by measuring the expression of collagen type II (COL II) and glycosaminoglycan (GAG). The constructs after being in vitro induced for 2 weeks were implanted to repair full thickness articular cartilage defects (8mm in diameter, deep to subchondral bone) in femur trochlea (the experimental group), while scaffold alone was implanted to serve as the control. Histologically, the generated neo-cartilage integrated well with its surrounding normal cartilage and subchondral bone in the defects of experimental group at 3 months post-implantation, whereas only fibrous tissue was filled in the defects of control group. Immunohistochemical and toluidine blue staining confirmed the similar distribution of COL II and GAG in the regenerated cartilage as the normal one. A vivid remolding process with post-operation time was also witnessed in the neo-cartilage as its compressive moduli increased significantly from 50.55% of the normal cartilage at 3 months to 88.05% at 6 months. The successful repair thus substantiates the potentiality of using chondrogenic induced ASCs and PGA/PLA scaffold for cartilage regeneration.

Evaluation of the viability and osteogenic differentiation of cryopreserved human adipose-derived stem cells

Human adipose-derived stem cells (ASCs) have the ability to differentiate into osteoblasts and thus the potential therapeutic use to tissue-engineer bone, so a reliable method for cell storage is necessary. The aim of this study was to determine whether a simple method of cryopreservation with 10% Me(2)SO as a protectant had an effect on proliferation potential and osteogenic differentiation of ASCs isolated from fresh human adipose tissue. ASCs were harvested from 6 human lipoaspirates and each was halved for either cryopreservation in liquid nitrogen for 2 weeks or for control culture. Cells from the second-passage were plated at a density of 5000cells/well in 24-well plates and cultured with or without osteogenic media for 14 days. Cell surface antigens were used to identify the cryopreserved ASCs by flow cytometry. The proliferation rate of both populations was evaluated using a cell DNA assay. To detect osteogenic differentiation of both the cryopreserved and non-cryopreserved populations, determination of osteoblastic protein production (alkaline phosphatase and osteocalcin) and excellular matrix calcification (calcium content) was applied. The expression of osteoblastic-associated genes was also analyzed using reverse-transcription polymerase chain reaction. These results demonstrate that cryopreservation has no effect on the phenotype, proliferation or osteogenic differentiation of human ASCs, showing cryopreserved human ASCs might be applied for bone tissue engineering.

Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration*

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

TISSUE-ENGINEERED BONE FORMATION WITH CRYOPRESERVED HUMAN BONE MARROW MESENCHYMAL STEM CELLS

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at -196 degrees C in liquid nitrogen with 10% Me(2)SO as cryoprotectant for 24h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10mM sodium beta-glycerophosphate, 50muM l-ascorbic acid, and 10nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.

Tissue-engineered bone formation using human bone marrow stromal cells and novel β-tricalcium phosphate

In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel beta-tricalcium phosphate (beta-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous beta-TCP scaffolds with pores of 300-500 microm in size were prepared by the polymeric sponge method. beta-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous beta-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In beta-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in beta-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous beta-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.

In vitro engineering of fibrocartilage using CDMP1 induced dermal fibroblasts and polyglycolide.

This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation ☆

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.

Comparative investigation of viability, metabolism and osteogenic capability of tissue-engineered bone preserved in sealed osteogenic media at 37 ?C and 4 ?C

Preservation of tissue-engineered (TE) bone is one of the key problems needed to be solved for its clinic application and industrialization. Traditional cryopreservation has been restricted because of the damages caused by ice formation and solution. Hypothermic preservation at 4 °C has been widely used for the preservation of transplanted organ despite potential negative effects on viability of cells and tissue. 37 °C is the best temperature for maintaining cellular bioactivities. However, 37 °C also has a potential negative effect on preserved cells due to consumption of nutrients and accumulation of by-products. No studies have reported which temperature is more suitable for the preservation of TE bone constructs. The current study explored the feasibility of preservation of TE bone constructs in sealed osteogenic media at 37 °C and 4 °C. Human bone marrow stromal cells (hBMSCs) were seeded into partially demineralized bone matrix (pDBM) scaffolds and cultured for 7 days to form TE bone constructs. The constructs were preserved in sealed osteogenic media at either 37 °C or 4 °C for 5, 7, 9 and 11 days, respectively. Growth kinetics, viability, metabolism and osteogenic capability were evaluated to explore the feasibility of preservation at 37 °C and 4 °C. The constructs cultured in osteogenic media at humidified 37 °C/5%CO2 served as the positive control. The results demonstrated that all the constructs preserved at 4 °C showed negative osteogenic capability at all time points with a much lower level of growth kinetics, viability and metabolism compared to the positive control. However, the constructs preserved at 37 °C showed good osteogenic capability within 7 days with a certain level of growth kinetics, viability and metabolism, although an obvious decrease in osteogenic capability was observed in the constructs preserved at 37 °C over 9 days. These results indicate that the preservation of TE bone constructs is feasible at 37 °C within 7 days in sealed osteogenic media.