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Impact Statement: The presented translational research work represents the important progress and evolution of tissue engineering (TE) researches in China from proof-of-concept to actual application of TE technology for health management and biotechnology development in the past 15 years. It has been made with national governmental support of "863" project grant, which gathers multidisciplinary people in China to work together towards the translation of basic/applied research achievements into clinical reality and the fulfillment of unmet medical needs. It also truly reflects the global trend of fast development in this field from China's perspective.

Tendons are subjected to high strength dynamic mechanical forces in vivo. Mechanical strength is an essential requirement for tendon scaffold materials. A composite scaffold was used in this study to provide mechanical strength, which was composed of an inter part of nonwoven polyglycolic acid (PGA) fibers and an outer part of the net knitted with PGA and polylactic acid (PLA) fibers in a ratio of 4:2. This study compared three different approaches for in vivo tendon engineering, that is, cell-free scaffold and allogeneic and autologous cell seeded scaffolds, using a rabbit Achilles tendon repair model. Dermal fibroblasts were, respectively, isolated from the dermis of regular rabbits or green fluorescence protein transgenic rabbits as the autologous and the allogeneic cell sources, respectively. The cell scaffolds and cell-free scaffolds were implanted to bridge a partial segmental defect of rabbit Achilles tendon. The engineered tendons were harvested at 7 and 13 months postsurgery for various examinations. The results showed that all three groups could achieve in vivo tendon regeneration similarly with slightly better tissue formation in autologous group than in other two groups, including better scaffold degradation and relatively thicker collagen fibrils. There were no statistically significant differences in mechanical parameters among three groups. This work demonstrated that allogeneic fibroblasts and scaffold alone are likely to be used for tendon tissue engineering.

Synthetic polymers such as polyglycolic acid (PGA) fibers are the traditional tissue engineering scaffolds that are widely used for engineering a variety of soft tissues. However, the major disadvantage of this polymer material is its released acidic degradation products that trigger inflammatory response and fibrotic process, which affects the biocompatibility and the quality of the engineered tissues. In this study, the effect of hyaluronic acid (HA) coating on improving PGA biocompatibility was explored. The results showed that 1% HA solution could better coat PGA fibers than other tested concentrations of HA, and coated PGA exhibited less inflammatory reaction upon in vivo subcutaneous implantation. In vitro characterization demonstrated that HA coating could enhance cell adhesion to the scaffold and reduce gene expression of IL-1, IL-6, IL-8, and α-SMA. It also decreased the acidity of degradation products in vitro. Furthermore, coated PGA could engineer better cartilages in vitro with higher content of total collagen and glycosaminoglycan, as well as higher gene expression levels of collagen II, aggrecan, and Sox9. Collectively, the data indicate that HA coating can significantly enhance the biocompatibility of this traditional scaffold material, which also enhances the quality of engineered tissues.

The potential of increasing bone mass and preventing fractures in osteoporosis using stem cell therapy is currently an area of intense focus. However, there are very little data available regarding the postfracture bony defect healing efficacy under osteoporotic conditions. This study aims to investigate whether critical-sized segmental bone defects in a rabbit model of osteoporosis could be repaired using an allogenic stem cell-based tissue engineering (TE) approach and to investigate the potential influence of osteoporosis on the treatment efficacy. Rabbit fetal bone marrow mesenchymal stem cells (BMSCs) were harvested and expanded in vitro. Decalcified bone matrix (DBM) scaffolds were then seeded with allogenic fetal BMSCs and cultivated in osteogenic media to engineer BMSC/DBM constructs. Critical-sized radial defects were created in ovariectomized (OVX) rabbits and the defects were repaired either by insertion of BMSC/DBM constructs or by DBM scaffolds alone. Also, nonovariectomized age-matched (non-OVX) rabbits were served as control. At 3 months post-treatment under the osteoporotic condition (OVX rabbits), the BMSC/DBM constructs inserted within the defect generated significantly more bone tissue when compared to the DBM scaffold as demonstrated by the X-ray, microcomputed tomography, and histological analyses. In addition, when compared to a normal nonosteoporotic condition (age-matched non-OVX rabbits), the defect treatment efficacy was adversely affected by the osteoporotic condition with significantly less bone regeneration. This study demonstrated the potential of allogenic fetal BMSC-based TE strategy for repairing bone defects in an osteoporotic condition. However, the treatment efficacy could be considerably compromised in the OVX animals. Therefore, a more sophisticated strategy that addresses the complicated pathogenic conditions associated with osteoporosis is needed.

Mesenchymal stem cells (MSCs) usually lose their stemness during in vitro expansion as they are deprived of their niche environment. Cell-extracellular matrix (ECM) interaction is known to play important roles in preserving the stemness of the cells in their stem cell niche environment. Previously, coating with bone marrow MSC (BMSC)-derived ECM was found able to maintain the differentiation potential of in vitro cultured MSCs. This study aimed to determine if this ECM coating could also maintain the stemness of cultured murine adipose-derived stem cells (ASCs) using a regular culture flask as a control. Cells were expanded in ECM-coated and ECM-noncoated flasks for two and four passages and then harvested for various analyses. The results showed that ASCs exhibited fibroblast-like spindle morphology in ECM-coated flasks, whereas ASCs gradually spread and enlarged in the ECM-noncoated flasks. After three and five passages, both groups of cells exhibited similar cytokinetics in the MSC culture medium (MesenPRO RS™ Medium). However, when cultured in Dulbecco's modified Eagles medium (DMEM) plus 10% fetal bovine serum, coating group cells exhibited more potent proliferation than control group cells with a significant difference in both passages 3 and 5 (p<0.01). When seeded at low density (500 cells/10-cm dish), coating group cells formed significantly more and larger sized cell colonies than control group cells with significant difference in cell colony numbers between two groups (p<0.05). In addition, coated colony cells were much smaller and more compactly arranged compared to control colony cells. Furthermore, ASCs expanded in coated flasks exhibited greater potentials for adipogenic, osteogenic, and chondrogenic differentiations than the cells expanded in regular flasks. Quantitatively, the Oil Red O staining area, Alizarin staining area, and Toluidine Blue staining area were all significantly larger than the respective staining areas of control cells (p<0.05). Real-time polymerase chain reaction also revealed significantly higher gene expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein (C/EBP), Runx2, osteocalcin, Sox9, collagen II, and aggrecan in ECM-coated group cells than in control group cells (p<0.05). Collectively, these results suggest that human BMSC decellular ECM coating helps to preserve the stemness of cultured murine ASCs.

The reconstruction of large craniofacial defects remains a significant clinical challenge. The complex geometry of facial bone and the lack of suitable donor tissue often hinders successful repair. One strategy to address both of these difficulties is the development of an in vivo bioreactor, where a tissue flap of suitable geometry can be orthotopically grown within the same patient requiring reconstruction. Our group has previously designed such an approach using tissue chambers filled with morcellized bone autograft as a scaffold to autologously generate tissue with a predefined geometry. However, this approach still required donor tissue for filling the tissue chamber. With the recent advances in biodegradable synthetic bone graft materials, it may be possible to minimize this donor tissue by replacing it with synthetic ceramic particles. In addition, these flaps have not previously been transferred to a mandibular defect. In this study, we demonstrate the feasibility of transferring an autologously generated tissue-engineered vascularized bone flap to a mandibular defect in an ovine model, using either morcellized autograft or synthetic bone graft as scaffold material.

Mesenchymal stem cell (MSC) has great potential for both regenerative medicine and immunotherapy due to its multipotency and immunomodulatory property. The derivation of MSCs from human tissues involves an invasive procedure and the obtained MSCs often suffer from inconsistent quality. To overcome these issues, the approaches of deriving a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs) were established. However, few studies compared the immunological characteristics of MSCs derived from hESCs with tissue-derived MSCs or demonstrated differences and the underlying mechanisms. Here, we differentiated H9 hESCs into MSC-like cells (H9-MSCs) through an embryoid body outgrowth method and compared the immunological characteristics of H9-MSCs with bone marrow-derived MSCs (BMSCs). Both sources of derived cells exhibited typical MSC morphologies and surface marker expressions, as well as multipotency to differentiate into osteogenic and adipogenic lineages. A immunological characterization study showed that H9-MSCs and BMSCs had similar immunoprivileged properties without triggering allogeneic lymphocyte proliferation as well as equivalent immunosuppressive effects on T-cell proliferation induced by either cellular or mitogenic stimuli. Flow cytometry analysis revealed a lower expression of human major histocompatability complex class II molecule human lymphocyte antigen (HLA)-DR and a higher expression of coinhibitory molecule B7-H1 in H9-MSCs than in BMSCs. Interferon gamma (IFN-γ) is a proinflammatory cytokine that can induce the expression of HLA class II molecules in many cell types. Our results showed that pretreatment of H9-MSCs and BMSCs with IFN-γ did not change their immunogenicity and immunosuppressive abilities, but increased the difference between H9-MSCs and BMSCs for their expression of HLA-DR. Further detection of expression of molecules involved in IFN-γ signaling pathways suggested that the lower expression of HLA-DR in H9-MSCs could be partially attributed to the lower expression and the less nuclear translocation of its transcriptional factor CIITA. The present study provides evidence that the hESC-derived MSCs share similar immunogenicity and immunosuppressive abilities with BMSCs, but differ in the expression profile of immunological markers and the responsiveness to certain inflammatory cytokines, which suggests that H9-MSCs could be a safe and efficient candidate for MSC treatment in patients with inflammatory disorders.

Ectopic ossification of mesenchymal stem cell (MSC) regenerated cartilage has greatly restricted its application in repairing subcutaneous cartilage defects (such as nasal or auricular). Different from MSCs, chondrocytes can maintain stable chondrogenic phenotype in ectopic microenvironment, which was speculated to be related with the existence of antiangiogenic factors such as Chondromodulin-I (Chm-I). Therefore, the purpose of this study was to illustrate whether Chm-I was indispensable for stable ectopic chondrogenesis by chondrocyte, which may help to solve the problem of MSC ectopic ossification in the future. The current study demonstrated that Chm-I knockout did not obviously influence articular cartilage development in situ. However, native articular cartilage from Chm-I knockout (Chm-I(-/-), KO), but not wild-type (WT) mice, showed obvious ossification after subcutaneously implanted into nude mice for 16 days. Interestingly, cell morphology, cartilage-specific matrix expression, and pellet culture demonstrated that Chm-I knockout had no obvious influence on the phenotype, function, and chondrogenic ability of chondrocytes in vitro, except that cells in the WT group proliferated a little faster than those in the KO group. Nevertheless, Chm-I knockout directly interfered with in vivo ectopic cartilage regeneration when chondrocytes were subcutaneously injected into nude mice with matrigel. Moreover, Chm-I knockout obviously compromised ectopic stability of in vitro regenerated cartilage after subcutaneous implantation. These findings indicated that Chm-I was an indispensable factor for ectopic cartilage regeneration and the maintenance of cartilage homeostasis, which may provide a clue for solving the stability problem of MSC regenerated cartilage in ectopic niche. In addition, this study also provides a novel model based on tissue engineering strategy to properly evaluate the function of other targeted genes.

Macromolecular crowding (MMC) has been shown to have a beneficial effect on production and maturation of extracellular matrices in a monolayer cell culture model. To explore its potential in tissue engineering, a mixture of Ficoll 70 and Ficoll 400 is used to examine its MMC effect on cartilaginous matrix production of monolayer cultured porcine chondrocytes as well as on in vitro engineered cartilage formation using porcine chondrocytes and polyglycolic acid-unwoven fibers. The results showed that the production of total collagens and glycosaminoglycans production was enhanced by MMC in monolayer cultured cells (two-dimensional model), but the matrix production and tissue formation were significantly inhibited in the in vitro engineered cartilage (three-dimensional model) by the macromolecules. Further mechanism study on this phenomenon will be important for MMC applications in regenerative medicine.

Introduction: Human hair follicle outer root sheath (hORS) cells are known to contain hair follicle stem cells and play an important role in healing large size wounds, and thus can serve as the cell source for skin engineering. This study investigated the effect of low oxygen tension culture on hORS cell proliferation potential and functional maintenance during in vitro expansion., Materials and Methods: Spared postsurgery scalp tissues were donated by 15 patients aged 20-45 (13 men and 2 women) and were randomly divided into three groups, and isolated hORS cells were combined into three pooled cell samples. They were cultured either in 4% O(2) or 21% O(2) and were analyzed for cell proliferation, colony forming efficiency (CFE), and their ability in forming engineered skin in vitro., Results: The results showed that freshly isolated hORS cells expressed CD200 (22.88±8.76), cytokeratin 15 (CK15) (62.57±4.72), CD29 (22.53±2.49/strong and 29.80±4.09/dim), and CD49f (28.07±15.76/strong and 49.73±5.65/dim). When exposed in 4% O(2), hORS cells proliferated significantly faster than the cells in 21% O(2) for the first three passages (p<0.05), could better maintain cobblestone morphology, respectively, generate 3.63-folds more and 23.26-folds more cell yields after one and three passages. Additionally, enhanced CFE with significantly higher total and holoclone colony numbers were found in the 4% O(2) group than in the 21% O(2) group (p<0.05) for the first three passages along with better maintained CK15 expression. Furthermore, hORS cells expanded in 4% O(2) could form better epidermal structure of in vitro engineered skin comparing to the skin engineered by the control cells., Conclusion: The low oxygen culture method of hORS cells is simple, low cost, less labor intensive, and less biosafety concern, which may potentially be applied in skin engineering and clinical application.

Induced pluripotent stem (iPS) cells are a potential cell source for regenerative medicine. However, the tumorigenicity of iPS cells is a big concern for clinical application. In addition to the genetic manipulation of the reprogramming process and the greater risk of tumor formation, it is unclear whether iPS cells with normal development potential are still tumorigenic. Here, we investigated 3 mouse iPS cell lines, including one line that is able to generate full-term mice via tetraploid blastocyst complementation. We found that a small number of undifferentiated iPS cells could be steadily isolated and expanded after long-term differentiation of cells in vitro or in vivo. The residual undifferentiated iPS cells could be expanded and redifferentiated, and undifferentiated pluripotent stem cells could again be isolated after further rounds of differentiation, suggesting that residual undifferentiated iPS cells could not be eliminated by extended cell differentiation. The residual undifferentiated cells could form teratomas in vivo, indicating that they are a potential tumorigenic risk during transplantation. These findings prompt us to reconsider the strategies for solving the tumorigenic problem of iPS cells, not only focusing on improving the reprogramming process.

Human parthenogenetic embryonic stem cells (hpESCs) established from artificially activated oocytes have a wider immune-matching ability because of their homozygosity in the major histocompatibility complex alleles. Whether these cells possess the differentiation capacity similar to regular human embryonic stem cells (hESCs) derived from fertilized eggs is unclear. The aims of this study were to determine whether hpESCs could be differentiated into multipotent mesenchymal stem cell (MSC)-like cells in vitro and then compare these cells with those derived from hESCs. MSC-like cells were obtained from both hpESCs and hESCs, which exhibited similar cell surface marker expression profiles. Further analyses revealed that cells derived from hpESCs possessed stronger osteogenic but weaker adipogenic potentials compared with cells derived from hESCs. This is the first work that demonstrates the differentiation of hpESCs into multipotent MSC-like cells. These hpESCs could be a potential source for cell-based therapies.

Due to the lack of an appropriate cell source, external ear reconstruction based on tissue engineering techniques has not yet been translated to clinical applications. It has been reported that the chondrocytes from microtic cartilage can be used for cartilage regeneration. However, in our prior study, we found the passaged microtic chondrocytes became less proliferative and decreased expression of cartilage-specific markers, which would lead to the limited quantity and quality of cells from microtic cartilage to engineer an entire ear. To make full use of these cells, it is important to know whether they can provide the chondrogenic niche to promote the chondrogenesis of BMSCs. In this study, through the co-transplantation of BMSCs with microtic chondrocytes subcutaneously into nude mice, we showed that microtic chondrocytes have the potential to promote ectopic chondrogenesis of BMSCs. These results indicate that the early passage of chondrocytes from the microtic cartilage might have the same potential to promote chondrogenesis of BMSCs as normal chondrocytes. The co-culture of microtic chondrocytes with BMSCs potentially offers a practical approach to autologous ear regeneration.

Vascular endothelial cells (ECs) and most hematopoietic cells express platelet endothelial cell adhesion molecule-1 (PECAM-1), which is the cell surface protein also expressed in mouse embryonic stem (ES) cells. To better understand how PECAM-1(+) ES cells differentiate into PECAM-1(+) hematopoietic cells/ECs, 3 cell surface markers, PECAM-1, stage-specific embryonic antigen-1 (SSEA-1), and Flk-1, were utilized to dissect the developmental process during ES cell differentiation in vitro. Undifferentiated ES cells expressed PECAM-1, with a majority of them coexpressing SSEA-1. During ES cell differentiation, expression of PECAM-1 decreased to give rise to PECAM-1⁻/SSEA-1(+) cells, which represented epiblast stem cells. Subsequently, Flk-1-expressing cells developed from PECAM-1⁻/SSEA-1(+) cells, becoming SSEA-1⁻/Flk-1(+) through the downregulation of SSEA-1 expression. Following this, a second wave of PECAM-1 expression, which represented the mature hematopoietic cells/ECs, developed from Flk-1(+) cells. Also, a small portion of PECAM-1(+)/SSEA-1(+) cells, which represented the residual undifferentiated ES cells, were consistently observed in long-term differentiated embryoid bodies. This work revealed a sequential change in PECAM-1, SSEA-1, and Flk-1 expression during ES cell differentiation; therefore, they could be valuable cell surface markers for isolating cells at distinct developmental stages in ES cell differentiation.

This study aimed to investigate the chondrogenic transdifferentiation potential of human dermal fibroblasts (DFs) by stimulation with cartilage-derived morphogenetic protein 1 (CDMP1). Using CDMP1 (100 ng/mL) we induced human DFs at passage 5 in both monolayer and micromass culture. Chondrogenic-specific markers were detected via immunochemistry, reverse transcription-polymerase chain reaction, and Western blot analysis in the collected specimens. The expression profile of adhesion molecules including integrin alpha5, beta1, and N-cadherin of DFs accompanying with chondrogenesis was further investigated. After 7 days of induction in monolayer culture, DFs acquired the polygonal chondrocyte-like shape with positive expression of chondrogenic-specific markers. Such a phenotypic transition of DFs was lost at 14 days. However, in micromass culture the chondrogenic transdifferentiation of DFs can be maintained even at 14 days. No chondrogenesis was detected in DFs without CDMP1 treatment under both culture conditions. By neutralization assay with blocking antibodies, it was further revealed that integrin alpha5 expression was in direct proportion to the degree of chondrogenic differentiation. Based on our findings, it can be ascertained that DFs are capable of transdifferentiating into a chondrogenic lineage by stimulation with CDMP1 in vitro. The integrin alpha5 mediating cell-cell and cell-matrix interactions is required for maintaining the chondrogenic phenotype of DFs.

Smooth muscle cells (SMCs) play an essential role in maintaining the structural and functional integrity of blood vessel and thus is a critical element for blood vessel construction via tissue engineering approach. Adipose-derived stem cells (ASCs) represent a reliable source of mesenchymal stem cells with multidifferentiation potential. In this study, the feasibility of differentiation of human ASCs (hASCs) into cells with phenotypic and functional properties of SMCs was explored. hASCs isolated from human lipoaspirate were expanded to passage 5 and then induced with administration of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-4 (BMP4) either alone or in combination with culture medium. Expression of SMC-related markers including alpha-SM actin (alpha-SMA, SM22alpha, calponin, and SM myosin heavy chain) were detected by immunofluorescent staining, reverse transcription (RT)-polymerase chain reaction, and western blot analysis. It was found that only under the circumstance of a combined stimulation with TGF-beta1 and BMP4, both early and mid markers (alpha-SMA, SM22alpha, calponin) as well as a late marker (SM myosin heavy chain) of SMC differentiation were identified to similar levels as those in human umbilical artery SMCs. More importantly, these SM differentiated cells showed the function of contracting collagen matrix lattice when they were entrapped inside. The contractile function of differentiated hASCs was further enhanced by direct exposure to 60 mM KCl, consistent with what occurred in human umbilical artery SMCs. These results provide evidence that ASCs possess the potential to differentiate into contractile SM-like cells when stimulated by TGF-beta1 and BMP4 together. SMCs differentiated from hASCs may provide an abundant source as seed cells for blood vessel engineering.

The osteogenic differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) has been documented previously, and partially demineralized bone matrix (pDBM) represents a promising candidate for bone tissue engineering scaffolds. In this study, pDBM scaffolds derived from porcine cancellous bone were evaluated for their ability to support human UCB-MSCs osteogenic differentiation in vitro and bone-forming capacity in vivo to assess the potential use of UCB-MSCs in bone tissue engineering applications. MSCs were isolated from full-term human UCB and expanded, and their cell surface antigen markers and multilineage capability to differentiate into osteoblasts, chondrocytes, and adipocytes were analyzed. The in vitro proliferation and osteogenic differentiation of UCB-MSCs loaded onto the three-dimensional pDBM scaffolds were determined. Critical-sized full-thickness circular defects (5 mm in diameter) created bilaterally in the parietal bones of athymic rats were treated with one of the following: osteogenically induced UCB-MSC/pDBM composites (Group A, n = 8), noninduced UCB-MSC/pDBM composites (Group B, n = 8), pDBM alone (Group C, n = 8), or left untreated (Group D, n = 8). Microcomputed tomography analysis showed that new bone was formed in Group A at 6 weeks postimplantation, and greater bone volume and density were found after 12 weeks. In other groups, new bone formation was not evident after 6 weeks, and no bone union was found at 12 weeks. Histological examination revealed that the defect was repaired by tissue-engineered bone in Group A at 12 weeks, and fibrous union was observed in Groups B, C, and D. These results demonstrate that pDBM can support osteogenic differentiation of human UCB-MSCs in vitro and in vivo, and UCB-MSCs may serve as an alternative cell source for bone tissue engineering and regeneration.

Human adipose-derived stem cells (hASCs) offer great promise for bone tissue engineering because of their osteogenic differentiation potential. At molecular levels, this study investigated the contribution of one of the main members of mitogen-activated protein kinases (MAPKs), extracellular signal-related kinase (ERK), to hASC osteogenic differentiation and the regulation of ERK for the balance between osteogenesis and adipogenesis in hASCs in vitro. As analyzed using western blot, ERK activation in osteo-induced hASCs was initiated at day 7, peaked at day 10, and declined from day 14 to basal levels. As detected using histochemical and biochemical methods, alkaline phosphatase (ALP) activity in hASCs experienced a process similar to that of ERK activation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as revealed by an ALP activity assay, extracellular calcium deposition detection, osteocalcin (OCN) secretion examination, and real-time polymerase chain reaction (PCR) analysis for expression of osteogenesis-relative genes: core binding factor alpha 1, collagen type I, ALP, and OCN. Blockage of ERK phosphorylation in osteo-induced hASCs by PD98059 supplemented with dexamethasone (Dex) led to adipogenic differentiation, as confirmed by Nile Red staining to detect intracellular lipid droplets and real-time PCR analysis for expression of adipogenesis-relative genes: peroxisome proliferator-activated receptor gamma 2 and fatty acid-binding protein. These findings indicated a potential mechanism for the function of ERK in hASC osteogenic differentiation, especially the regulation of ERK in association with Dex for the balance between osteogenesis and adipogenesis, pointing out the significance of ERK signaling pathway for ASCs as a promising cell source for bone tissue engineering.

Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.

Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.

In vivo niche is known to play important roles in terminal differentiation of implanted bone marrow stromal cells (BMSCs). This study explored the feasibility of repairing articular osteochondral defects using autologous BMSCs and biodegradable polymers. BMSCs from 18 hybrid pigs' marrows were either treated with dexamethasone (40 ng/mL) alone or chondrogenically induced with dexamethasone and transforming growth factor-beta1 (10 ng/mL). The cells were seeded respectively onto polylactic acid (PLA)- coated polyglycolic acid (PGA) scaffolds. Four osteochondral defects in each animal were created at non-weightbearing areas of knee joints (2/each side) and were respectively repaired by a chondrogenically induced BMSC-PGA/PLA construct in experimental group (Exp), by a dexamethasone-treated BMSC-PGA/PLA construct in control 1 group (Ctrl 1), by a PGA/PLA construct alone in control 2 group (Ctrl 2), or left unrepaired in control 3 group (Ctrl 3). To trace the implanted cells, green fluorescent protein (GFP)- labeled BMSCs were implanted in 2 animals. Gross view and histology showed that Exp and Ctrl 1 (with cell implantation) achieved better reparative results than Ctrl 2 and Ctrl 3 (without cell implantation) in terms of the reparative level and the restoration of the histological structure. In addition, 6-month results were better than 3-month results in all 4 groups. In Exp, 11 of 16 defects were completely repaired by hyaline cartilage and cancellous bone. In Ctrl 1, 11 of 16 defects were repaired by fibrocartilage and cancellous bone, although the repair with hyaline cartilage and cancellous bone was observed in 5 of 16 defects. In contrast, no obvious repair or only fibrotic tissue was observed in Ctrl 2 and Ctrl 3. The compressive moduli of repaired cartilage in Exp reached 80.27% of the normal amount at 6 months, with a high level of glycosaminoglycan (GAG) content (no statistical difference from normal). In Ctrl 1, the compressive moduli and GAG content were 62.69% and 78.03% of normal levels, respectively. More importantly, GFP-labeled cells were detected in the engineered cartilage and the repaired subchondral bone. These results strongly indicate that the implanted BMSCs can differentiate into either chondrocytes or osteoblasts and repair articular osteochondral defects by forming engineered cartilage and engineered bone.

Alveolar bone resorption caused by periodontal diseases remains a difficult clinical problem to treat. Our purpose here was to develop protocols for repairing experimental horizontal alveolar bone defects. The procedure entailed isolating bone marrow stromal cells (BMSC). They were expanded and induced in vitro into osteogenic cells in a defined medium. Induced BMSCs were mixed with calcium alginate to form a gel form of cell-scaffold construct for developing engineered bone. A horizontal alveolar bone defect was created in 15 mongrel dogs, which was 5 mm high on each of two buccal sides at the location of mandibular premolar 3, 4, and molar 1. Without bias, the animals were separated into the following groups: (1) cell-scaffold construct as the experimental group; (2) calcium alginate alone as the control group A; (3) untreated as the control group B. Block sections of the defects were collected at 4, 12, and 24 weeks postsurgery, respectively, and processed for gross and histological observation as well as x-ray examination. The results showed that in vitro induced BMSCs exhibited an osteogenic phenotype. Histologically, bone nodule structure was observed in the tissue of the experimental group at 4 weeks postsurgery and the engineered bone became more mature after 12 weeks, which was similar to normal bone. At 12 weeks postsurgery, the height of repaired alveolar bone reached 2.43 +/- 0.93 mm, 0.98 +/- 0.87 mm, 0.78 +/- 0.75 mm for the experimental group, control groups A and B, respectively, with a significant difference between the experimental and control groups (p < 0.01). The average level of buccal alveolar ridge in experimental group, control groups A and B reached 48.59%, 19.74%, and 15.76% of the height of normal alveolus, respectively, with a significant difference between the experimental group and two control groups (p < 0.01). We thus conclude that BMSCs can be induced to become osteogenic and can be used as seed cells to engineer bone tissue and repair experimental alveolar bone defects.

Although there are many reports of in vivo tendon engineering using different animal models, only a few studies involve the short-term investigation of in vitro tendon engineering. Our previous study demonstrated that functional tendon tissue could be engineered in vivo in a hen model using tenocytes and polyglycolic acid (PGA) fibers. This current study explored the feasibility of in vitro tendon engineering using the same type of cells and scaffold material. Tenocytes were extracted from the tendons of a hen's foot with enzyme digestion and cultured in DMEM plus 10% FBS. Unwoven PGA fibers were arranged into a cord-like construct and fixed on a U-shape spring, and tenocytes were then seeded on PGA fibers to generate a cell-PGA construct. In experimental group 1, 22 cell-scaffold constructs were fixed on the spring with no tension and collected at weeks 4 (n = 7), 6 (n = 7) and 10 (n = 8); in experimental group 2, five cell-scaffold constructs were fixed on the spring with a constant strain and collected after 6 weeks of culture. In the control group, three cell-free scaffolds were fixed on the spring without tension. The collected engineered tendons were subjected to gross and histological examinations and biomechanical analysis. The results showed that tendon tissue could be generated during in vitro culture. In addition, the tissue structure and mechanical property became more mature and stronger with the increase of culture time. Furthermore, application of constant strain could enhance tissue maturation and improve mechanical property of the in vitro engineered tendon (1.302 +/- 0.404 Mpa with tension vs 0.406 +/- 0.030 Mpa without tension at 6 weeks). Nevertheless, tendon engineered with constant strain appeared much thinner in its diameter than tendon engineered without mechanical loading. Additionally, its collagen fibers were highly compacted when compared to natural tendon structure, suggesting that constant strain may not be the optimal means of mechanical load. Thus, application of dynamic mechanical load with a bioreactor to the construction of tendon tissue will be our next goal in this series of in vitro tendon engineering study.

Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord-like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering.

Tissue engineering can generate bone tissue and has been shown to provide a better means of repairing weight-bearing bone defect. Previous studies, however, have heretofore been limited to the use of nonosteogenically induced bone marrow stromal cells (BMSCs) or the application of slow-degradation scaffolds. In this study, weight-bearing bone was engineered using osteogenically induced BMSCs. In addition, coral was used as a scaffold material, due to its proper degradation rate for the engineering and repair of a goat femur defect. A 25 mm long defect was created at the middle of the right femur in each of 10 goats. The rates of defect repair were compared in an experimental group of ten goats receiving implants containing osteogenically induced BMSCs and in the control group of goats (n = 10) receiving just coral cylinders. In the experimental group, bony union was observed by radiographic and gross view at 4 months, and engineered bone was further remodeled into newly formed cortexed bone at 8 months. There was increased gray density of radiographic rays in the repaired area, which was significantly different (p < 0.05) from that of the control group. H&E staining demonstrated that trabecular bone was formed at 4 months. Moreover, irregular osteon was observed at 8 months. Most importantly, the tissue-engineered bone segment revealed a similarity to the left-side normal femur in terms of bend load strength and bend rigidity, showing no significant difference (p > 0.05). In contrast, the coral cylinders of the control group showed no bone formation. Furthermore, almost complete resorption of the carrier had occurred, being evident at 2 months in the control group. H&E staining demonstrated that a small amount of residual coral particle was surrounded by fibrous tissue at 4 months whereas the residues disappeared at 8 months. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and coral can ideally heal critical-sized segmental bone defects in the weight-bearing area of goats.

We determined whether a polyglycolic acid (PGA) scaffold bearing an adherent corneal stromal cell insert could be integrated into the ultrastructure of rabbit corneal stroma without compromising tissue transparency. Stromal cells were isolated from 10 newborn rabbits and expanded by tissue culture. After reaching confluence, the cells were harvested and mixed with nonwoven PGA fibers to form cell-scaffold constructs. After 1 week of culturing, they were implanted into the corneal stroma of female rabbit recipients. Green fluorescent protein (GFP) expression in transplanted corneal stromal cells was monitored at the protein level to determine cellular origin in the reconstructed stroma. Eight weeks after implantation, transmission electron microscopy and histological evaluation were performed on stromal tissue. Insertion of PGA scaffold alone served as a sham control. After stromal implantation, implants gradually became transparent over an 8-week period. During this time stromal histology was gradually restored, as collagen fibril organization approached that of their normal counterpart. GFP-labeled corneal stromal cells were preponderant in the regions bearing inserted scaffolds, suggesting that they were derived from the implants rather than from neighboring corneal stromal cells. No reconstructed stroma developed in regions where naked PGA was implanted instead. We conclude that intrastromal implantation of PGA fiber scaffold implants bearing corneal stromal cells is a useful procedure for corneal stromal tissue reconstruction because, over an 8-week period, the implants become progressively more transparent. Marked losses of translucence during this period combined with restoration of ultrastructure indicate that the implants provide the functions needed for deturgescing initially swollen stroma.

Tissue engineering started in late 1980s and is now well established and progressing rapidly in Western developed countries. However, the development of tissue-engineering research in China remains relatively unknown to the international society of tissue engineering. Although involved in all areas of tissue-engineering research, including the creation of new scaffold materials, in vitro studies of seed cells, application of growth factors, and modification of seed cells and scaffold materials, China has put special emphasis on tissue construction in large mammalian animals in order to establish a solid scientific basis for clinical application of engineered tissues. To provide a closer view of tissue-engineering research in China, this article reviews our experience in tissue construction and tissue repair using immunocompetent animals such as sheep, pig, and dog as well as hen and rabbit. The engineered tissues include bone, cartilage, tendon, skin, blood vessels, cornea, and peripheral nerves.

Large full-thickness defects of articular cartilage remain a major challenge to orthopedic surgeons because of unsatisfactory results of current therapy. Many methods, such as chondrectomy, drilling, cartilage scraping, arthroplasty, transplantation of chondrocytes, periosteum, perichondrium, as well as cartilage and bone, have been tried to repair articular cartilage defects. However, the results are far from satisfactory. In this study, we applied a tissue-engineering approach to the repair of articular cartilage defects of knee joints in a porcine model. Using isolated autologous chondrocytes, polyglycolic acid (PGA), and Pluronic, we have successfully in vivo-engineered hyaline cartilage and repaired articular cartilage defects. The surface of the repaired defects appeared smooth at 24 weeks postrepair. Histological examination demonstrated a typical hyaline cartilage structure with ideal interface healing between the engineered cartilage and the adjacent normal cartilage and underlying cancellous bone. In addition, glycosaminoglycan (GAG) levels in the engineered cartilage reached 80% of that found in native cartilage at 24 weeks postrepair. Biomechanical analysis at 24 weeks demonstrated that the biomechanical properties of the tissue-engineered cartilage were improved compared with those at an earlier stage. Thus, the results of this study may provide insight into the clinical repair of articular cartilage defects.