Aijuan He

The influence of Gelatin/PCL ratio and 3-D construct shape of electrospun membranes on cartilage regeneration

Scaffolds play an important role in directing three-dimensional (3-D) cartilage regeneration. Our recent study reported the potential advantages of electrospun gelatin/polycaprolactone (GT/PCL) membranes in regenerating 3-D cartilage. However, it is still unknown whether the changes of GT/PCL ratio have significant influence on 3-D cartilage regeneration. To address this issue, the current study prepared three kinds of electrospun membranes with different GT/PCL ratios (70:30, 50:50, 30:70). Adhesion and proliferation of chondrocytes on the membranes were examined to evaluate biocompatibility of the membranes. Cartilage with different 3-D shapes was engineered to further evaluate the influences of GT/PCL ratio on cartilage regeneration. The current results demonstrated that all the membranes with different GT/PCL ratios presented good biocompatibility with chondrocytes. Nevertheless, the high PCL content in the membranes significantly hampered early 3-D cartilage formation at 3 weeks in vivo. Unexpectedly, at 12 weeks, all the cylinder-shaped constructs formed mature cartilage-like tissue with no statistical differences among groups. To our surprise, ear-shaped cartilage regeneration obtained quite different results again: the high PCL content completely disrupted cartilage regeneration even at 12 weeks, and only the least PCL content group formed homogeneous and continuous cartilage with a satisfactory shape and elasticity similar to human ear. All these results indicated that the high PCL content was unfavorable for 3-D cartilage regeneration, especially for the cartilage with a complicated shape, and that GT/PCL 70:30 might be a relatively suitable ratio for ear-shaped cartilage regeneration. The research models established in the current study provide detailed information for cartilage and other tissue regeneration based on electrospun GT/PCL membranes.

Long-term functional reconstruction of segmental tracheal defect by pedicled tissue-engineered trachea in rabbits.

Due to lack of satisfactory tracheal substitutes, reconstruction of long segmental tracheal defects (>6 cm) is always a major challenge in trachea surgery. Tissue-engineered trachea (TET) provides a promising approach to address this challenge, but no breakthrough has been achieved yet in repairing segmental tracheal defect. The longest survival time only reached 60 days. The leading reasons for the failure of segmental tracheal defect reconstruction were mainly related to airway stenosis (caused by the overgrowth of granulation tissue), airway collapse (caused by cartilage softening) and mucous impaction (mainly caused by lack of epithelium). To address these problems, the current study proposed an improved strategy, which involved in vitro pre-culture, in vivo maturation, and pre-vascularization of TET grafts as well as the use of silicone stent. The results demonstrated that the two-step strategy of in vitro pre-culture plus in vivo implantation could successfully regenerate tubular cartilage with a mechanical strength similar to native trachea in immunocompetent animals. The use of silicone stents effectively depressed granulation overgrowth, prevented airway stenosis, and thus dramatically enhanced the survival rate at the early stage post-operation. Most importantly, through intramuscular implantation and transplantation with pedicled muscular flap, the TET grafts established stable blood supply, which guaranteed maintenance of tubular cartilage structure and function, accelerated epithelialization of TET grafts, and thus realized long-term functional reconstruction of segmental tracheal defects. The integration of all these improved strategies finally realized long-term survival of animals: 60% of rabbits survived over 6 months. The current improved strategy provided a promising approach for long-term functional reconstruction of long segmental tracheal defect.

Induction of transient tenogenic phenotype of high-density cultured human dermal fibroblasts

Abstract Previous study showed that high-density culture supported phenotype maintenance of in vitro expanded tenocytes. This study explored the possibility of inducing the tenogenic phenotype of dermal fibroblasts by high-density monolayer culture. Human fibroblasts were seeded either in high-density (2.5 × 106 per 10 cm dish) or at low-density (0.36 × 106 per 10 cm dish). A preliminary tenogenic phenotype was observed in high-density cultured cells after one passage with significantly enhanced tenogenic gene expression. With continued cultivation to passage 3, scleraxis (SCX), tenomodulin (TNMD), collagen I, III, VI, decorin and tenascin-c were all significantly upregulated in high-density cultured dermal fibroblasts as opposed to low-density cells. High-density culture also led to relatively elongated cell shape, whereas cells appeared in spread shape in low-density culture. In addition, cytochalasin D treatment disrupted the cellular cytoskeleton and resulted in inhibition of density-induced tenogenic gene expression. However, high-density cultured fibroblasts failed to induce other lineage differentiations (osteogenic, chondrogenic and adipogenic). It also failed to induce tenogenic phenotype in high-density cultured chondrocytes. Mechanism studies revealed enhanced gene expression of growth and differentiation factors (GDF) 5, 6, 7 and 8 and transforming growth factor-β (TGF-β)1 in the high-density group and enhanced protein production of both GDF8 and TGF-β1. Moreover, BMP/GDF signaling inhibitor (LDN193189) and TGF-β signaling inhibitor (LY2109761) could both abrogate the density induced phenotype. In conclusion, high-density culture was able to induce transient tenogenic phenotype of dermal fibroblasts likely via cell morphology change and production of pro-tenogenic factors.

Repair of osteochondral defects with in vitro engineered cartilage based on autologous bone marrow stromal cells in a swine model

Functional reconstruction of large osteochondral defects is always a major challenge in articular surgery. Some studies have reported the feasibility of repairing articular osteochondral defects using bone marrow stromal cells (BMSCs) and biodegradable scaffolds. However, no significant breakthroughs have been achieved in clinical translation due to the instability of in vivo cartilage regeneration based on direct cell-scaffold construct implantation. To overcome the disadvantages of direct cell-scaffold construct implantation, the current study proposed an in vitro cartilage regeneration strategy, providing relatively mature cartilage-like tissue with superior mechanical properties. Our strategy involved in vitro cartilage engineering, repair of osteochondral defects, and evaluation of in vivo repair efficacy. The results demonstrated that BMSC engineered cartilage in vitro (BEC-vitro) presented a time-depended maturation process. The implantation of BEC-vitro alone could successfully realize tissue-specific repair of osteochondral defects with both cartilage and subchondral bone. Furthermore, the maturity level of BEC-vitro had significant influence on the repaired results. These results indicated that in vitro cartilage regeneration using BMSCs is a promising strategy for functional reconstruction of osteochondral defect, thus promoting the clinical translation of cartilage regeneration techniques incorporating BMSCs.

Chondrogenic differentiation and three dimensional chondrogenesis of human adipose-derived stem cells induced by engineered cartilage-derived conditional media

Due to lack of optimal inductive protocols, how to effectively improve chondrogenesis of adiposederived stem cells (ASCs) is still a great challenge. Our previous studies demonstrated that the culture media derived from chondrocyte-scaffold constructs (conditional media) contained various soluble chondrogenic factors and were effective for directing chondrogenic differentiation of bone marrow stem cells. Nevertheless, it remains unclear whether the conditional media can induce ASCs towards chondrogenic differentiation, especially for three-dimensional (3D) cartilage formation in a preshaped scaffold. In this study, it demonstrated that the conditional media derived from chondrocyte-scaffold constructs could promote ASCs to differentiate into chondrocyte-like cells, with similar expression of type II collagen to those induced by chondrogenic growth factors. Moreover, the expression level of chondrocyte-specific genes, such as SOX9, type II collagen, and COMP, was even higher in conditional medium group (CM) than that in optimized chondrogenic growth factor group (GF), indicating that the conditional media can serve as an effective inducer for chondrogenic differentiation of ASCs. Most importantly, the conditional media could also induce ASC-scaffold constructs to form 3D cartilage-like tissue with typical lacunae structures and positive expression of cartilage specific matrices, even higher contents of GAG and type II collagen were achieved in CM group compared to GF group. The current study establishes a simple, but stable, efficient, and economical method for directing 3D cartilage formation of ASCs, a strategy that may be more closely applicable for repairing cartilage defects.

In Vitro Regeneration of Patient-specific Ear-shaped Cartilage and Its First Clinical Application for Auricular Reconstruction

Microtia is a congenital external ear malformation that can seriously influence the psychological and physiological well-being of affected children. The successful regeneration of human ear-shaped cartilage using a tissue engineering approach in a nude mouse represents a promising approach for auricular reconstruction. However, owing to technical issues in cell source, shape control, mechanical strength, biosafety, and long-term stability of the regenerated cartilage, human tissue engineered ear-shaped cartilage is yet to be applied clinically. Using expanded microtia chondrocytes, compound biodegradable scaffold, and in vitro culture technique, we engineered patient-specific ear-shaped cartilage in vitro. Moreover, the cartilage was used for auricle reconstruction of five microtia patients and achieved satisfactory aesthetical outcome with mature cartilage formation during 2.5 years follow-up in the first conducted case. Different surgical procedures were also employed to find the optimal approach for handling tissue engineered grafts. In conclusion, the results represent a significant breakthrough in clinical translation of tissue engineered human ear-shaped cartilage given the established in vitro engineering technique and suitable surgical procedure. This study was registered in Chinese Clinical Trial Registry (ChiCTR-ICN-14005469).

Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet

In vivo niche plays an important role in regulating differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. This study explored the feasibility that chondrocyte sheet created chondrogenic niche retained chondrogenic phenotype of BMSC engineered cartilage (BEC) in subcutaneous environments. Porcine BMSCs were seeded into biodegradable scaffolds followed by 4weeks of chondrogenic induction in vitro to form BEC, which were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS, SIS group), or nothing (Blank group) respectively and then implanted subcutaneously into nude mice to trace the maintenance of chondrogenic phenotype. The results showed that all the constructs in Sheet group displayed typical cartilaginous features with abundant lacunae and cartilage specific matrices deposition. These samples became more mature with prolonged in vivo implantation, and few signs of ossification were observed at all time points except for one sample that had not been wrapped completely. Cell labeling results in Sheet group further revealed that the implanted BEC directly participated in cartilage formation. Samples in both SIS and Blank groups mainly showed ossified tissue at all time points with partial fibrogenesis in a few samples. These results suggested that chondrocyte sheet could create a chondrogenic niche for retaining chondrogenic phenotype of BEC in subcutaneous environment and thus provide a novel research model for stable ectopic cartilage regeneration based on stem cells. STATEMENT OF SIGNIFICANCE In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by high-density culture of chondrocytes in vitro could cearte a chondrogenic niche in subcutaneous environment and efficiently retain the chondrogenic phenotype of in vitro BMSC engineered cartilage (vitro-BEC). Furthermore, cell tracing results revealed that the regenerated cartilage mainly derived from the implanted vitro-BEC. The current study not only proposes a novel research model for microenvironment simulation but also provides a useful strategy for stable ectopic cartilage regeneration of stem cells.

Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique

Tissue-engineered trachea provides a promising approach for reconstruction of long segmental tracheal defects. However, a lack of ideal biodegradable scaffolds greatly restricts its clinical translation. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration owing to natural tubular structure, cartilage matrix components, and biodegradability. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. To address these problems, a laser micropore technique (LMT) was applied in the current study to modify trachea sample porosity to facilitate decellular treatment and cell ingrowth. Decellularization processing demonstrated that cells in LMT treated samples were more easily removed compared with untreated native trachea. Furthermore, after optimizing the protocols of LMT and decellular treatments, the LMT-treated DTM (LDTM) could retain their original tubular shape with only mild extracellular matrix damage. After seeding with chondrocytes and culture in vitro for 8 weeks, the cell-LDTM constructs formed tubular cartilage with relatively homogenous cell distribution in both micropores and bilateral surfaces. In vivo results further confirmed that the constructs could form mature tubular cartilage with increased DNA and cartilage matrix contents, as well as enhanced mechanical strength, compared with native trachea. Collectively, these results indicate that LDTM is an ideal scaffold for tubular cartilage regeneration and, thus, provides a promising strategy for functional reconstruction of trachea cartilage. STATEMENT OF SIGNIFICANCE Lacking ideal biodegradable scaffolds greatly restricts development of tissue-engineered trachea. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. By laser micropore technique (LMT), the current study efficiently enhanced the porosity and decellularized efficacy of DTM. The LMT-treated DTM basically retained the original tubular shape with mild matrix damage. After chondrocyte seeding followed by in vitro culture and in vivo implantation, the constructs formed mature tubular cartilage with matrix content and mechanical strength similar to native trachea. The current study provides an ideal scaffold and a promising strategy for cartilage regeneration and functional reconstruction of trachea.

Repair of articular cartilage defects with acellular cartilage sheets in a swine model

Acellular cartilage sheets (ACSs) have been demonstrated as a good biomaterial for cartilage regeneration as a result of their natural cartilage matrix components, cartilage-specific structures, and good biocompatibility. However, it remains unknown whether allogeneic ACSs could promote cartilage regeneration and repair cartilage defects in a large animal model. The current study explored the feasibility of repairing articular cartilage defects using ACS scaffold with or without autologous bone marrow stromal cells (BMSCs) in a swine model. According to the current results, ACSs retained natural cartilage structure, primary cartilage matrices, and cartilage-specific growth factors. After cell seeding, ACSs presented good biocompatibility with BMSCs, which produced abundant extracellular matrix (ECM) proteins to cover the lacuna structures. In vivo results indicated that ACSs alone could induce endogenous host cells to regenerate cartilage and achieve generally satisfactory repair of cartilage defects at 6 months post-operation, including good interface integration and cartilage-specific ECM deposition. After combination with autologous BMSCs, BMSC-ACS constructs achieved more satisfactory repair of cartilage defects even without in vitro pre-induction of chondrogenesis. More importantly, all defects in both BMSC-ACS and ACS-only groups showed enhanced cartilage regeneration compared with BMSC-polyglycolic acid and blank groups, which mainly exhibited fibrogenesis in defect areas. Collectively, the current results indicate that ACSs can efficiently repair articular cartilage defects by promoting chondrogenic differentiation of BMSCs or inducing endogenous chondrogenesis in situ, thus serving as a good cartilage regeneration scaffold for recovery of articular function.

Cell yield, chondrogenic potential and regenerated cartilage-type of chondrocytes derived from ear, nasoseptal, and costal cartilage

Functional reconstruction of large cartilage defects in subcutaneous sites remains clinically challenging because of limited donor cartilage. Tissue engineering is a promising and widely accepted strategy for cartilage regeneration. To date, however, this strategy has not achieved a significant breakthrough in clinical translation owing to a lack of detailed preclinical data on cell yield and functionality of clinically applicable chondrocytes. To address this issue, the current study investigated the initial cell yield, proliferative potential, chondrogenic capacity, and regenerated cartilage type of human chondrocytes derived from auricular, nasoseptal, and costal cartilage using a scaffold‐free cartilage regeneration model (cartilage sheet). Chondrocytes from all sources exhibited high sensitivity to basic fibroblast growth factor within 8 passages. Nasoseptal chondrocytes presented the strongest proliferation rate, whereas auricular chondrocytes obtained the highest total cell amount using comparable cartilage sample weights. Importantly, all chondrocytes at fifth passage showed strong chondrogenic capacity both in vitro and in the subcutaneous environment of nude mice. Although some significant differences in histological structure, cartilage matrix content and cartilage type specific proteins were observed between the in vitro engineered cartilage and original tissue; the in vivo regenerated cartilage showed mature cartilage features with high similarity to their original native tissue, except for minor matrix changes influenced by the in vivo environment. The current study provides detailed preclinical data for choice of chondrocyte source and thus promotes the clinical translation of cartilage regeneration approach.