Injectable Cartilage Microtissue Regenerated by Autologous Chondrocytes for Nasal Augmentation: A 5-Year Follow-Up Study.

BACKGROUND: A lack of ideal filling materials is a critical limitation in current rhinoplasty. Cartilage sheet regeneration by autologous chondrocytes is expected to provide an ideal source of material. However, the inability to perform minimally invasive transplantation of cartilage sheets has greatly limited the clinical application of this material. In this article, the authors propose the concept of injectable cartilage microtissue (ICM) based on cartilage sheet technology, with the aim of achieving minimally invasive augmentation rhinoplasty in clinical practice. METHODS: Approximately 1.0 cm2 of posterior auricular cartilage was collected from 28 patients. Isolated chondrocytes were expanded, then used to construct autologous cartilage sheets by high-density seeding and in vitro culture in chondrogenic medium with cytokines (eg, transforming growth factor beta-1 and insulin-like growth factor-1) for 3 weeks. Next, ICM was prepared by granulation of the cartilage sheets; it was then injected into a subcutaneous pocket for rhinoplasty. RESULTS: ICM was successfully prepared in all patients, and its implantation efficiently raised the nasal dorsum. Magnetic resonance imaging confirmed that regenerative tissue was present at the injection site; histologic examinations demonstrated mature cartilage formation with typical cartilage lacunae and abundant cartilage-specific deposition of extracellular matrix. Excellent or good postoperative patient satisfaction results were achieved in 83.3% of patients over 5 years of follow-up. Obvious absorption of grafts occurred in only two patients (8.3%). CONCLUSIONS: These results demonstrated that ICM could facilitate stable cartilage regeneration and long-term maintenance in the human body; the implantation of ICM enabled natural augmentation of the depressed nasal dorsum. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Comparative effectiveness analysis of deep low heat burns in the shin surgical approaches: Outcomes and cost for wound rehabilitation.

A variety of surgical techniques exist for deep burn wounds in the shin at low temperature reconstruction after appropriate debridement, but limited high-quality data exist to inform treatment strategies. Using multi-institutional data, the authors evaluated the length of healing time, cost, and outcomes of three common surgical reconstructive modalities. All subjects with deep burn wounds in the shin caused by low temperature who received direct suture repair, skin grafting, or local flap reconstruction were retrospectively reviewed (from 2015.01 to 2021.03). Mean operation time, mean blood loss in operation, postoperative healing time, whether there is scar depression after operation were the primary outcomes; patient satisfaction score, Vancouver scar scale (VSS) score and average costs were secondary outcomes. Two hundred subjects (68 suture, 87 skin-grafting, and 45 local flap coverage patients) were evaluated. Matched patients (n = 200; 3/groups) were analysed. The average operation time, average operation blood loss, and postoperative healing time were statistically significant differences (P < 0.05). Readmissions and reoperations were greater for direct suture and local flaps, if achievable, direct suture provided success at low cost. Skin grafting was effective with large burn wounds but at higher costs and longer length of stay. Local flaps successfully treated smaller burn wounds unable to suture directly, with less pigmentation and scars, even suitable for older patients. Deep low heat burn wounds in the shin healing can be performed effectively using multiple modalities with varying degrees of success and costs. Direct suture or local skin flap reconstruction, if achievable, provides successful coverage at minimal costs, no skin contractures, and reducing length of hospital stay.

Regeneration of trachea graft with cartilage support, vascularization, and epithelization.

The repair and functional reconstruction of long-segment tracheal defects is always a great challenge in the clinic. Finding an ideal substitute for tracheal transplantation is the only way to solve this problem. The current study proposed a series of novel strategies for constructing a bionic living trachea substitute. For the issue of tubular cartilage support, cartilage sheet technique based on high-density culture of chondrocytes was adopted to avoid the inflammatory reaction triggered by the materials and thus formed mature cartilage-like tissue in autologous goat model. For the issue of epithelialization, the autologous transplantation of oral mucosal epithelium was used to realize mucosa coverage of the constructed trachea lumen. Finally, the flat trapezius fascia flap with double blood supply was separated by microsurgical techniques to achieve stable pre-vascularization of both the regenerated cartilage and the grafted epithelium simultaneously. By integrating the above strategies, the vascularized and epithelialized tracheal substitute with tubular cartilage support was successfully constructed in a goat model. The reconstructed trachea possessed a multiple layer structure of muscle-cartilage-fascia-mucosa comparable to the native trachea, and thus might realize stable survival and long-term airway function maintenance, providing a promising tracheal substitute for the repair and permanent functional reconstruction of long-segment tracheal defects. STATEMENT OF SIGNIFICANCE: The repair of long-segment tracheal defects is always a great challenge in the clinic. Finding an ideal substitute for tracheal transplantation is the only way to solve this problem. In the current study, by technical integration of cartilage regeneration, microsurgery, and oral mucosa transplantation, a complex tracheal substitute with satisfactory vascularization, epithelialization, and tubular cartilage support was successfully constructed in a goat autologous model. The reconstructed trachea substitute possessed a multiple layer structure of muscle-cartilage-fascia-mucosa exactly similar to native trachea, and thus might realize stable survival and long-term airway function maintenance. The current study provides feasible strategies and ideal tracheal substitutes for permanent functional reconstruction of long-segmental trachea defects.

Porous decellularized trachea scaffold prepared by a laser micropore technique.

Rapid development of tissue engineering technology provides new methods for tracheal cartilage regeneration. However, the current lack of an ideal scaffold makes engineering of trachea cartilage tissue into a three-dimensional (3-D) tubular structure a great challenge. Although a decellularized trachea matrix (DTM) has become a recognized scaffold for trachea cartilage regeneration, it is difficult for cells to detach from or penetrate the matrix because of its non-porous structure. To tackle these problems, a laser micropore technique (LMT) was applied in the current study to enhance trachea sample porosity, and facilitate decellularizing treatment and cell ingrowth. Furthermore, after optimizing LMT and decellularizing treatment parameters, LMT-treated DTM (LDTM) retained its natural tubular structure with only minor extracellular matrix damage. Moreover, compared with DTM, the current study showed that LDTM significantly improved the adherence rate of cells with perfect cell biocompatibility. Moreover, the optimal implantation cell density for chondrogenesis with LDTM was determined to be 1 × 108 cells/ml. Collectively, the results suggest that the novel LDTM is an ideal scaffold for trachea tissue engineering.

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 2.5years 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).

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.

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.

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.

Prolonged in vitro precultivation alleviates post-implantation inflammation and promotes stable subcutaneous cartilage formation in a goat model.

Synthetic biodegradable scaffolds such as polylactic acid coated polyglycolic acid (PLA-PGA) are especially suitable for engineering shaped cartilage such as auricle, but they induce a serious inflammatory reaction particularly in the immunologically aggressive subcutaneous site, leading to resorption of the engineered autologous cartilage. Our previous study in a rabbit model has demonstrated 2 weeks of in vitro precultivation could significantly alleviate the post-implantation inflammation induced by PLA-PGA engineered cartilaginous grafts, but reproduction of this result failed in a preclinical goat model. The aims of the current study were to investigate whether prolonged in vitro precultivation could form a mature cartilaginous graft to resist the acute host response and promote stable subcutaneous cartilage formation in a preclinical goat model. Goat chondrocytes were seeded onto PLA-PGA scaffolds, in vitro precultivated for 2, 4, 8, and 12 weeks, and then implanted subcutaneously in autologous goats for 1 and 8 weeks. The in vitro engineered cartilage (vitro-EC) was examined histologically (hematoxylin and eosin, safranin-O, collagen II). The 1 week explants were examined histologically and stained for CD3, CD68, collagen I, and apoptosis. The 8 week explants were evaluated by histology, wet weight, volume, glycosaminoglycan (GAG) quantification and Young's modulus. With prolonged in vitro time, the quality of vitro-EC improved and the amount of scaffold residue decreased; more pronounced cartilage formation with fewer immune cells (CD3 and CD68 positive), apoptotic cells, and less collagen I expression were observed in explants that had been in vitro precultivated for a longer period. The subcutaneously regenerated neocartilage became more mature after prolonged implantation. These results suggested that prolonged in vitro precultivation allowed formation of a mature cartilaginous graft to resist the acute host response and promoted stable subcutaneous cartilage formation in autologous goats. These findings may provide useful reference for engineering auricle, trachea, nose, and eyelid shaped cartilage, for example.

Convenient three-dimensional cell culture in supermolecular hydrogels.

A convenient three-dimensional cell culture was developed by employing high swelling property of hybrid hydrogels coassembled from C2-phenyl-based supermolecular gelators and sodium hyaluronate. Imaging and spectroscopic analysis by scanning electron microscopy (SEM), atomic force microscopy (AFM), transform infrared (FT-IR) spectra confirm that the hybrid gelators can self-assemble into nanofibrous hydrogel. The high swelling property of dried gel ensures cell migration and proliferation inside bulk of the hydrogels, which provides a facial method to study disease models, the effect of drug dosages, and tissue culture in vitro.

Regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with BMSCs.

Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction.

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.