Passaged Articular Chondrocytes From the Superficial Zone and Deep Zone Can Regain Zone-Specific Properties After Redifferentiation.

BACKGROUND: Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage. HYPOTHESIS: Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone-derived chondrocytes will express superficial zone-specific proteoglycan-4, and only passaged deep zone-derived chondrocytes will express deep zone-specific collagen type X. STUDY DESIGN: Controlled laboratory study. METHODS: Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media. RESULTS: Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes. CONCLUSION: The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage. CLINICAL RELEVANCE: The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.

Lobectomy Demonstrates Superior Survival Than Segmentectomy for High-Grade Non-Small Cell Lung Cancer: The National Cancer Database Analysis.

BACKGROUND: Current recommendations for segmentectomy for non-small cell lung cancer (NSCLC) include size ≤2 cm, margins ≥ 2 cm, and no nodal involvement. This study further stratifies the selection criteria for segmentectomy using the National Cancer Database (NCDB). METHODS: The NCDB was queried for patients with high-grade (poorly/undifferentiated) T1a/b peripheral NSCLC (tumor size ≤2 cm), who underwent either lobectomy or segmentectomy. Patients with pathologic node-positive disease or who received neoadjuvant/adjuvant treatments were excluded. Propensity score analysis was used to adjust for differences in pretreatment characteristics. RESULTS: 11 091 patients were included with 10 413 patients (93.9%) treated with lobectomy and 678 patients (6.1%) underwent segmentectomy. In a propensity matched pair analysis of 1282 patients, lobectomy showed significantly improved median survival of 88.48 months vs 68.30 months for segmentectomy, P = .004. On multivariate Cox regression, lobectomy was associated with significantly improved survival (hazard ratio (HR): .81, 95% CI .72-.92, P = .001). Subgroup analysis of propensity score matched patients with a Charlson-Deyo comorbidity score (CDCC) of 0 also demonstrated a trend of improved survival with lobectomy. DISCUSSION: Lobectomy may confer significant survival advantage over segmentectomy for high-grade NSCLC (≤2 cm). More work is needed to further stratify various NSCLC histologies with their respective grades allowing more comprehensive selection criteria for segmentectomy.

Treatment quality and outcomes vary with hospital burden of uninsured and Medicaid patients with cancer in early non-small cell lung cancer.

Objectives: Safety-net hospitals deliver a significant level of care to uninsured patients, Medicaid-enrolled patients, and other vulnerable patients. Little is known about the impact of safety-net hospital status on outcomes in non-small cell lung cancer. We aimed to compare treatment characteristics and outcomes between hospitals categorized according to their relative burden of uninsured or Medicaid-enrolled patients with non-small cell lung cancer. Methods: We queried the National Cancer Database for patients with clinical stage I and II non-small cell lung cancer presenting from 2004 to 2018. We categorized hospitals on the basis of their relative burden of uninsured or Medicaid-enrolled patients with non-small cell lung cancer into low-burden (<8.2%), medium-burden (8.2%-12.0%), high-burden (12.1%-16.8%), and highest burden (>16.8%) quartiles. We investigated the impact of care at these hospitals on outcomes while controlling for sociodemographic, clinical, and facility characteristics. Results: We identified 204,189 patients treated at 1286 facilities. There were 592 low-burden, 297 medium-burden, 219 high-burden, and 178 highest burden hospitals. Patients at highest burden hospitals were more likely to be younger, male, Black, and Hispanic (P < .01), and to reside in rural, low-income, and low-educated regions (P < .01). Patients at these facilities had a greater likelihood of not receiving surgery, undergoing an open procedure, undergoing a regional lymph node examination involving less than 10 lymph nodes, having a length of stay more than 4 days, and not receiving treatment (P < .05). Conclusions: Our results indicate reduced treatment quality and higher mortality in patients undergoing surgery for early non-small cell lung cancer at hospitals with an increased burden of uninsured or Medicaid-enrolled patients with non-small cell lung cancer. There is a need to raise the standard of care to improve outcomes in vulnerable populations.

Conversion to Thoracotomy in Non-Small Cell Lung Cancer: Risk Factors and Perioperative Outcomes.

Objective: We aimed to identify predictors of conversion to thoracotomy and test the hypothesis that conversion is associated with inferior perioperative outcomes in non-small cell lung cancer (NSCLC). Methods: We queried the National Cancer Database for patients with stage I to III NSCLC undergoing minimally invasive surgery (MIS) during 2010 to 2016. We compared clinicopathologic factors between patients undergoing MIS with and without conversion. We fitted multivariable regression models to identify independent predictors of conversion and compare perioperative outcomes between the 2 groups. Results: A rising trend in the use of MIS was accompanied by a declining trend in the rate of conversion to thoracotomy. A total of 11.3% of the 83,219 cases were converted. Conversion was associated with a higher Charlson-Deyo score, squamous histology, nodal involvement, high tumor grade, tumor size ≥5 cm, and a higher T stage (P < 0.05). Successful MIS without conversion was predicted by advanced age, sublobar resection, robotic approach, and treatment at an academic high-volume facility (P < 0.05). Conversion was linked to longer hospital stays, higher 30-day and 90-day mortality, and unplanned readmission (P < 0.05), irrespective of the type of MIS approach. Conclusions: Conversion rates for video-assisted and robot-assisted thoracoscopic surgery have seen a decline in recent years. Irrespective of the type of MIS approach, conversion was associated with inferior perioperative outcomes. The robotic approach and treatment at an academic high-volume facility were associated with a lower likelihood of conversion. Early recognition of the individual risk factors for conversion may help to counsel patients about the likelihood of, and detriments associated with, conversion and ultimately reduce conversion rates.

Social determinants and facility type impact adherence to best practices in operable IIIAN2 lung cancer.

OBJECTIVES: We aimed to identify patient- and facility-specific predictors of collective adherence to 4 recommended best treatment practices in operable IIIAN2 non-small-cell lung cancer (NSCLC) and test the hypothesis that collective adherence is associated with superior survival. METHODS: We queried the National Cancer Database for clinical stage IIIAN2 NSCLC patients undergoing surgery during 2010-2015. The following best practices were examined: performance of an anatomic resection, performance of an R0 resection, examination of regional lymph nodes and administration of induction therapy. Multivariable regression models were fitted to identify independent predictors of guideline-concordance. RESULTS: We identified 7371 patients undergoing surgical resection for IIIAN2 lung cancer, of whom 90.8% underwent an anatomic resection, 88.2% received an R0 resection, 92.5% underwent a regional lymph node examination, 41.6% received induction therapy and 33.7% received all 4 best practices. Higher income, private insurance and treatment at an academic facility were independently associated with adherence to all 4 best practices (P < 0.01). A lower level of education and residence in a rural county were associated with a lack of adherence (P < 0.05). Adherence to all 4 practices correlated with improved survival (P < 0.01). CONCLUSIONS: National adherence to best treatment practices in operable IIIAN2 lung cancer was variable as evidenced by the majority of patients not receiving recommended induction therapy. Socioeconomic factors and facility type are important determinants of guideline-concordance. Future efforts to improve outcomes should take this into account since guideline concordance, in the form of collective adherence to all 4 best practices, was associated with improved survival.

Travelling to a High-Volume Center Confers Improved Survival in Stage I Non-small Cell Lung Cancer.

BACKGROUND: The association of hospital volume with outcomes has been assessed previously for patients with non-small cell lung cancer (NSCLC), but there are limited data on the cumulative effect of travel burden and hospital volume on treatment decisions and survival outcomes. We used the National Cancer Database to evaluate this relationship in early-stage NSCLC. METHODS: Outcomes of interest were compared between 2 propensity-matched groups with stage I NSCLC: patients in the bottom quartile of distance travelled who underwent surgery at low-volume centers (Local) and those in the top quartile of distance travelled who received surgery at high-volume centers (Distant). Outcomes included type of resection (anatomic or nonanatomic), time to resection (< or ≥8 weeks), number of lymph nodes examined (< or ≥10 nodes) and R0 resection. RESULTS: We identified 3325 Local patients who travelled 2.3 miles (interquartile range [IQR]: 1.4-3.3 miles) to centers that treated 10.5 (IQR: 6.5-16.5) stage I NSCLCs/year and 3361 Distant patients who travelled 40.0 miles (IQR: 29.1-63.4 miles) to centers treating 56.9 (IQR: 40.1-84.7) stage I NSCLCs/year. Local patients were less likely to receive surgery <8 weeks post-diagnosis, have ≥10 lymph nodes examined during surgery, and undergo an R0 resection (all P < .01). Distant patients had shorter hospital stays and superior median survival, both P < .01. CONCLUSIONS: Patients travelling longer distances to high-volume centers receive better and more timely surgical care, leading to shorter hospital stays and improved survival outcomes. Regionalization of lung cancer care by improving travel support to larger treatment facilities may help improve early-stage NSCLC outcomes.

Effect of nutrient metabolism on cartilaginous tissue formation.

A major shortcoming in cartilage tissue engineering is the low biosynthetic response of chondrocytes. While different strategies have been investigated, a novel approach may be to control nutrient metabolism. Although known for their anaerobic metabolism, chondrocytes are more synthetically active under conditions that elicit mixed aerobic-anaerobic metabolism. Here, we postulate this metabolic switch induces HIF-1α signaling resulting in improved growth. Transition to different metabolic states can result in the pooling of metabolites, several of which can stabilize HIF-1α by interfering with PHD2. Chondrocytes cultured under increased media availability accelerated tissue deposition with the greatest effect occurring at 2 ml/106 cells. Under higher media availability, metabolism switched from anaerobic to mixed aerobic-anaerobic. Around this transition, maximal changes in PHD2 activity, HIF-1α expression, and HIF-1 target gene expression were observed. Loss-of-function studies using YC-1 confirmed the involvement of HIF-1. Lastly, targeted metabolomic studies revealed that intracellular lactate and succinate correlated with PHD2 activity. This study demonstrates that cartilaginous tissue formation can be regulated by nutrient metabolism and that this response is mediated through changes in HIF-1α signaling. By harnessing this newly identified metabolic switch, engineered cartilage implants may be developed without the need for sophisticated methods which could aid translation to the clinic.

Invasive thymoma - Which patients benefit from post-operative radiotherapy?

BACKGROUND: The aim of this study is to identify patients with thymoma who should receive post-operative radiotherapy. METHODS: The Surveillance, Epidemiology, and End Results database was queried for stage IIB-IV thymoma patients diagnosed during 1988-2015. We analyzed the prognostic implications of various clinical-pathological factors by comparing the outcomes of those who received surgery with and without post-operative radiotherapy. RESULTS: A total of 1120 patients were identified; 62% received post-operative radiotherapy and 38% underwent surgery alone. In a propensity-matched cohort of 812 patients, no survival difference was seen in World Health Organization A, AB, B1, B2, or B3 tumors with the addition of post-operative radiotherapy to surgery (p>0.05). Post-operative radiotherapy also did not improve survival over surgery alone for tumors ≥ or < less than the 4 cm, 7 cm, 10 cm, and 13 cm cutoffs, all p>0.05. Post-operative radiotherapy was an independent, positive prognostic indicator only in the subgroup with stage III disease and in those receiving chemotherapy in addition to post-operative radiotherapy, both p<0.05. CONCLUSIONS: Patients with stage III thymoma are most likely to benefit from the addition of post-operative radiotherapy to surgical treatments. Tumor size or World Health Organization histology alone should not be criteria for determining the need for post-operative radiotherapy in locally advanced thymoma. Masaoka-Koga stage, which has traditionally been used to help make such decisions, appears to be the most reliable determinant of the use of post-operative radiotherapy.

Novel composite trachea grafts using 3-dimensional printing.

Objective: Porcine-derived small intestine submucosa (SIS) extracellular matrix (ECM) surgical patches claim to have greater regenerative properties compared with dermal extracellular matrices. We hypothesized that using SIS-ECM in a bioengineered composite tracheal graft would allow better incorporation into the native tissue. Methods: Two types of size-matched polycaprolactone support scaffolds were designed: rigid and flexible. The SIS-ECM was wrapped around the polycaprolactone supports lining the inside and outside of the graft. The grafts were implanted in 4 Yorkshire pigs, replacing an ∼2 cm segment of native trachea. Airway patency was evaluated with computed tomography scans and explanted grafts were examined grossly and histologically. Results: All animals survived through the immediate postoperative period. Generally, extraluminal examination showed a smooth transition between native and graft without significant volumetric loss. Animals that received the flexible design survived ∼10 days longer than those that received the rigid design; however, severe perianastomotic intraluminal granulation tissue was observed. The rigid design had less significant intraluminal granulation tissue development at the distal anastomosis, but partial dehiscence had occurred at the proximal anastomosis interrupting graft incorporation. Conclusions: The generally good extraluminal graft incorporation in our composite tracheal graft highlights some increased regenerative capabilities of SIS-ECM. However, the presence of intraluminal granulation tissue indicates that its use as an off-the-shelf, unaltered substrate in an airway graft is still not ideal. Further research must be conducted to determine whether a modification of the substrate is possible to enhance luminal airway incorporation and to exert control over the mechanisms responsible for granulation tissue development.

Patient-specific 3-dimensional lung modelling: next-generation imaging helps guide precise surgical resection.

Recent strides have allowed the consolidation of routine imaging modalities with highly accurate reconstruction software to aid the operating surgeon generate patient-specific three-dimensional models. In this preliminary report, we describe our initial experience using a patient-specific reconstruction software to guide surgical resection for 2 patients with non-small-cell lung cancer. Digital imaging and communications in medicine data from patient chest CT scans was configured into IQQA BodyImaging Lung software to generate highly accurate maps of airways, vessels and segments as well as estimates of lung volumes. Models generated aided us in planning appropriate lung cancer resection procedures.

Anatomic resection has superior long-term survival compared with wedge resection for second primary lung cancer after prior lobectomy.

OBJECTIVES: The extent of surgical resection for early-stage second primary lung cancer (SPLC) in patients with a previous lobectomy is unclear. We sought to compare anatomic lung resections (lobectomy and segmentectomy) and wedge resections for small peripheral SPLC using a population-based database. METHODS: The Surveillance, Epidemiology and End Results database was queried for all patients with ≤2 cm peripheral SPLC diagnosed between 2004 and 2015 who underwent prior lobectomy for the first primary and surgical resection only for the SPLC. American College of Chest Physicians guidelines were used to classify SPLC. Kaplan-Meier analysis and multivariable Cox regression were used to compare overall survival. RESULTS: A total of 356 patients met the inclusion criteria with 203 (57%) treated with wedge resection and 153 (43%) treated with anatomic resection. Significantly better median survival was observed with anatomic resection than with wedge resection using a Kaplan-Meier analysis (124 vs 63 months; P < 0.001). With multivariable Cox regression, improved long-term survival was observed for anatomic resection (hazard ratio: 0.44, confidence interval: 0.27-0.70; P = 0.001). Improvement in survival was demonstrated with wedge resection when lymph node sampling was done. Lastly, we calculated the average treatment effect on the treated with inverse probability weighting for a subgroup of patients and found that those with wedge resection and lymph node sampling had shorter long-term survival times. CONCLUSIONS: Anatomic resections may provide better long-term survival than wedge resections for patients with early-stage peripheral SPLC after prior lobectomy. Significant improvement in survival was observed with wedge resection for SPLC when adequate lymph node dissection was performed.

Successes and Failures in Tracheal Bioengineering: Lessons Learned.

BACKGROUND: Controversy in tracheal reconstruction using grafts and bioengineered constructs highlights the importance of animal studies before human application. Small animal models help to refine designs but do not adequately model sizes relevant to human anatomy. We have conducted extensive large animal studies and summarize our findings in 26 consecutive transplants. METHODS: We pooled 26 large animal studies together to investigate common elements related to successes and failures. In general the engineered tracheal graft consisted of a decellularized extracellular matrix surgical patch supported by a 3-dimensional-printed plastic polymer scaffold. Circumferential graft coverage ranged from 50% to 100%, spanning the length of 4 to 6 tracheal rings. Some grafts included embedded stem cells. Control grafts were fabricated without the support scaffold. At death grafts were harvested and examined grossly and through histology. RESULTS: The support scaffold prevented graft malacia and collapse. Luminal epithelialization was most extensive in grafts with smaller circumferential coverage. Smaller circumferential coverage was also associated with longest animal survival. Chondrogenesis was only observed in grafts with embedded stem cells. Survival time was shortest in 100% circumferential grafts. Granulation tissue was an issue for all graft designs. CONCLUSIONS: Large animal models capture challenges and complexities relevant to human anatomy. Development of granulation tissue remains a challenge, especially in circumferential grafts. Significant additional research is needed to investigate granulation tissue formation and to provide actionable insight into its management.

Lobectomy is superior to segmentectomy for peripheral high grade non-small cell lung cancer ≤2 cm.

BACKGROUND: Current practice guidelines recommend the following criteria for segmentectomy for non-small cell lung cancer (NSCLC): size ≤2 cm, margins ≥2 cm and no lymph node involvement. We sought to further stratify the selection criteria for segmentectomy for small peripheral high-grade tumors. METHODS: This retrospective database study was conducted using the Surveillance, Epidemiology and End Results (SEER) database. We queried for patients with high-grade (poorly differentiated/undifferentiated) pathological (p)T1a/b peripheral NSCLC (tumor size ≤2 cm), who underwent either lobectomy or segmentectomy between 2004 and 2015. Patients with node-positive disease or those who received any form of induction or adjuvant treatments were excluded. RESULTS: A total of 4,332 patients met the inclusion criteria, with 3,977 patients (91.8%) treated with lobectomy and 355 patients (8.2%) who underwent segmentectomy. In a propensity matched pair analysis of 640 patients, lobectomy (n=320) showed significantly improved 5-year survival of 45.9% vs. 33.8% for segmentectomy (n=320), P<0.01. In a multivariate Cox regression analysis, lobectomy was associated with significantly improved survival (HR: 0.84, 95% CI: 0.714-0.989, P=0.036). Interestingly, married status, adenocarcinoma histology, number of lymph nodes sampled were associated with better survival (P<0.05), while advanced age and male gender had worse survival outcomes (P<0.05). CONCLUSIONS: For small peripheral NSCLC ≤2 cm and high grades of tumor differentiation, lobectomy is associated with better long-term survival outcomes as compared to segmentectomy. Additional data is needed to further stratify various NSCLC histologies with their respective grades to allow for better selection for segmentectomy.

Surgical Advances in the Management of the Silent Sinus Syndrome: Our Experience and Review of the Literature.

Silent sinus syndrome is a rare disorder characterized by ipsilateral enophthalmos and hypoglobus caused by collapse of the orbital floor in the presence of asymptomatic long-term maxillary sinusitis. The basic principles in the management are to address the diseased sinus and reconstruct the orbit concurrently or separately. Failure to accurately restore the orbit volume can have significant consequences on the patient. In this article, we provide an update on current practices and highlight our experience of using computer-assisted planning and patient-specific implant in managing this syndrome.

Engineering of scaffold-free tri-layered auricular tissues for external ear reconstruction.

OBJECTIVES: Current strategies for external ear reconstruction can lead to donor site morbidity and/or surgical complications. Tissue-engineered auricular tissues may provide readily available reconstructive materials that resemble native auricular tissue, which is composed of a cartilaginous region sandwiched between two perichondrial layers. We previously developed scaffold-free bi-layered auricular tissues, consisting of a perichondrial layer and a cartilaginous layer, by cultivating chondrocytes and perichondrial cells in a continuous flow bioreactor. Here, we aimed to improve construct properties and develop strategies to engineer tri-layered auricular constructs that better mimic native auricular tissue. STUDY DESIGN: Experimental study. METHODS: Different concentrations of insulin-like growth factor (IGF)-1 and insulin were supplemented during bioreactor culture to determine conditions for engineering bi-layered constructs. We also investigated two methods of engineering tri-layered constructs. Method 1 used Ficoll separation to isolate perichondrial cells, followed by the seeding of isolated perichondrial cells onto the opposing side of the bi-layered constructs. Method 2 involved the growth of the bi-layered constructs in osteogenic culture medium. RESULTS: The combination of 10 nM IGF-1 and 100 nM insulin led to increased collagen content in the engineered bi-layered constructs. For developing tri-layered constructs, method 2 yielded thicker constructs with better mechanical and biochemical properties compared to method 1. In addition, the presence of the perichondrial layers protected the engineered constructs from tissue calcification. CONCLUSION: Auricular tissues with a biomimetic microstructure can be created by growing chondrocytes and perichondrial cells in a continuous flow bioreactor, followed by cultivation in osteogenic medium. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E272-E283, 2019.

Characterization of a novel decellularized bone marrow scaffold as an inductive environment for hematopoietic stem cells.

Due to the increasing demand for a bone marrow study model, we developed a natural scaffold from decellularized bovine bone marrow (DeBM). The obtained bioscaffold was analyzed after the decellularization process; histological staining, scanning and transmission electron microscopy confirmed the preservation of its native 3D-architecture; including blood vessels and cell niches as well as the integrity of important components of the extracellular matrix; Collagen III, IV and fibronectin. In addition to biochemical composition, physical properties of the bone marrow were also conserved. We evaluated the suitability of this bio-scaffold as a tridimensional culture platform. Seeding experiments with umbilical cord-derived hematopoietic stem cells and human bone marrow stromal cell line HS5 demonstrated that this scaffold is capable of supporting hematopoietic and stromal cell adhesion and proliferation without the need of exogenous factors. DeBM provided an inductive environment for the repopulation of the bone marrow inducing the expression of SDF-1, HGF and SCF by seeded stromal cells. The presence of these potent hematopoietic chemoattractants would be crucial for ex vivo long-term culture of HSCs, and for recreating the natural microenvironment of the bone marrow for bioengineering applications. We conclude that the decellularization process succeeded in preserving the 3D structure and mechanical properties of the bone marrow. The resulting scaffold is suitable for cell culture, representing an advantageous bone marrow experimental model, and potentially an effective platform for CD34+ HSC expansion and differentiation for clinical applications.

Stochastic Resonance with Dynamic Compression Improves the Growth of Adult Chondrocytes in Agarose Gel Constructs.

Dynamic mechanical stimulation has been an effective method to improve the growth of tissue engineering cartilage constructs derived from immature cells. However, when more mature cell populations are used, results are often variable due to the differing responses of these cells to external stimuli. This can be especially detrimental in the case of mechanical loading. In previous studies, multi-modal mechanical stimulation in the form of stochastic resonance was shown to be effective at improving the growth of young bovine chondrocytes. Thus, the aim of this study was to investigate the short-term and long-term effects of stochastic resonance on two groups of bovine chondrocytes, adult (> 30 month) and juvenile (~ 18 months). While the juvenile cells outperformed the adult cells in terms of their anabolic response to loading, combined mechanical loading for both age groups resulted in greater matrix synthesis compared to compressive loading alone. In the adult cells, potential pathological tissue formation was evident with the presence of cell clustering. However, the presence of broad-band mechanical vibrations (alone or with compressive loading) appeared to mitigate this response and allow these cells to attain a growth response similar to the juvenile, unstimulated cells. Therefore, the use of stochastic resonance appears to show promise as a method to improve the formation and properties of tissue engineered cartilage constructs, irrespective of cell age.

Comparisons of Auricular Cartilage Tissues from Different Species.

OBJECTIVES: Tissue engineering of auricular cartilage has great potential in providing readily available materials for reconstructive surgeries. As the field of tissue engineering moves forward to developing human tissues, there needs to be an interspecies comparison of the native auricular cartilage in order to determine a suitable animal model to assess the performance of engineered auricular cartilage in vivo. METHODS: Here, we performed interspecies comparisons of auricular cartilage by comparing tissue microstructure, protein localization, biochemical composition, and mechanical properties of auricular cartilage tissues from rat, rabbit, pig, cow, and human. RESULTS: Human, pig, and cow auricular cartilage have smaller lacunae compared to rat and rabbit cartilage ( P < .05). Despite differences in tissue microstructure, human auricular cartilage has similar biochemical composition to both rat and rabbit. Auricular cartilage from pig and cow, alternatively, display significantly higher glycosaminoglycan and collagen contents compared to human, rat, and rabbit ( P < .05). The mechanical properties of human auricular cartilage were comparable to that of all 4 animal species. CONCLUSIONS: This is the first study that compares the microstructural, biochemical, and mechanical properties of auricular cartilage from different species. This study showed that different experimental animal models of human auricular cartilage may be suitable in different cases.

Bioengineering pediatric scaffold-free auricular cartilaginous constructs.

OBJECTIVE: The use of exogenous materials as scaffolds in cartilage tissue engineering has limited the clinical application of resultant constructs due to the risk of postoperative complications. In an effort to minimize such complications, we aim to generate human, scaffold-free auricular cartilaginous constructs. STUDY DESIGN: Laboratory study using pediatric auricular cartilage. METHODS: Remnant, normal pediatric auricular cartilage samples that would have otherwise been discarded were collected and digested to free cells. Harvested cells were cultured and expanded in vitro for two passages and plated as micromass cultures. The culture medium was replaced with a chemically defined chondrogenic medium, and cellular monolayers surrounding micromass cultures were continuously scraped off. Constructs were allowed to mature for a period of 8 weeks. RESULTS: Micromass constructs showed mechanical stability and structurally resembled native auricular tissue, with a perichondrium-like layer of cells surrounding the inner cartilaginous zone. Constructs accumulated equivalent sulphated glycosaminoglycan and 50% of collagen content compared to native auricular cartilage by mass, while displaying 156% more cellularity. CONCLUSIONS: High-density micromass cultures of pediatric auricular chondrocytes can generate stable cartilaginous constructs following prolonged chondrogenic inductions in vitro. This technique is an essential step toward the development of three-dimensional constructs to recreate clinically applicable auricular cartilaginous constructs. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E153-E158, 2017.

Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

OBJECTIVE: When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFß) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFß supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture. METHODS: Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFß. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically. RESULTS: Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFß3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers. CONCLUSIONS: This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.