Standardization of Microcomputed Tomography for Tracheal Tissue Engineering Analysis.

Tracheal tissue engineering has become an active area of interest among clinical and scientific communities; however, methods to evaluate success of in vivo tissue-engineered solutions remain primarily qualitative. These evaluation methods have generally relied on the use of photographs to qualitatively demonstrate tracheal patency, endoscopy to image healing over time, and histology to determine the quality of the regenerated extracellular matrix. Although those generally qualitative methods are valuable, they alone may be insufficient. Therefore, to quantitatively assess tracheal regeneration, we recommend the inclusion of microcomputed tomography (µCT) to quantify tracheal patency as a standard outcome analysis. To establish a standard of practice for quantitative µCT assessment for tracheal tissue engineering, we recommend selecting a constant length to quantify airway volume. Dividing airway volumes by a constant length provides an average cross-sectional area for comparing groups. We caution against selecting a length that is unjustifiably large, which may result in artificially inflating the average cross-sectional area and thereby diminishing the ability to detect actual differences between a test group and a healthy control. Therefore, we recommend selecting a length for µCT assessment that corresponds to the length of the defect region. We further recommend quantifying the minimum cross-sectional area, which does not depend on the length, but has functional implications for breathing. We present empirical data to elucidate the rationale for these recommendations. These empirical data may at first glance appear as expected and unsurprising. However, these standard methods for performing µCT and presentation of results do not yet exist in the literature, and are necessary to improve reporting within the field. Quantitative analyses will better enable comparisons between future publications within the tracheal tissue engineering community and empower a more rigorous assessment of results. Impact statement The current study argues for the standardization of microcomputed tomography (µCT) as a quantitative method for evaluating tracheal tissue-engineered solutions in vivo or ex vivo. The field of tracheal tissue engineering has generally relied on the use of qualitative methods for determining tracheal patency. A standardized quantitative evaluation method currently does not exist. The standardization of µCT for evaluation of in vivo studies would enable a more robust characterization and allow comparisons between groups within the field. The impact of standardized methods within the tracheal tissue engineering field as presented in the current study would greatly improve the quality of published work.

Biodegradable electrospun patch containing cell adhesion or antimicrobial compounds for trachea repair in vivo.

Difficulty breathing due to tracheal stenosis (i.e. narrowed airway) diminishes the quality of life and can potentially be life-threatening. Tracheal stenosis can be caused by congenital anomalies, external trauma, infection, intubation-related injury, and tumors. Common treatment methods for tracheal stenosis requiring surgical intervention include end-to-end anastomosis, slide tracheoplasty and/or laryngotracheal reconstruction. Although the current methods have demonstrated promise for treatment of tracheal stenosis, a clear need exists for the development of new biomaterials that can hold the trachea open after the stenosed region has been surgically opened, and that can support healing without the need to harvest autologous tissue from the patient. The current study therefore evaluated the use of electrospun nanofiber scaffolds encapsulating 3D-printed PCL rings to patch induced defects in rabbit tracheas. The nanofibers were a blend of polycaprolactone (PCL) and polylactide-co-caprolactone (PLCL), and encapsulated either the cell adhesion peptide, RGD, or antimicrobial compound, ceragenin-131 (CSA). Blank PCL/PLCL and PCL were employed as control groups. Electrospun patches were evaluated in a rabbit tracheal defect model for 12 weeks, which demonstrated re-epithelialization of the luminal side of the defect. No significant difference in lumen volume was observed for the PCL/PLCL patches compared to the uninjured positive control. Only the RGD group did not lead to a significant decrease in the minimum cross-sectional area compared to the uninjured positive control. CSA reduced bacteria growth in vitro, but did not add clear value in vivo. Adequate tissue in-growth into the patches and minimal tissue overgrowth was observed inside the patch material. Areas of future investigation include tuning the material degradation time to balance cell adhesion and structural integrity.

Reinforced Electrospun Polycaprolactone Nanofibers for Tracheal Repair in an In Vivo Ovine Model.

Tracheal stenosis caused by congenital anomalies, tumors, trauma, or intubation-related damage can cause severe breathing issues, diminishing the quality of life, and potentially becoming fatal. Current treatment methods include laryngotracheal reconstruction or slide tracheoplasty. Laryngotracheal reconstruction utilizes rib cartilage harvested from the patient, requiring a second surgical site. Slide tracheoplasty involves a complex surgical procedure to splay open the trachea and reconnect both segments to widen the lumen. A clear need exists for new and innovative approaches that can be easily adopted by surgeons, and to avoid harvesting autologous tissue from the patient. This study evaluated the use of an electrospun patch, consisting of randomly layered polycaprolactone (PCL) nanofibers enveloping 3D-printed PCL rings, to create a mechanically robust, suturable, air-tight, and bioresorbable graft for the treatment of tracheal defects. The study design incorporated two distinct uses of PCL: electrospun fibers to promote tissue integration, while remaining air-tight when wet, and 3D-printed rings to hold the airway open and provide external support and protection during the healing process. Electrospun, reinforced tracheal patches were evaluated in an ovine model, in which all sheep survived for 10 weeks, although an overgrowth of fibrous tissue surrounding the patch was observed to significantly narrow the airway. Minimal tissue integration of the surrounding tissue and the electrospun fibers suggested the need for further improvement. Potential areas for further improvement include a faster degradation rate, agents to increase cellular adhesion, and/or an antibacterial coating to reduce the initial bacterial load.

Comparison of Platelet-Rich Plasma, Stromal Vascular Fraction (SVF), or SVF with an Injectable PLGA Nanofiber Scaffold for the Treatment of Osteochondral Injury in Dogs.

Stromal vascular fraction (SVF) contains a small number of mesenchymal stem cells and has been used as a treatment for osteoarthritis and cartilage injury. Due to limited evidence of successful cartilage regeneration with injected stem cell therapies, there is interest in combining cellular therapies with injectable scaffolding materials to increase intra-articular residence times of stem cells and improve tissue regeneration. However, the safety of intra-articular injection of SVF combined with injectable scaffolds is unestablished. Also, it is unclear if SVF therapy is superior to more easily prepared biologics, such as platelet-rich plasma (PRP). The purpose of this study was to assess the safety of SVF when combined with an injectable poly(L-lactide-co-glycolide) nanofiber scaffold and to provide a comparison of SVF therapy to PRP. A total of 12 Beagles had osteochondral defects created in both medial femoral condyles and 4 dogs each were allocated to treatment groups of SVF (n = 4), SVF plus PLGA scaffolding (n = 4), or leukoreduced PRP (n = 4). One knee in each dog received treatment, and the contralateral knee was sham treated with saline. Dogs were assessed over a 6-month period, and outcome measures included functional, radiographic, biochemical, and histological assessments. PRP treatment resulted in improvements in lameness scores and objective kinetic assessments of function. There were no statistically significant improvements in function, cartilage biochemical composition, or histology for SVF-treated knees. The combination of SVF and the injectable PLGA scaffold had worse outcomes than other groups including sham treatment based upon functional, biochemical, and histological assessments, raising concerns over the safety of this scaffold for intra-articular injection.

Mechanical evaluation of gradient electrospun scaffolds with 3D printed ring reinforcements for tracheal defect repair.

Tracheal stenosis can become a fatal condition, and current treatments include augmentation of the airway with autologous tissue. A tissue-engineered approach would not require a donor source, while providing an implant that meets both surgeons' and patients' needs. A fibrous, polymeric scaffold organized in gradient bilayers of polycaprolactone (PCL) and poly-lactic-co-glycolic acid (PLGA) with 3D printed structural ring supports, inspired by the native trachea rings, could meet this need. The purpose of the current study was to characterize the tracheal scaffolds with mechanical testing models to determine the design most suitable for maintaining a patent airway. Degradation over 12 weeks revealed that scaffolds with the 3D printed rings had superior properties in tensile and radial compression, with at least a three fold improvement and 8.5-fold improvement, respectively, relative to the other scaffold groups. The ringed scaffolds produced tensile moduli, radial compressive forces, and burst pressures similar to or exceeding physiological forces and native tissue data. Scaffolds with a thicker PCL component had better suture retention and tube flattening recovery properties, with the monolayer of PCL (PCL-only group) exhibiting a 2.3-fold increase in suture retention strength (SRS). Tracheal scaffolds with ring reinforcements have improved mechanical properties, while the fibrous component increased porosity and cell infiltration potential. These scaffolds may be used to treat various trachea defects (patch or circumferential) and have the potential to be employed in other tissue engineering applications.

The effects of electrospun substrate-mediated cell colony morphology on the self-renewal of human induced pluripotent stem cells.

The development of xeno-free, chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard, various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal, i.e., proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly, when surface chemistry of the substrates was uniformly controlled by collagen conjugation, the stiffness of substrate was inversely related to the sphericity, a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture, implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall, we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal.

Hemoglobin regulates the migration of glioma cells along poly(ε-caprolactone)-aligned nanofibers.

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2 )-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2 . Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2 , while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2 , Hb did not impact aggregate dispersion. Also at 1% O2 , aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on nanofibers.

Comparison of the mechanical characteristics of polymerized caprolactam and monofilament nylon loops constructed in parallel strands or as braided ropes versus cranial cruciate ligaments of cattle.

OBJECTIVE: To compare the mechanical characteristics of polymerized caprolactam and monofilament nylon loops with those of the cranial cruciate ligament (CCL) in cattle. SAMPLE: 6 femorotibial joints harvested from 3 cows and suture constructs made from No. 8 polymerized caprolactam, 80-lb test monofilament nylon fishing line, and 450-lb test monofilament nylon fishing line. PROCEDURES: Joints were cleared of soft tissue structures except the CCL, connected to a load frame, and loaded to failure while measuring force and elongation. Synthetic constructs tested in a similar manner included single-stranded and 3-stranded No. 8 polymerized caprolactam, 3- and 6-stranded 80-lb test monofilament nylon fishing line, and 3- and 6-stranded 450-lb test monofilament nylon fishing line. RESULTS: The CCL ruptured at a mean ± SD force of 4,541 ± 1,417 N with an elongation of 2.0 ± 0.3 cm. The tensile strength of 3-stranded 450-lb test monofilament nylon fishing line was similar to that of the CCL, rupturing at loads of 5,310 ± 369 N (braided strands) and 6,260 ± 239 N (parallel strands). Elongation was greater for braided constructs. CONCLUSIONS AND CLINICAL RELEVANCE: The 3-stranded cords of 450-lb test monofilament nylon fishing line most closely approximated the strength of the CCL. Marked increases in elongation occur when large-sized materials are constructed in braided configurations, and this elongation would likely not provide stability in CCL-deficient stifle joints. Additional studies are needed to determine whether any of these materials are suitable CCL replacements in cattle.

Cell attachment to hydrogel-electrospun fiber mat composite materials.

Hydrogels, electrospun fiber mats (EFMs), and their composites have been extensively studied for tissue engineering because of their physical and chemical similarity to native biological systems. However, while chemically similar, hydrogels and electrospun fiber mats display very different topographical features. Here, we examine the influence of surface topography and composition of hydrogels, EFMs, and hydrogel-EFM composites on cell behavior. Materials studied were composed of synthetic poly(ethylene glycol) (PEG) and poly(ethylene glycol)-poly(ε-caprolactone) (PEGPCL) hydrogels and electrospun poly(caprolactone) (PCL) and core/shell PCL/PEGPCL constituent materials. The number of adherent cells and cell circularity were most strongly influenced by the fibrous nature of materials (e.g., topography), whereas cell spreading was more strongly influenced by material composition (e.g., chemistry). These results suggest that cell attachment and proliferation to hydrogel-EFM composites can be tuned by varying these properties to provide important insights for the future design of such composite materials.

Glioma cell migration on three-dimensional nanofiber scaffolds is regulated by substrate topography and abolished by inhibition of STAT3 signaling.

A hallmark of malignant gliomas is their ability to disperse through neural tissue, leading to long-term failure of all known therapies. Identifying new antimigratory targets could reduce glioma recurrence and improve therapeutic efficacy, but screens based on conventional migration assays are hampered by the limited ability of these assays to reproduce native cell motility. Here, we have analyzed the motility, gene expression, and sensitivity to migration inhibitors of glioma cells cultured on scaffolds formed by submicron-sized fibers (nanofibers) mimicking the neural topography. Glioma cells cultured on aligned nanofiber scaffolds reproduced the elongated morphology of cells migrating in white matter tissue and were highly sensitive to myosin II inhibition but only moderately affected by stress fiber disruption. In contrast, the same cells displayed a flat morphology and opposite sensitivity to myosin II and actin inhibition when cultured on conventional tissue culture polystyrene. Gene expression analysis indicated a correlation between migration on aligned nanofibers and increased STAT3 signaling, a known driver of glioma progression. Accordingly, cell migration out of glioblastoma-derived neurospheres and tumor explants was reduced by STAT3 inhibitors at subtoxic concentrations. Remarkably, these inhibitors were ineffective when tested at the same concentrations in a conventional two-dimensional migration assay. We conclude that migration of glioma cells is regulated by topographical cues that affect cell adhesion and gene expression. Cell migration analysis using nanofiber scaffolds could be used to reproduce native mechanisms of migration and to identify antimigratory strategies not disclosed by other in vitro models.

Organ-derived coatings on electrospun nanofibers as ex vivo microenvironments.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by irreversible scarring. Collagen deposition, myofibroblast expansion, and the development of fibroblastic foci are the hallmark pathological events. The origin and mechanism of recruitment of myofibroblasts, the key cell contributing to these events, is unknown. We hypothesize that the fibrotic lung microenvironment causes differentiation of arriving bone marrow-derived cells into myofibroblasts. Therefore, a method of isolating the effects of fibrotic microenvironment components on various cell types was developed. Electrospun nanofibers were coated with lung extracts from fibrotic or non-fibrotic mice and used to determine effects on bone marrow cells from naïve mice. Varying moduli nanofibers were also employed to determine matrix stiffness effects on these cells. At structured time points, bone marrow cell morphology was recorded and changes in fibrotic gene expression determined by real-time PCR. Cells plated on extracts isolated from fibrotic murine lungs secreted larger amounts of extracellular matrix, adopted a fibroblastic morphology, and exhibited increased myofibroblast gene expression after 8 and 14 days; cells plated on extracts from non-fibrotic lungs did not. Similar results were observed when the nanofiber modulus was increased. This ex vivo system appears to recapitulate the three-dimensional fibrotic lung microenvironment.

Effects of orthopedic implants with a polycaprolactone polymer coating containing bone morphogenetic protein-2 on osseointegration in bones of sheep.

OBJECTIVE: To determine elution characteristics of bone morphogenetic protein (BMP)-2 from a polycaprolactone coating applied to orthopedic implants and determine effects of this coating on osseointegration. ANIMALS: 6 sheep. PROCEDURES: An in vitro study was conducted to determine BMP-2 elution from polycaprolactone-coated implants. An in vivo study was conducted to determine the effects on osseointegration when the polycaprolactone with BMP-2 coating was applied to bone screws. Osseointegration was assessed via radiography, measurement of peak removal torque and bone mineral density, and histomorphometric analysis. Physiologic response was assessed by measuring serum bone-specific alkaline phosphatase activity and uptake of bone markers. RESULTS: Mean +/- SD elution on day 1 of the in vitro study was 263 +/- 152 pg/d, which then maintained a plateau at 59.8 +/- 29.1 pg/d. Mean peak removal torque for screws coated with polycalprolactone and BMP-2 (0.91 +/- 0.65 dN x m) and screws coated with polycaprolactone alone (0.97 +/- 1.30 dN.m) did not differ significantly from that for the control screws (2.34 +/- 1.62 dN x m). Mean bone mineral densities were 0.535 +/- 0.060 g/cm(2), 0.596 +/- 0.093 g/cm(2), and 0.524 +/- 0.142 g/cm(2) for the polycaprolactone-BMP-2-coated, polycaprolactone-coated, and control screws, respectively, and did not differ significantly among groups. Histologically, bone was in closer apposition to the implant with the control screws than with either of the coated screws. CONCLUSIONS AND CLINICAL RELEVANCE: BMP-2 within the polycaprolactone coating did not stimulate osteogenesis. The polycaprolactone coating appeared to cause a barrier effect that prevented formation of new bone. A longer period or use of another carrier polymer may result in increased osseointegration.